Everything

· Tracks for intracellular movement · Hollow tubes, 25nm diameter · Move organelles, vesicles, chromosomes · Adjacent dimers of α-tubulin and β-tubulin · Each dimer made by 8-9 genes · Dimers form protofilaments, which form filaments · Grow from microtubule organising centres (MTOCs) such as centrosomes and basal bodies · Modified in cilia and flagella · Chemotherapy target (mitosis – movement of chromosomes) · Have + end where β-tubulin is exposed · Have – end where α-tubulin is exposed · GTP-tubulin is added to + end; this is where polymerisation happens · GDP-tubulin falls off at – end; this is where depolymerisation happens · The – end is capped by the ** gamma tubulin ring complex **to keep polymerisation at + end only · Has two associated motor proteins, kinesins and dyenins · 8nm steps (breadth of one dimer) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Walks on single protofilament · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">A + end directed motor (moves towards cell periphery) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Hydrolyses ATP; ATPase accelerates upon binding to microtubule (some mutations inhibit this acceleration) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">A minus end directed motor (moves into cell) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Hydrolyses ATP; activated by dynactin · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Binds to side or end of microtubules · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Increases or decreases microtubule stability · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">MAPs in neurones are tau and MAP2 o <span style="font-family: 'Times New Roman',serif; font-size: 10pt; line-height: 115%;">Tau binds to microtubules in axons o <span style="font-family: 'Times New Roman',serif; font-size: 10pt; line-height: 115%;">MAP2 binds to microtubules in cell bodies and dendrites o <span style="font-family: 'Times New Roman',serif; font-size: 10pt; line-height: 115%;">Both bundle and stabilise microtubules o <span style="font-family: 'Times New Roman',serif; font-size: 10pt; line-height: 115%;">MAP2 produces more highly-spaced bundles than tau · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Kinesin mutations results in ** trafficking defects **. Problem in neurones because mitochondria need to be trafficked to synapses. · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Tuba1a mutation: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lissencephaly (smooth brain) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Microcephaly (small head) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Seizures, spasms o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Average life expectancy < 2 years o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Absence of folds in cortex o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Due to defects in embryonic neural migration · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Kif5a mutation in kinesin-1 of neurone o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Hereditary spastic paraplegia o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Progressive weakness in limbds o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Onset around 24 years o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Frequency around 3 in 100,000 o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Autosomal dominat o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mis-sense mutation – single amino acid in motor domain of kinesin so it cannot move along microtubule · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">N265S mutation o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Autosomal dominant o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Peripheral neuropathy · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">KIF1Bβ mutation o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Trafficking kinesin in neurones o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">CMT disease type 2A · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutations in tau o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cannot associate with microtubules in axons o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Microtubules fall apart because not stabilised o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Organelles cannot be trafficked o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Taus self-assemble into plaques o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Associated with hyper-phosphorylation of tau o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Caspase (cysteine protease, involved in apoptosis) normally activated by tau o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Caspase cleaved at D421 causes truncated tau which recruits full-length tau o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Leads to ** neuronal dysfunction ** and ** no apoptosis ** · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Tubulin, kinesin, dyenin and MAPs · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Especially cause neuronal disease · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Drug that inhibits kinesins – called monastrol – used in cancer treatment because monaster is the star-shaped formation at the end of prophase · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Polymers of actin monomers · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Actin monomers have ATP-binding cleft · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Monomers are 42kDa (375 amino acids) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">α-actin found in muscle cells · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">β and γ-actin found in non-muscle cells · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Actin filament is helical o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">2 stranded right-handed helix or   o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Shallow left-handed genetic helix · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Track for the protein myosin · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Required for: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Muscle contraction o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cell crawling o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Wound healing o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Chemotaxis in bacteria/white blood cells · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cardiac muscle: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutations in sarcomeric proteins (actin, myosin, tropomyosin) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Hypertrophic or dilated cardiac myopathy o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutations in heavy chain of myosin = 30-40% of HCM cases · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Skeletal muscle: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Interferes with actin polymerisation and sarcomere assembly o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">In diaphragm = respiratory failure · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Smooth muscle: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">α-actin mutation (ACTA2) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">degeneration of middle layer of aorta · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Blood diseases, deafness, blindness · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Deafness: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">DFNA20/26 = mutation in γ-actin causing progressive hearing loss o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Myo7a – mutation in myosin 7 causing Usher’s syndrome – deafblind o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Myosin 7 needed for proper formation of stereocilia · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Bacteria such as lysteria can hijack actin cytoskeleton · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Actin cytoskeleton involved in endo- and exocytosis: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Griscelli disease = patchy skin pigmentation due to improper transport of melanosomes <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Size in between microtubules and actin filaments · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Rope-like · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">10nm diameter · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Tough and durable · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Maintain cell shape · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Filaments fibrous and elongated · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Not dynamic (no motor proteins) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Maintain nuclear shape = ** nuclear lamina ** · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Different names in different cells: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Epithelial cells = keratin o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Muscle cells = desmin o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Fibroblasts = vimentin o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nerve cells = neurofilaments · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Formed of polypeptides, which form dimers, then tetramers, then filaments · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Required for: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cell-cell adhesion (desmosomes) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cell-ECM adhesion (hemi-desmosomes or focal adhesions) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutations in keratin o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Skin blistering o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Because skin layers don’t attach to each other and to ECM · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutations in desmin o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Myopathies – desminopathy o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Hypertrophic or dilated cardiac myopathy, heart arrhythmia · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutations in neurofilaments o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">CMT disease types 1 and 2E o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Parkinson’s Disease o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Amyotrophic lateral sclerosis I   · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutations in nuclear lamina o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">CMT disease o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Dilated myopathy o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Limb girdle muscular dystrophy o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Progeria (big head, premature ageing) <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Roles include o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Energy production o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Apoptosis o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Protect nucleus from high Ca2+= o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Platform for molecular signalling · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Have circular DNA · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Maternally inherited · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">37 genes on 16,569 base pairs o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">13 protein-coding o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">2 rRNA-coding o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">22 tRNA-coding (cellular tRNA cannot penetrate membrane) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Have own ribosomes (70S) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mitochondrial genome o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Heavy strand has two promoters IH1 and IH2 o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Light strand has one promoter IL   o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Promoter-associated elements found in D-loop · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mitochondrial proteome o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">1000 proteins o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">13 mtDNA-encoded (respiratory enzymes) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Rest are nuclear-encoded and imported · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Energy generation: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">5 complexes § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">NADH dehydrogenase § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Fumarate reductase § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cytochrome bc1 complex § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cytochrome c oxidase § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">ATP synthetase o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Electrons donated from NADH (complex I) and FADH2 (complex II) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Protons pumped from matrix to I.M. space on complexes I, III and IV   o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">ATP released into cytosol, and ADP+Pi imported, using ANT = adenine nucleotide transporter o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">UDAC = non-specific voltage-dependent anion channel (since ADP is anionic due to negative phosphates) <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">TOM complex = translocase outer membrane · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">TIM complex = translocase inner membrane · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Transit peptide needed for import o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">N-terminal o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Positively charged (basic) amino acids · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cytosolic chaperones guide protein to TOM complex · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">TOM receptors recognise transit peptide and translocate protein · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">General import pore (GIP) guides protein through outer membrane · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">TIM complex: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">TIM17+23 = import pore o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">TIM44 = peripheral subunit · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Matrix and inter-membrane space have a potential difference – provides energy for translocation across TIM · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Matrix Hsp70 + TIM44 + MgeI chaperone: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">ATP-dependent motor for complete import · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Once inside the matrix, mitochondrial processing peptidase (MPP) cleaves transit peptide · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Protein found in inner mitochondrial membrane · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Arranged in rings (PHB complexes) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">16-20 alternating Phb1/2 · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Role unclear o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Possibly mitochondrial scaffold, or chaperone for respiratory proteins · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Oxygen ions, hydrogen peroxide · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Normally used in cell signalling · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Environmental stress increases concentration of ROS · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Leads to DNA and cell damage = ** oxidative stress ** · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Result of oxidative metabolism, or ionising radiation · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">ROS can damage mitochondrial proteins, impairing ATP synthesis · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">ROS can induce mitochondrial permeability transition pore (PTP) of inner membrane, making it accessible to small molecules · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">ROS = modulatable redox signal = reversibly affects activity in mitochondria, nucleus, cytosol <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Alzheimers and Parkinsons often caused by too much apoptosis of neurones · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Caused by mitochondria – many mitochondrial diseases are neurodegenerative · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Apoptosis