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PROTEIN DEGRADATION ^1,2

General principles Protease classes Autophagy Chaperones Cysteine Ferroptosis Lysosomal Metalloproteinases Mitochondrial Muscle Serine Ubiquitin-Proteasome Also see   Apoptosis   Caspases

• Peptide bond: Planar amide bond between α-carboxyl group & α-amino group of 2 adjacent amino acids
• Proteolysis
  • Definition: Biochemical degratation of protein through hydrolysis of peptide bonds
  • Targets: Abnormally folded proteins; Short-lived proteins; Other cell contents
    • Protein folding
      • Propensity is intrinsic property of peptide
      • Molecular chaperones: Minimize abnormal folding & aggregation of proteins
      • Mutant proteins
        • Commonly abnormally folded
        • ± Increased tendency to aggregate
  • Intracellular proteolytic systems
    • Lysosomal
      • Steps
        • Uptake (Autophagy) into lysosome: Secretory vesicles; Cytoplasm; Organelles
        • Enzymatic degradation
    • Non-lysosomal
      • Steps
        • Tagging of protein to be degraded (e.g. by ubiquitination)
        • Recognition by proteolytic systems
          • Exposure of peptide sequences, or
          • Distinction of unfolded protein segments
      • Example: Proteasome
  • End products
    • Partial hydrolysis → Oligopeptides
    • Complete hydrolysis → Free amino acids
  • Functions
    • Elimination of damaged or unneeded proteins
    • Activation of protein precursors: Partial proteolysis
    • Regulation of levels of intracellular proteins: Complete proteolysis
      • Proteolysis of specific protein
      • Proteolysis of proteins that regulate functions & levels of other proteins
• Classes of proteases (peptidases)
  • Serine
  • Cysteine: Includes most lysosomal proteases
  • Threonine: Proteasome multicatalytic endopeptidase
  • Metalloproteinases
  • Aspartic: Pepsin
  • Acid: Renin; HIV protease
  • Ubiquitin-Proteasome
  • Mitochondrial
• Diseases & Mechanisms
  

  PROTEOLYSIS: DISEASE & REGULATORY MECHANISMS
  Disease or regulatory mechanism	Example
  ↓ Proteolytic activation of enzyme	Factor IX Chapel Hill
  ↓ Proteolytic inactivation of enzyme	Factor V Leiden
  ↑ Protein expression 2° stabilized mRNA	Hyperprothrombinemia
  ↓ Enzyme activity; Disulfide bond Δ	Factor VII
  ↓ Enzyme activity; Loss of highly conserved arginine	Limb Girdle MD 2A
  ↓ Enzyme activity; Structural change near catalytic cysteine	Pycnodysostosis
  Altered protein folding	Cystic fibrosis
  Chaperone disorder	Desmin myopathy
  Gene promoter inactivation	Myoclonus-Epilepsy
  Docking site change on cysteine protease inhibitor	Myoclonus-Epilepsy
  Deletion of binding site for ubiquitination enzymes	Liddle syndrome
  Degranulation from immune cells	MMP-9: IL8 effect on neutrophils
  Glycosylation	N-linked sugars ↓ serine protease activity
  Response to inflammation	↑ matrilysin & gelatinase B in GBS nerves
  Autophagy

• General classes & disorders
  • Pancreatic enzymes
    • Trypsin
      • Cleavage site is positively charged Lys and Arg side chains
      • Disease (Trypsinogen-1 ): Hereditary pancreatitis
    • Chymotrypsin: Cleavage site is large hydrophobic side chains (Phe, Tyr, Trp)
    • Elastase : Cleavage site is small hydrophobic side chains such as Ala
    • Enterokinase B
      • Activates trypsinogen & chymotrypsinogen
      • Disease: Pancreatic insufficiency
  • Clotting factors
    • Diseases → Hemorrhagic disorders
      • Prothrombin
      • Factor V : Serine protease cofactor
      • Factor VII
      • Factor VIII : Serine protease cofactor
      • Factor IX
      • Factor X
      • Factor XI : Hemophilia B
    • Diseases → Thrombophilia
      • Antithrombin
      • Protein C
      • Protein S : Cofactor for Protein C
      • Plasminogen
      • Tissue plasminogen activator
      • Hyperprothrombinemia
      • APC resistance : Factor V Leiden
  • Complement factors
    • C1q α chain : Autoimmune skin & renal disorders
    • C1q β chain
    • C1q γ chain
    • C1r : Autoimmune arthralgias, rhinobronchitis, discoid lupus,nephritis
    • C1s
    • C2 : Autoimmune lupus, polymyositis, Henoch-Schonlein purpura
    • C3
    • C4
    • Properdin
    • Factor D
  • Viral proteases
  • LONP1
• Mechanisms of action
  • Uses serine hydroxyl to catalyze peptide bond hydrolysis
  • Regulated by highly specific serine protease inhibitors (serpins)
• Serine protease inhibitors
  • Naturally occurring
  • Highly specific
  • Disease-related proteins
    • α-1-antitrypsin
      • Inhibits trypsin & several other serine proteases
      • Disease: Cirrhosis with emphysema
      • Mutations not always associated with disease; May constitute risk factors
    • C1 esterase inhibitor
      • Initiating component of complement cascade
      • Disease: Angioneurotic edema; Episodic swelling of Respiratory & GI tracts
    • Plasminogen activator inhibitor-1
      • Disease: Deficiency → ↑ Dissolution of blood clots (fibrinolysis) → Excessive bleeding

• Use cysteine thiol to catalyze peptide bond hydrolysis
• Regulation by natural inhibitors
• Most lysosomal proteases
• Classes
  • Cathepsins
    • Location: Often lysosomal
    • Actions
      • 1°: Digest proteins engulfed into intracellular lysosomes
      • Cancer metastasis: Digest basement membrane ® Blood borne tumor cells can invade tissues
  • Calpains
    • Ca⁺⁺ activated neutral proteases
    • Non-lysosomal
  • Caspases
    • Cleavage specificity: Internal aspartate residues
    • Proteolysis mediates signal pathway downstream of Fas receptor
    • Trigger apoptosis
• Diseases
  • Cysteine proteases
    • Calpain-1: SPG 76
    • Loss of CAPNS1 (reduces both CAPN1 & CAPN2 ) in mice: Myopathy, severe
    • Calpain-3, Large subunit: Limb girdle muscular dystrophy 2A (LGMD 2A)
    • Cathepsin K: Pycnodysostosis
    • Calpain 10
      • Polymorphisms in intron 3 predispose to NIDDM
  • Cysteine protease inhibitors
    • Cystatin B: Myoclonus epilepsy
  • Cathepsins
    • Cathepsin C : Papillon-Lefevre ; Haim-Munk
    • Cathepsin L: Upregulated in muscle atrophy

• Lysosome formation: From Phagocytosis, Endocytosis, Autophagy
• Enzymes: Acid proteases (peptidases)
  • Cathepsin B , D , H , L S : Papain family of cysteine thiol proteases
  • Cathepsin C (Dipeptidyl aminopeptidase I)
    • Removes dipeptides from amino terminus of proteins
    • Activates granule serine proteases in bone marrow-derived effector cells
    • Activates T-lymphocyte granzymes A & B : Agents of T cell-mediated cell killing
  • Other hydrolases with less activity
  • Inhibitor of lysosomal enzymes: Cystatin C
• Location of lysosomal enzymes: Endosomes & Lysosomes
  • Lysosomal enzyme received by Golgi complex from rough endoplasmic reticulum in cis region
  • Golgi complex sorts the lysosomal enzyme in the trans region: Phosphate attached to a mannose residue
  • Mannose-6 phosphate forms a sorting signal: Moves through cisternae to trans region & binds to a specific receptor
  • Budding forms
  • "Cage" or "coat" made of clathrin forms around the bud (to strengthen it)
  • Vacuole moves to lysosome
  • AP3 complex
    • Adaptin β-3A is 1 of 4 subunits
    • Facilitates trafficking of vesicles from trans-Golgi network or endosomal compartments to lysosome
    • Acts via tyrosine and dileucine signals on proteins of lysosomes & other intracellular organelles
  • Acidic environment in lysosome: Removes phosphate & dissociates hydrolase from the receptor
  • Receptor is then recycled back to the Golgi complex
• Functions
  • Degradation
    • Substrates: Extracellular proteins; Bacteria; Intracellular organelles
    • 10% to 20% of proteins in mammalian cells
  • Peptide generation for immune cell presentation
  • Regulation of cell membrane proteins
• Lysosomal Diseases
  • Pepstatin-insensitive lysosomal peptidase: Neuronal Lipofuscinosis - Late infantile
  • Cathepsin C : Papillon-Lefevre syndrome {Keratosis palmoplantaris with periodontopathia}
  • CHS1: Chediak-Higashi
  • Cardiomyopathy, Retinal degeneration & Epilepsy: CLN3
  • CTSD: Ataxia; Pigmentary retinopathy; Cognitive decline
  • Hermansky-Pudlak syndromes
    • HPS1 gene
    • HPS2: Adaptin β-3A ; Also Pearl mouse
  • Lysosomal membrane protein defects
    • Glycogen storage, Type 2b: LAMP-2
    • Cystinosis, Nephropathic : Cystinosin (CTNS)
    • Sialic acid storage diseases: Sialin (SLC17A5)
      • Infantile sialic acid storage disorder
      • Salla disease
  • Myoclonus epilepsy & Demyelinating neuropathy: SCARB2
  • HMSN 2B
  • Methylmalonic aciduria & Homocystinuria: ABCD4
  • Parkinson-Spasticity: ATP6AP2
  • Storage disorders
    • Glycogen: Acid maltase
    • LAMP-2: Danon
    • Mucolipidosis IV: MCOLN1
    • Mucopolysaccharidosis
    • Toxic: Amiodarone; Chloroquine; Perhexiline
    • Inclusions: Cockayne
    • Aging: Lipofuscin accumulation
    • VMA21: XMEA
    • Niemann-Pick, Type C: NPC1