regulated by   o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Intrinsic factors (mitochondrial) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Extrinsic factors (death factors) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">These come together to stimulate caspases – apoptotic enzymes · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mitochondrial-directed (intrinsic) apoptosis: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Increased permeability of PTP caused by high levels of ROS o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Dissipation of H+ gradient across inner membrane, which was made by ETC o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">H2O accumulates in matrix, causing swelling and rupturing of outer membrane o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Apoptotic agents released: § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cytochrome C   § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Apoptosis protease activating factor (APAF-1) § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Pro-caspase G   § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Ca2+ (stimulates caspase) § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">These agents are also stimulated by Aβ plaques in Alzheimers · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Viral RNA detected by intracellular pattern recognition receptors (PRRs) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Convergence of these and other signalling molecules to outer mitochondrial membrane o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Rapid activation of antiviral cytokines o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Including type 1 interferons, which signal antibody production o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">NF-KB also induces transcription of immunity genes o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Interferon/NF-KB can be assayed using reporter genes <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">MAVS receptor on mitochondria and peroxisomes recognises viral RNA · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Peroxisomes respond ** first ** o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Rapid response o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Interfereon-** independent ** o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Defence factors o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Short-term protection · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mitochondria have ** delayed response ** o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Interferon-** dependent ** o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Amplifies and stabilises the antiviral response <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Storage, replication and transcription of DNA · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Response to cellular stress (e.g. production of interferons) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleus visualised by fluorescence microscopy (resolution 200nm) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Field-emission scanning electron microscopy for 3-5nm structures (e.g. chromatin, nuclear pore complexes) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleus has ‘cytoskeleton’ – which changes structure in response to stress · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">DNA is organised on a scaffold called nuclear matrix · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">DNA is wrapped around positively-charged histone octamers · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">10nm chromatin fibres – nucleosome – then further condensation to 30nm fibres · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Chromatin structural changes to regulate gene expression · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Gene insulators = regions of looped DNA to prevent the spread of chromatin remodelling · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nuclear proteome is highly dynamic o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">For example, more replicative enzymes present during S-phase o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Analyse proteome in health vs. disease to see which proteins are over/under-expressed · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nuclear pore complex = not empty hole, network of mirror-imaged protein fibrils · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Small proteins (less than 50kDa) can enter down concentration gradient by diffusion · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">When protein is too large / moving against concentration gradient = need energy from GTP · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Can characterise which proteins are energy-dependent by: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cooling cell to 15C o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Ran-GTP is inhibitied o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">GTP not hydrolysed, proteins not translocated · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Import of proteins through importins o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Ran-GDP bound o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">GDP dissociates once inside nucleus o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Ran-GEF exchanges GDP for GTP · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Export of proteins through exportins o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Ran-GTP bound o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">GTP hydrolysed to release Pi   o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Ran-GAP involved in GTP hydrolysis · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Import **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> requires a ** nuclear localisation signal (NLS), ** which are ** basic: ** o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Recogniton requires energy o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Found on protein surface o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Can be masked by phosphorylation o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Pat4 = 4 basic amino acids o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Pat7 = 7 basic amino acids o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Bipartite = arginine + lysine · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Export signals **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> are ** hydrophobic: ** o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Leu/Val – XXX – Leu/Val – XX – Leu/Val o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Also found on protein surface o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">There are approx. 10 export pathways o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">All require Ran-GTP and an exportin · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Import and export signals can be characterised by deleting amino acids and then monitoring translocation with enhanced GFP · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleus is home of the cell cycle: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Positively regulated by cyclin-dependent kinases (CDKs), which are themselves activated by phosphorylation by a cyclin complex = maturation promoting factors (MPFs) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Negatively regulated by CDK inhibitors · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Oncogenes cause uncontrolled cell division when disrupted – problem in gene therapy · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">CDK2 and cyclin B are two pro-cell cycle proteins. Their targets: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Histone 1: § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">H1 phosphorylation § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Dissociates from DNA § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Forced condensation of chromosomes § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Start of prophase o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lamins: § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Proteoglycans in nuclear envelope § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Phosphorylation prevents