• Properties
  • Neutral
  • Contain Zinc
• Matrix metalloproteinases (MMP; Matrixins; Family 10B) ⁶
  • General: Zinc dependent endopeptidases
  • Properties
    • Secreted: Except for membrane-types
      • Extracellular activity
      • Propeptide contains signal domain
      • MMPs 1-13 & 18-20
    • Collagenase structure: 5 protein domains
    • Structural regions of MMPs
      • Prodomain: Contain PRCGVPD motif; Maintains latency in zymogens
      • Active site zinc-binding domain
        • Includes HEXGHXXGXXH sequence motif
        • Histidines: Zinc binding
        • Glu (E): Acts as a catalytic base
      • Catalytic domain
    • Other structural domains
      • Proline rich hinge region
      • Hemopexin homology at carboxyterminus
        • Present in all but MMP7
        • Promotes interaction with substrate
      • Fibronectin binding domain: MMP2 & MMP9
      • Transmembrane domain: MMPs 14-17
    • Role in tissue remodeling & repair during development and inflammation
      • Degrade extracellular matrix proteins
      • Activated MMP enzymes can mount a nucleophilic/peptidolytic attack at neutral pH in extracellular space
      • MMP-13 (collagenase-3) & MMP-15 (MT2-MMP) expressed selectively in rheumatoid arthritis synovial fluid
  • Regulation of matrix metalloproteinase activity
    • Regulation at several levels
      • Gene transcription: Primary regulation
      • Pro-enzyme activation
      • Activity of tissue inhibitors of metalloproteinases (TIMPs)
        • TIMP mechanism: Block MMP activity by binding to Zinc binding site
        • 4 TIMP family members
          • TIMP-1 : Expression regulated at transcription by cytokines & growth factors; Soluble
          • TIMP-2 : Constitutively expressed; Soluble; Frequent allelic deletion in breast cancer
          • TIMP-3
            • Regulated in cell cycle dependent manner; Sequestered in pericellular matrix
            • Disease: Sorsby fundus dystrophy
          • TIMP-4 : Expression highest in heart; Soluble; May inhibit tumor invasion
        • TIMPs Produced by Keratinocytes, fibroblasts, osteoblasts & endothelial cells
      • Release from stores
        • MMP-8 & MMP-9: Secretory granules of neutrophils & eosinophils
        • MMP-7: Secretory epithelial cells of skin, GI & respiratory systems
    • Enzyme activation
      • Secreted in latent precursor (zymogen) form
      • Proteolytic cleavage of amino-terminal domain exposes active catalytic site
      • ? By removal of cysteine switch at position 73
    • Typically absent from normal adult tissues
  • Classes
    • Collagenase
      • Types: Interstitial (MMP-1) ; 2 (MMP-8; Neutrophil) ; 3 (MMP-13)
      • Action: Initiate degradation of native collagens I, II, III, VII
      • Cleavage products: Denatured at body temperature to gelatin; Further degraded by MMP2 & MMP9
      • MMP13
        • Broadest substrate specificity
        • Expressed in regions with rapid extracellular matrix remodelling
        • Pathological associations
          • Chronic inflammation (Osteoarthritis; Rheumatoid synovium; Ulcers)
          • Malignant tumor invasion
    • Gelatinases
      • Degrade: Denatured collagens; Basement membrane collagen IV
      • A (MMP-9) : Induced with
        • Tissue remodelling: Development; Wound healing; Tumor invasion
        • Response to cytokines, growth factors & mitogens
        • Upregulated in: CIDP (T-cells); Non-systemic vasculitic neuropathy (T-cells)
      • B (MMP-2)
        • Broadly expressed
        • Upregulated in: Polymyositis; CIDP (T-cells); Non-systemic vasculitic neuropathy (T-cells)
        • Disease: Idiopathic multicentric osteolysis
        • Likely effector of endometrial menstrual breakdown
        • MMP-2 & Integrin αV/β3: Functionally associated on surface of angiogenic blood vessels
    • Stromelysins
      • 1 (MMP-3) & 2 (MMP-10)
        • Expressed by epithelial cells & carcinomas
        • Degrade broad range of extracellular matrix substrates
      • 3 (MMP-11)
        • Expressed by fibroblasts
        • Cleaves serine protease inhibitors
    • Stromelysin-like MMPs
      • Macrophage Metallo-elastase (MMP-12) :
        • Expressed in macrophages & Stromal cells
        • Primarily degrades elastin
      • Matrilysin (MMP-7) : Broad substrate specificity
    • Membrane-type MMPs (MT-MMP)
      • Types
        • 1 (MMP-14)
          • Provides activation mechanism for proMMP-2 & proMMP-13
          • Degrades Collagen I, Fibronectin, Laminin, Fibrin, Gelatin & Cartilage proteoglycan core protein
          • Function: Modeling of skeletal & extraskeletal connective tissues
        • 2 (MMP-15)
          • Provides activation mechanism for proMMP-13
          • Degrades Fibronectin, Laminin, & Tenascin
          • Highest levels in liver, placenta, testis, colon, & intestine
        • 3 (MMP-16)
          • ? Membrane bound
          • Degrades Collagen III, Fibronectin, Gelatin
          • Expressed in placenta, heart, & some carcinoma cell lines
        • 4 (MMP-17)
          • Expressed in brain, leukocytes, colon, ovary, testis & breast cancer
      • Contain C-terminal insertion with a transmembrane domain: Integrated into cell membrane
      • Furin (Golgi-associated MMP activator) recognition domain: Between propeptide & catalytic domain
    • Other MMPs
      • MMP-19
      • MMP-20 (Enamelysin)
      • MMP-23A
      • MMP-23B
  • External link: R&D
• Thimet oligopeptidase (M3 family)
  • Mitochondrial intermediate peptidase (MIPEP)
    • Required for maturation of iron-containing proteins
    • Disease: COXPD31
  • Thimet oligopeptidase 1 : Cytoplasmic; Degrades small peptides
• Disease-relations
  • Angiotensin-converting enzyme (CD143)
    • Polymorphism: Insertion (I) or Deletion (D) of 287 base pair DNA fragment in intron 16
      • Insertion is Alu repetitive sequence
      • DD genotype associated with
        • Cardiovascular complications: Myocardial infarction, Strokes, Diabetic nephropathy
      • II genotype associations
        • Better endurance performance; Frequent in high altitude mountaineers
  • Clostridial neurotoxins: Botulinum toxin; Tetanus
  • Paraplegin: Hereditary spastic paraparesis 7 (Recessive)
  • Friedreich's ataxia: Mitochondrial intermediate peptidase interacts with frataxin
  • POEMS syndrome: Increased serum MMP1, 2, 3, 9 & TIMP
  • Alzheimer's: Thimet oligopeptidase 1 possibly involved in amyloidogenic processing of APP
  • McLeod syndrome: Kell protein
  • Tumor cell invasion: MMP-14; MMP-11 (Epithelial)
  • Emphysema: MMP-12 lysis of elastin activated by cigarette smoke
  • Inflammatory myopathies: Over-expression by some cells
  • Lumbar disc disease: MMP-9
  • Demyelinating disorders
    

    Extracellular proteolysis in demyelinating disorders: Mechanisms
    Proteolysis of myelin proteins	Myelin degradation
    Generation of remnant antigens	↓ Tolerance
    Proteolysis of basement membrane	Blood-brain barrier damage
    Proteolysis of extracellular matrix	↑ Cell migration
    ↑ Cytokine activation from precursors	↑ Inflammation
    Proteolytic activation of zymogens	↑ Proteolysis
    Proteolysis of cell surface molecules	Cell death

  • MMP-2: Idiopathic multicentric osteolysis
  • Weill-Marchesani Syndrome, Autosomal Recessive : ADAMTS10

UBIQUITIN-PROTEASOME PROTEIN DEGRADATION ^3-5,7

Classes of proteases Degradation pathway Diagrams Diseases Related cellular processes

Ubiquitination

26S Proteasome

• Proteasome degradation: Overall process
  • General: Degrades proteins
    • Unfolded
    • Short-lived
  • Multienzymatic
  • Requires
    • ATP at multiple steps
    • Neutral or slightly basic pH
  • Protein targeted for proteasome degradation
    • Targeting process: Binding to polyubiquitin chain
      • Protein-ubiquitin linkage type: Covalent
      • Polyubiquitin chain
        • Terminal residue (G76) of one ubiquitin molecule linked lysine (K) residue of next
        • Most common lysine linkage is via K48
        • Polyubiquitin chain assembly can also occur at 6 other lysine residues in ubiquitin
      • Protein-ubiquitin linkage not associated with proteasomal degradation
        • Mono-ubiquitination
        • Binding to polyubiquitins linked via K63
          • K63 ubiquitin-modified inclusions preferentially cleared by autophagy
    • Degradation process
      • Usually by proteasome
      • Covalently bound ubiquitin occasionally targets proteins for lysosomal degradation
    • Some proteins may not require ubiquitination for proteasome degradation
      • Short-lived enzyme ornithine decarboxylase
      • Many denatured polypeptides (e.g. casein)
  • Proteasome localization: Nucleus & Cytoplasm
  • Proteins cleaved
    • At selected amino acid sites: Arg, Phe, Tyr, Leu, Glu, Asp adjacent to leaving group
    • By different β subunits with specific activities, acting together
      • Chymotrypsin-like: After large hydrophobic residues; Rate-limiting site
      • Trypsin-like: After basic residues
      • Caspase-like (Peptidylglutamyl-peptide): After acidic residues; Aspartyl > Glutamate
      • ?? After branched-chain or small neutral amino acids
    • End products: Oligopeptides (4 to 24 residues) small enough to diffuse out of proteasome
  • Substrates: Most short-lived regulatory proteins & misfolded proteins degraded by this system
  • Inhibitors: Clastolactacystin β-lactone; Peptide-vinyl sulfones
  • Role: Proteasome is primary site for degrading proteins (80% to 90%) in mammalian cells
• Cofactor: Ubiquitin
  • One of the most conserved proteins known
  • Ubiquitin binding to proteins to be degraded
    • Covalent
    • Isopeptide bonds from the C-terminal glycine residue to e-amino groups of lysyl side chains
  • Most abundant ubiquitin-protein conjugate
    • Ubiquitin-Histone2A
    • Not degraded
    • Present primarily in actively transcribed chromosomal regions
  • Ubiquitin: Homologous proteins
    • BAG-1 : Binds to BCL-2; Inhibits apoptosis
    • Ubiquitin cross-reactive protein (UCRP)
• Ubiquitin binding process
  • E1 : Activates carboxyl end of ubiquitin by conversion to thiol ester
  • E2 : Carrier proteins (family)
  • E3 (Ubiquitin-protein ligase) :
    • Couples ubiquitin to protein substrate
    • Ubiquitin binding site: Carboxyl group
    • Protein substrate binding site: e-amino groups of lysines
    • Disorders
  • Protein specificity
    • Via E2 carriers & E3 ligase enzymes
    • Multiple E2 carriers: Each dedicated to one or few E3s
    • Multiple E3 enzymes: Each dedicated to one or few substrates
    • Specificity of E3-substrate interaction
      • Depends on cis-acting ubiquitination signal in substrate