polymerisation § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nuclear envelope breaks down, leading to spatial separation of chromosomes and onset of mitosis <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">5-30 per nucleus · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">0.2-1µm in size · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Sequester proteins involved in apoptosis, antiviral response, DNA repair, etc · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Modifies them and arranges them into complexes · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Integrity ensured by ** p **ro** m **yelocytic ** l **eukaemia (PML) proteins – without this structure, targeted proteins form a diffuse localisation, or concentrate into random microspeckles · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nuclear domain 10 (ND10) proteins form around the genes involved in immunity · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Disturbances in the balance of ND10: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Acute promyelocytic leukaemia (APL) – individuals have a genetic translocation where PML gene is fused to alpha retinoic acid receptor gene, leading to fragmented PML bodies. Treated with retinoic acid to restore normal PML bodies. o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Ageing progression o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Increased viral replication · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Because of role of PML-NBs in antiviral response, they do not appear in healthy/unstressed cells · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Unspliced RNA cannot be trafficked from nucleus · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Spliceosomes are small ribonucleoprotein particles (snRNPs) made of snRNA and protein. Catalyse introns removal. · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cajal bodies **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> are involved in snRNP assembly · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">0.3-1µm in diameter · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Contents of cajal bodies not separated from nucleoplasm by phospholipid bilayer · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">snRNPs are modified and stored in P and U bodies in cytoplasm · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nuclear GEMs **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> are adjacent to cajal bodies o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Contain proteins involved in splicing o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Disease – spinal muscular atrophy · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Dense with proteins so can be seen under light microscope · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Surrounded by histones (protein-protein, protein-nucleic acid interactions), rather than phospholipid bilayer · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Associated with many diseases: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Auto-immunity: lupus, scleroderma o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Viral infection and hi-jacking o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cancers: APL, nucleostemin, Bloom/Werner’s Syndrme · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Alterations of nuclear proteome, either genetically or virally induced, causes changes in the loss or sequestration of proteins by the nucleus = disease · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleolus involved in ribosome biogenesis. · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">rRNA trafficking: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Fibrillar centre (FC) – depot of rDNA genes o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Dense fibrillar component (DFC) – maturation of pre-mRNA transcripts o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Granular component (GC) – assembly of pre-ribosomal particles o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Peri-nucleolar region – before release · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">4500 proteins traffic to/reside in nucleus; release of proteins varies: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">G1 phase – lots of ribosome biogenesis o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Clusters into 1 single nucleolus at end of G2 phase o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Falls apart during mitosis when not needed · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleolin – **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">protein in nucleus o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">10% of nucleolar proteins o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">His ‘disordered’ regions with no defined tertiary structure § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Can bind to many proteins incl. viral proteins § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">These proteins interact with other proteins, e.g. telomerase, p53 · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleolus relatively easy to isolate intact (centrifugation) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Use quantitative proteomics to compare protein in health and disease o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Stain and hybridise antibodies to nucleolar extract containing nucleolin o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">2D gel electrophoresis and mass spectrometry of proteins o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Alternatively, N-terminal sequencing, or mRNA sequencing, or cDNA cloning · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleolar proteome is dynamic o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Some proteins found** mainly **in nucleolus – fibrillarin, nucleolin o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Some proteins found ** part-time **in nucleolus – ribosomal proteins o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Some proteins are ** time or condition dependent – ** p68/cell cycle factors · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">The stability of nucleolin is linked to the proliferative state of the cell o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Contact inhibition induces auto-catalytic breakdown o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cancer cells have more nucleoli (6 to 10) · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleolar localisation: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">8 amino acid sequence is necessary and sufficient in the coronavirus, avian coronavirus, N protein o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Signal contains basic amino acids o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">No real consensus o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleolus localisation signal also determines dynamic trafficking · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Invading viruses can hijack nucleolus (e.g. ribosome biogenesis) to promote its own lifestyle – e.g. HIV hijacks export factors in the nucleolus (this is a therapy target) <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Labyrinthine membrane-bound organelle · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Rough ER – protein synthesis and modification, first step in secretory pathway · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Smooth ER – lipid synthesis · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Signal peptide for entry of proteins to ER: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">N-terminal o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Hydrophobic amino acids o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Signal peptide translated before protein o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Either cleaved in ER, or retained as transmembrane domain (due to its hydrophobicity) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cytosolic particle – signal recognition particle (SRP) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">ribonucleoprotein particle o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">recognises signal peptide o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">pauses translation o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">translocates