      • e.g. 9-residue "destruction box" of mitotic cyclins
  • Repeat conjugation reactions:
    • Link 5 or more ubiquitins to each other & then to protein substrate
• Large 26S proteasome
  • Subunit composition : 20S Core; 19S Regulatory; 11S Regulator
    • One 20S (700kDa) core
      • Cylindrical stack of 4 7-membered rings
      • Upper & lower (outer) rings are each composed of 7 α subunits
        • α subunits may constitute a physical barrier
          • Limits access of cytosolic proteins into inner proteolytic chamber
          • Forms narrow pore (? variably open): Only allows unfolded proteins into 20S core
        • α subunits may have regulatory functions
          • α subunits required as scaffold in self-assembly of β-rings
          • α subunits are sites for binding of PA28 (11S) or PA700 (19S) regulatory complexes
          • Some α subunits contain nuclear localization signal
      • Two middle rings are each composed of 7 β subunits
        • Mediate proteolysis
        • ~6 of 14 β subunits contain functionally active nucleophilic threonine residues
      • Subunit array & activities
        

        20S Proteasome subunit array α     zeta    30    C8    27    C3    C9 XAPC7 β    MB1   C5   HSB   δ   Z   C10   C7 β   C7  C10  Z  δ  HSB  C5  MB1 α  XAPC7 C9 C3 27 C8 30 zeta

        MB1: May confer trypsin & chymotrypsin-like activity       Inhibited by lactacystin Z: May cleave behind large amino acid residues δ: Peptidylglutamyl-peptide hydrolase (caspase-like) activity Other associated β subunits: LMP7; LMP2; MECL1       γ-interferon inducible

      • Associates with regulatory proteasome activator proteins: 19S; 11S (PA28)
      • Alternative 20S proteasome activator
    • Two 19S (700kDa) regulatory complexes (PA700)
      • Constituents
        • Six AAA ATPase superfamily proteins: External link
        • One deubiquitinating enzyme
        • Polyubiquitin recognition proteins (S5a or other)
      • Associates with 20S complex in ATP-dependent process
      • Required for degradation of ubiquitinated proteins
    • Associated with 11S regulator (PA28) complex
      • Conical structure: Can also form cap at either end of 20S particle
      • Does not employ ubiquitin
      • Involved in the presentation of endogenous antigens by MHC class I molecules
      • Expressed constitutively in antigen-presenting cells
      • Expression is upregulated by interferon γ
      • PA28 γ-subunits
        • Expression suppressed by IFN γ
        • Can assemble into complex that stimulates primarily trypsin-like activity of proteasomes
  • General actions
    • Degrades peptides after ubiquitin binding
    • Also degrades some proteins without ubiquitin: Ornithine decarboxylase
  • Steps in degradation
    • Encounter 19S complex
      • Recognize substrate's ubiquitin tag
      • ? deubiquitination
      • Substrate protein is unfolded
        • Catalyzed by ATP requiring enzymes
      • Translocation of protein to 20S core
    • 20S core proteasome
      • Protein progressively cut into 6 to 12 amino acid peptides
      • Catalyzed by hydroxyl of threonine at N-terminal of inner-ring subunits
      • Active sites in proteasome with differing functions: Peptide cleavage after
        • Large hydrophobic amino acids
        • Basic amino acids
        • Acidic amino acids
    • 11S regulator
      • Stimulates peptidase activities of 20S proteasome
      • Inhibits degradation of ubiquitinated substrates
    • Ubiquitin-independent proteasome targeting
      • Antizyme binds to substrate protein (e.g. ODC)
      • Carboxyl terminus of substrate modified
• End products of proteasome degradation process
  • Peptide products of degradation
    • Hydrolyzed to amino acids by cytosolic exopeptidases
    • Used by antigen presenting cells
  • Ubiquitin
    • Chain disassembled
    • Ubiquitin reutilized
• External link: Nottingham

• Normal processes
  • Signal transduction: Gα
  • Regulatory kinases & phosphatases: IκB kinase complex; c-Mos; c-jun; p53
  • Cell-cycle progression: Cyclin dependent kinases - Mitotic, G1 & S-phase
  • Receptor-mediated endocytosis
    • Transmembrane receptors & transporters: Ste2p ± Ste6; Non-proteasome
    • Membrane receptors coupled to tyrosine kinases
    • T-cell receptor
    • Cystic fibrosis transmembrane conductance regulator
  • Histones: Stable ubiquitination without degradation
  • Transcription: c-fos; c-jun
  • Organelle biogenesis & spermatogenesis
  • Protein degradation
  • Cell death & Apoptosis: Neurons; Thymocytes
    • Proximal to activation of interleukin-1β converting enzyme (ICE) family of proteases
  • Generation of peptides for presentation by MHC class I cell surface molecules
• Ubiquitin-mediated proteolysis: Disease processes
  • Ubiquitinated protein accumulation
    • Dementias: Alzheimer's & Pick's
    • Inclusion body myositis & myopathies
    • Polyglucosan body myopathy
    • ALS with SOD1 mutation
  • Oncologic processes
    • Abnomal substrates: Resistance to ubiquitination
    • Correlation with malignant transformation & loss of function
    • Cervical cancer
      • Human papilloma virus expression of E6 protein: Ubiquitin protein ligase
      • Increased degradation of tumor suppressor protein p53
    • Other neoplasms
      • BRCA1 : Breast
      • VHL : Pheochromocytoma; Hemangioblastoma; Paraganglioma
  • Hereditary disorders ⁹
    • Ubiquitin protein ligase (E3) associated
      • Ataxia
        • Ataxia & Deafness: RNF220
        • Ataxia, Dementia & Hypogonadotropism: RNF216
        • Ataxia, Retardation & Hypogonadotropism: CHIP
        • SCA3: Ataxin-3
        • RIDDLE syndrome: RNF168
      • CNS, Other
        • ALS-FTD: CCNF
        • Angelman syndrome: UBE3A
        • X-linked Mental Retardation Syndrome: CUL4B
        • Mental retardation ± Chorioathetosis: HUWE1 duplication (with HSD17B10 )
          • 17-Beta-Hydroxysteroid Dehydrogenase X Deficiency
          • 3-Hydroxyacyl-CoAa dehydrogenase II deficiency
          • HSD10 Mitochondrial disease (HSD10MD)
        • Mulibrey nanism: TRIM37
        • Lafora disease 2B: NHLRC1
        • SPPRS: HACE1
      • Myopathy
        • LGMD 2H : TRIM32
        • Polyglucosan body myopathy: RBCK1
        • Cardiomyopathy + Myopathy: TRIM63 (MuRF1)
        • Atrophy & Cachexia
      • Neuropathy
        • CMT 2P (Recessive or Dominant): LRSAM1
        • AR-CMT, Early onset: TRIM2
        • HMSN2 with Giant Axons: DCAF8
        • dHMN1: UBE3C
      • Cold induced sweating 3/ Crisponi like: KLHL7
    • E1 protein
      • Ataxia + Cataract, Recessive: UBA5
    • Sodium channel (SCNN1B): Liddle syndrome
      • Mutation removes binding site for an E3 ubiquitinating enzyme (Nedd4 )
    • Parkin
      • Protein function: Ubiquitin protein ligase
      • Disorder: Parkinsonism, Juvenile, Autosomal recessive
    • Ubiquitin carboxyl-terminal esterase L1 (UCHL1; PGP 9.5)
      • Protein function: Deubiquitinating enzyme
      • Disorders
        • Parkinson's disease
        • Spastic ataxia: Neurodegeneration with Optic atrophy, Childhood-onset (NDGOA) ; UCHL1
        • Gracile axonal dystrophy (gad) mouse
          • Recessive
          • Ataxia: Sensory then motor
          • Pathology: 'Dying-back' terminal axonal degeneration; Spheroid bodies in nerve terminals
    • UBE1
      • Infantile Spinal Muscular Atrophy with Arthrogryposis (XL-SMA)
      • Gigaxonin (GAN) (Interacts with UBE1): Giant axonal neuropathy
    • UBAP1: SPG80
    • pVHL
      • Protein function: Ubiquitin protein ligase
      • Disorder: von Hippel Lindau syndrome
    • Ubiquitin-proteasome pathway substrates
      • Presenilin-1 : Alzheimer's, early onset
      • Tau : Fronto-temporal dementia
      • α-synuclein : Parkinson's, Autosomal dominant
      • Huntingtin (HTT) : Huntington disease ; Lopes-Maciel-Rodan Syndrome (LOMARS)
      • PrP : Prion disease
      • CFTR : Cystic fibrosis
      • p53 : Cancer
    • Ubiquitin-proteasome pathway cofactor
      • Ubiquitin : Alzheimer's, late onset
    • Proteasome subunit, Beta-type, 8 (PSMB8)
      • JMP syndrome