ribosome to signal recognition particle receptor (SRPR) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">At same time as SRP binds to SRPR, the ribosome binds to SEC61 translocon o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nascent polypeptide is translated and translocated into the ER via the SEC61 translocon · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Proteins modified in the ER: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Oxidising environment allows formation of disulphide bonds between cysteine o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">N-linked (asparagine-linked) glycosylation § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Oligosaccharide composed of 2 N-acetylglucosamines, 9 mannoses and ** 3 terminal glucoses ** § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">After glycosylation, 2 terminal glucoses removed § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">1 glucose remains § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Chaperone calnexin and associated protein ERp57 bind to oligosaccharide and free cysteines to prevent aggregation of unfolded proteins § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">If protein is correctly folded, it exits the ER   § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Protein incorrectly folded – recognised by glucosyl transferase – re-adds a terminal glucose § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Allows Calnexin-ERp57 to recognise again, second attempt at folding § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">If protein takes too long to fold then it enters to ER-associated degradation (ERAD) pathway · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mannose residue trimmed off · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Tags protein for degradation · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Retro-translocation to cytosol via SEC61 · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Poly-ubiquination of lysine residues in cytosol · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Degradation by proteasome complex <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">ER residents: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Proteins that belong to ER lumen or membrane o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Contain retrieval motifs in case they escape to cis-Golgi o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Retrieval of membrane proteins: § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cytoplasmic (C-terminal) di-lysine motif = KKXX § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Interacts with coatamer complex in cis-Golgi § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Returns in COPI (retrograde) vesicle o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Retrieval of luminal proteins: § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">C-terminal KDEL motif (lys-asp-glu-leu) § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">KDEL-receptor in cis-Golgi § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">COPI vesicle and retrograde transport § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">In ER, KDEL-receptor dissociates and returns to cis-Golgi · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Defects in protein folding and/or assembly · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Proteins needed in cell are subsequently destroyed via the ERAD pathway · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cystic fibrosis: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">1000+ mutations o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Affects cystic transmembrane conductance regulator (CFTR) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">An ATP-binding cassette protein o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">0.5% of Caucasian population o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Transports Cl- ions, H­2O follows, mucus kept hydrated o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">ΔF508 (deletion of phenylalanine residue 508) found in at least one gene copy in 90% of sufferers o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Autosomal recessive trinucleotide deletion o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutant protein CAN transport Cl- ions, but retained in ER and degraded by ERAD before it can reach membrane · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Osteogenesis imperfecta: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutations in collagen I prevent ER folding o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Degradation of pro-collagen I by ERAD o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Severe defects in bone formation · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">α1-antitrypsin deficiency: ** o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">α1-antitrypsin = inhibitor of trypsin destroyed by ERAD o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">trypsin destroys lung tissue o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">inhibitor needed to prevent this · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cholera toxin: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Causes Cl- and H2O movement into gut lumen; diarrhoea o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Toxin is endocytosed by cell o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Retrograde transport to ER   o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cholera toxin has 2 subunits § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">CTA and CTB § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">CTB binds to GMI receptor to induce endocytosis § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">CTA has C-terminal KDEL motif for retrograde transport to ER lumen o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Inside ER, CTA subunit dissociates § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">CTA1 and CTA2 § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">CTA1 unfolded, retro-translocated to cytosol § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Evades destruction – insufficient lysines for ubiquination § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Activates adenylate cyclase, raising cAMP levels § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Raises protein kinase A levels § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">PKA phosphorylates and activates CFTR, more Cl-=pumped out · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Ricin toxin: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Ricin is a 2 subunit toxin § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">A-chain = enzyme § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">B-chain = binds to cell-surface glycoproteins, induces endocytosis o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Transport to ER (** lacks KDEL motif **) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Subunits dissociate in ER   § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">A-chain unfolded, retro-translocated and escapes ERAD (lacks sufficient lysines for ubiquination) § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">A-chain affects 28S ribosomal RNA § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Depurinates by cleaving adenine A4324 from its ribose sugar § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Elongation factor can no longer bind § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Protein synthesis inhibited · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Viruses can hijack ER retrieval and ERAD systems: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Human cytomegalovirus (HCMV): § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Usual role of MHC class I antigens is to present a peptide fingerprint of the cell for detection by T cells § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Foreign fragments trigger attack and death of the infected cell § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">US2/11 proteins made by HCMV localised to ER membrane § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Target heavy chain of MHC for ERAD degradation § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">No detection of infection by T-cells o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Adenovirus – E3/19K § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Virally-encoded ER membrane protein (targeted by KKXX motif) § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Binds MHC molecules and anchors it to the lumen so virus evades immune response <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">A very important example of a disease caused by this first group of disorders is <span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">[|Parkinson's disease] <span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">. It is caused by a mutation in the parkin <span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">[|gene] <span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;">. [|Parkin] is a protein that functions in complex with CHIP as a ubiquitin ligase and overcomes the accumulation and aggregation of misfolded proteins. <span style="font-family: "Times New Roman","serif"; font-size: 12.0pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosome found in all mammalian cells except red blood cells · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Roughly spherical · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">200-400nm diameter · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Membrane bound · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Dense under electron microscope due to protein core · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Roughly 1% of cell volume · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Involved in hydrolytic degradation of macromolecules · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Those destined for degradation delivered to lysosomes by endocytic pathway and by autophagy · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Other functions include: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Apoptosis o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Repairing damage to plasma membrane · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosome lumen contains >45 different hydrolases, each with different substrates o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleases, proteases, glycosidases, sulphatases · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosomal hydrolases optimally active at pH4.5-5 · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cytosol is maintained at pH7.2 · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Vacuolar pump uses ATP to transport H+ ions into lysosomal lumen and keep pH down · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosome is cell’s recycling centre · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosome membrane has H+-coupled transporters that deliver the water-soluble products of hydrolysis to the cytosol · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">H+ vacuolar pump therefore needed for maintaining acidic pH and for H+-coupled transporters for water-soluble products of hydrolysis · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Endocytosis and the endocytic pathway: ** o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">1. Extracellular molecules in the fluid phase o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">2. Membrane proteins destined for degradation o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Molecules to be degraded sorted to late endosomes and then lysosomes o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Plasma proteins not degraded are recycled from endosomes to membrane by transferring receptor (usually used for iron import) · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Autophagy: ** o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">1. Molecules from cytosol o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">2. Whole organelles o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Removal of cell’s own components (e.g. hepatocytes mitochondria every 10 days) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Enhanced by cell starvation o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Macroautophagy: § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cytoplasmic compartment surrounded by a double membrane § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Autophagosomes fuse with lysosomal membrane, releasing their contents into it for degradation · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Phagocytosis o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Large extracellular species, e.g. microorganisms · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">>50 genetic diseases · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Defects in degradative function · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Accumulation of molecules in lysosome · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Link between accumulation and disease – often poorly understood · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Main causes: o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Inability to deliver hydrolases to lysosome o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Deficiencies in specific hydrolases o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Inability to deliver products of hydrolysis to cytosol · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">I-cell disease (mucolipidosis type II): **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">So-called because of the formation of intracellular inclusions o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Clinical features – facial and skeletal abnormalities, severe psychomotor retardation o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">No cure, treatment limited o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Autosomal recessive o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutation in GNPTA gene o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Usually encodes the enzyme N-acetylglucosiminidase-1-phosphotransferase o ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Molecular basis: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cells have reduced conc. of intracellular hydrolases § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Hydrolases secreted into surrounding culture medium § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">N-acetylglucosiminidase-1-phosphotransferase is a cis-Golgi enzyme that modifies the mannosylated glycans that are attached to newly-synthesised lysosomal hydrolases (by attaching a phosphate group) § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">This allows the hydrolase to be recognised by the mannose-6-phosphate receptor and enter the M6P pathway for transport to the lysosome (the receptor-ligand dissociates in low pH of lysosome) § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">No phosphotransferase enzyme present means no phosphorylation of lysosomal hydrolases so they cannot enter M6P pathway; the hydrolases are // secreted //, reduces degradative ability of lysosome · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Pompe disease: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Autosomal recessive mutation o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">In gene encoding the lysosomal hydrolase: α-D-glucosidase o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Clinical symptoms: progressive cardiac/skeletal myopathies o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Death within 1 year due to cardiorespiratory failure o ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Molecular basis: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Glycogen in muscle cells § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Small % of glycogen degraded in lysosomes (poss. by autophagy) § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosomal α-D-glucosidase hydrolyses glycogen à <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> glucose § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Deficiency causes abnormal glycogen accumulation § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Can be treated by intravenous infusions of mannose phosphate-modified (i.e. lysosome-directed) α-D-glucosidase § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">This ‘enzyme replacement therapy’ only works on cardiac muscle · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Fabry disease: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">X-linked o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutation in α-galactosidase o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Symptoms: facial abnormalities, problems with vasculature, progressive organ deterioration (life expt. 40-50 years) o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Caused by deposition of the glycolipid, globotriaosylceramide in capillary walls, nerves, kidney tubule and glomerular cells o ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Molecular basis: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">α-galactosidase removes terminal galactose from globotriaosylceramide (Gb3) § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">lack of this enzyme – Gb3 accumulation in lysosomes and lipid droplets in many cell types · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Infantile sialic acid storage disease: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Autosomal recessive o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Sialin gene o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Symptoms: facial abnormalities, mental retardation, enlarged heart and liver, life expt. 2 years o <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Salla disease (Finnish) is less severe, but causes disability and life expt. of ~50 years o ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Molecular basis: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Sialin = lysosomal membrane transporter § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Transports sialic acid from lysosome to cytosol § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Sialic acids = 9C monosaccharides § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Among the breakdown products of glycoproteins, glycolipids and glycosaminoglycans § <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Loss of sialin transport activity = accumulation of sialic acid in lysosomes · ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Summary of lysosomal storage diseases: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Involves action of caspases (cytosolic cysteine proteases) · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Increased membrane permeability of lysosomes in apoptotic cells · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Release of lysosomal proteins – cathepsins – which act on cellular targets · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Cathepsins not at their optimum pH in the cytosol, but can cleave proteins · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Also triggers mitochondrial/instrinsic pathways for apoptosis via Bid · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Exact role of lysosomes in apoptosis unknown – cathepsins may amplify apoptosis · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Plasma membrane – mechanical damage · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosomes act as a ‘reserve’ of membrane · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosomes exocytosed to surface where they act as ‘plasters’ · <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Exocytosis is induced by an influx of Ca2+ which acts on synatotagmin on the lysosomal membrane <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;"> <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * __<span style="font-family: 'Times New Roman',serif; font-size: 16pt; line-height: 115%;">Cell Bio of Disease __ **<span style="font-family: 'Times New Roman',serif; font-size: 16pt; line-height: 115%;">
 * __<span style="font-family: 'Times New Roman',serif; font-size: 14pt; line-height: 115%;">Cytoskeleton __ **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Microtubules: **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Kinesins: **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Dyneins: **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">MAPs (microtubule-associated proteins): **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Kinesins and disease **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Microtubule-associated proteins (MAPs) and disease **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mutations in the following microtubule-related proteins cause disease: **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Actin filaments (or microfilaments): **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Muscular diseases (myopathies) caused by actin mutations: **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Actin-based diseases in non-muscle cells: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Actin mutations affect muscle and non-muscle cells, but not so much neurones. **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Intermediate filaments: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Diseases of intermediate filaments: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * __<span style="font-family: 'Times New Roman',serif; font-size: 14pt; line-height: 115%;">Mitochondria __ **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Structure and roles: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Protein import/export and mitochondria: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Prohibitin: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Reactive oxygen species (ROS): **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mitochondria and apoptosis: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Mitochondria as a platform for anti-viral signalling: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Peroxisomes and mitochondria in anti-viral signalling: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * __<span style="font-family: 'Times New Roman',serif; font-size: 14pt; line-height: 115%;">Nucleus __ **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nuclear pore complex and localisation: **
 * __<span style="font-family: 'Times New Roman',serif; font-size: 14pt; line-height: 115%;">Sub-nuclear structures __ **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">PML nuclear bodies: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nuclear speckles – site of RNA splicing **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Nucleolus: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * __<span style="font-family: 'Times New Roman',serif; font-size: 14pt; line-height: 115%;">Endoplasmic reticulum __ **<span style="font-family: 'Times New Roman',serif; font-size: 14pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Trafficking to ER and processing within ER: **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Diseases associated with ER: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">ER and delivery of toxins to cytosol: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * __<span style="font-family: 'Times New Roman',serif; font-size: 14pt; line-height: 115%;">Lysosomes __ **<span style="font-family: 'Times New Roman',serif; font-size: 14pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Delivery to lysosome: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosomal storage diseases: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">
 * ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Defect ** || ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Disease  ** || ** <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Consequence  ** ||
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Trafficking of enzymes into lysosome || <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">I-cell disease  || <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Loss of activity of specific hydrolase  ||
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Missing lysosomal hydrolase activity || <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Fabry disease, Pompe disease  || <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Inability to hydrolyse specific molecules  ||
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Missing lysosomal membrane transporter || <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Infantile sialic acid disease/salla disease  || <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Inability to transport products to the cytosol  ||
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosomes in apoptosis: **
 * <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">Lysosomes in plasma membrane repair: **<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 115%;">