• Paraplegin
  • AAA protease
  • Hereditary spastic paraparesis 7 (Recessive)
  • Metalloproteinase
• Elastase 2
  • Granulocytes & erythroblasts
  • Serine protease
• ATPase family gene 3-like 2 (AFG3L2): SCA28
  • AAA protease complex

Protein Folding & Chaperones ⁸

• Protein folding
  • Folding propensity: Intrinsic property of peptide
  • Proteins in "non-functionally" (Unfolded/Partially) folded configurations
    • More likely to be degraded
  • Mutant proteins: Abnormal folding
    • Exposed for longer time in "non-functionally" folded configurations
    • Increased likelihood of spontaneous unfolding
    • More likely to be degraded
  • Disorders
    • Endoplasmic reticulum
      • Cystic fibrosis (δ508)
      • α-1-antitrypsin deficiency (E342K)
      • FICD: SPG92
    • Mitochondria
      • MCAD deficiency (K304E)
      • SCAD deficiency (R22W)
    • Cytosol
      • Phenyketonuria (A104D)
    • Other protein folding disorders
      • Abnormally folded protein
        • Not secreted in enough quantity for proper function: Cystic fibrosis
        • Becomes insoluble & deposits in tissues: Amyloid
      • Charlevoix-Saguenay - Spastic Ataxia: Sacsin protein
• Molecular Chaperones
  • Function: Protein homeostasis & Quality control
    • Assist client proteins to attain native conformation
    • Minimize: Abnormal folding & aggregation of proteins
    • High chaperone levels: Enhance rescue of folding defects
    • Target damaged or superfluous proteins to degradative pathways
  • Mechanism: Recognize
    • Amino acid domains with hydrophobic side chains
    • Domains normally sequestered inside appropriately folded protein
  • Types of chaperones
    • Folding helper
      • Mechanisms
        • Cycle between high & low affinity forms by conformational changes
        • Energy dependent: Cycling mediated by ATP & other mechanisms
      • Families (HSPA): Hsp70; Hsp90; Chaperonins; Lectin chaperones; Clp/Hsp100; AAA
      • Diseases
        • Hereditary spastic paraplegia (SPG7): Paraplegin
        • Peroxisome biogenesis disorder
    • Holding
      • Family: Small Heat shock proteins (sHsp; HSPB)
        • Structural featues: α-crystallin domain (ACD)
        • Assemble: Into large homo- and hetero-oligomeric complexes
        • ACD binds to IxI/V motif in C-terminal domain
      • Mechanisms
        • Hydrophobic surfaces mediate reversible binding of unfolded peptides
        • Energy independent
        • High client-binding capacity
        • Cannot actively refold client proteins
        • Some have sequestrase activity
          • Drive controlled aggregation of non-native proteins
          • Prevent overburdening of refolding machinery during cellular stress
        • BAG3
          • Dissociates HSPB1 oligomers
          • Modulates sHSP function
      • Role
        • Protect partilly folded/misfolded proteins from aggregation & degradation
        • Protection needed until folding helper chaperones become available
        • First-line response against non-native proteins
      • Diseases
        • Cataract: α-crystallin
        • Desmin-related myopathy: ab-crystallin
  • Co-Chaperones
    • Types
      • J-domain proteins (DNAJC12 , DNAJC5 )
      • BAG proteins
    • Actions
      • Assist chaperones in their functions
      • Mediate interactions of the different chaperone families
      • Affect fate of client proteins
  • Chaperone Associations: Protein turnover pathways
    • Ubiquitin–proteasome system (UPS): Soluble proteins
    • Autophagy–Lysosome system: Soluble proteins, Aggregates, Organelles
• Chaperone-related disorders
  • Myopathies
    • VCP (IBMPFD1)
    • BAG3 (MFM6)
    • CRYAB (HSBP5) (MFM2)
    • DNAJB4
    • DNAJB6 (LGMD D1)
    • LAMP-2 (Danon)
    • VMA21 (XMEA)
    • SIL1 (Marinesco-Sjögren)
  • Cardiomyopathy
    • DNAJC19 (MGCA5)
    • Calreticulin 3 (CMH19)
  • CMT
    • HSPB1 (CMT2F)
    • HSPB8 (CMT2L)
  • SMA: Distal
    • DNAJB2 (DSMA5)
    • HSPB1 (HMN2B)
    • HSPB3 (HMN2C)
    • HSPB8 (HMN2A)
  • ALS
    • DNAJC7
    • SIGMAR1 (ALS16)
  • Spastic paraparesis
    • Spastin (SPG4)
    • Paraplegin (SPG7)
    • HSPD1 (SGP13)
    • CCT5 (SPG + HSN)
    • FICD (SPG92)
  • CNS
    • NDUFAF2 (MC1DN10)
    • Frataxin (FA)
    • Sacsin (ARSACs)
    • CABC1 (SCAR9; COQ10D4)
    • STUB1 (SCAR16)

Autophagy ¹¹

• Action
  • Process of subcellular degradation
  • Targets cytoplasmic cargo for lysosomal degradation
• Activation
  • Biologic stress
  • Cell homeostasis
• Stages
  • Sequestration of cytoplasm in vesicle
  • Transport into lysosomal system
  • Degradation
• Types
  • Microautophagy
    • Sequestration of cytoplasm
    • Action
      • Protrusion, septation or invagination of lysosomal membrane
      • Direct engulfment of cytoplasm
    • Location: Surface of lysosome
  • Macroautophagy (Autophagy)
  • Chaperone-mediated autophagy
  • Secretory autophagy
• Substrates & Selective autophagy
  • Proteins: Folded or unfolded
  • Peptides & Secretory granules: Crinophagy
    • Marker: HOPS
    • Regulation: Prostaglandin E2
  • Protein complexes & aggregates: Aggrephagy
    • Markers: LC3-II, Alfy
    • Cargo: Protein aggregates & inclusions
    • Regulation: p62-Nrf2, NBR1
  • Ferritinophagy
    • Marker: NCOA4
  • Glycophagy
    • Marker: STBD1
    • Cargo: Glycogen
    • Regulation: cAMP, mTOR, GTPases
  • Lipophagy
    • Marker: LIPA
    • Cargo: Lipid droplets
    • Regulation: FoxO1; PRDX1; ATGL
  • Nucleic acids
    • RNautophagy
    • DNautophagy
    • Marker: LAMP2C
    • Regulation: Ubp3/Bre5
  • Organelles
    • Mitochondria: Mitophagy ¹²
      • Pathways
        • PINK1/PRKN
        • Receptor mediated: BNIP3; NIX; FUNDC1
      • Cargo: Damaged mitochondria
      • Regulation: Bnip3/Nox
    • Peroxisomes: Pexophagy
      • Marker: Atg36
      • Regulation: Dnm1,Vps1, Atg11, Atg30
    • Endoplasmic reticulum: Reticulophagy
      • Markers: Syntaxin 17, DFCP1
      • Cargo: Damaged or superfluous ER
      • Regulation: Syntaxin17/Atg14, Ypt1
    • Ribosomes: Ribophagy
    • Lipid droplets: Lipophagy
    • Myelin: Myelinophagy
      • Plasma membrane degradation
      • Occurs with: Wallerian degeneration
      • Autophagic cells: Schwann cells
      • Participates in initial fragmentation of myelin
      • Positively regulated by: JNK/c-Jun pathway, central regulator of Schwann cell reprogramming during injury
      • Steps
        1. Axon damage
        2. Myelin fragmentation
        3. Myelin fragments enclosed in phagophore
  • Pathogens: Xenophagy
    • Markers: CALCOCO2/NDP52
    • Regulation: AMPK, mTOR
  • Chaperone-Assisted Selective Autophagy (CASA)
• Associated processes & disorders
  • Myopathies
  • Cancer
  • Neurodegeneration
  • Major histocompatibility complex (MHC) class II antigen processing
  • Removal of bacterial & viral pathogens

• General: Direct translocation of cytosolic proteins with pentapeptide motif across lysosomal membrane
  • Requires actions of cytosolic & lysosomal chaperones to unfold substrates
  • Formation of protein complex: Between cytosolic "substrate" & "chaperone"
  • Protein requirement: Pentapeptide motif sequence (KFERQ (or similar) pentapeptide)
  • Chaperone: hsc70 (HSPA8)
  • Complex then interacts with: Lysosomal membrane receptor (LAMP2A)
  • Interaction leads to: Translocation into lysosome
• Internalization
  • Direct translocation across lysosomal membrane
  • Carried out within the lysosomal lumen
  • By an additional chaperone protein
• Functions
  • Primarily involved as a secondary response to nutrient depletion
  • Stimulated by: Oxidative stress, Starvation (Ketones) & Aging
• Diseases
  • Danon
  • Myofibrillar myopathies
  • ? Blocked in some forms of Parkinson's disease

General Morphology Double membrane sequestering vesicle (Autophagosome) Histologic marker: AMPDA & LC3 aggregates Ultrastructure Proteins Stages Membrane sequestration of cytoplasm & organelles Organelle: Double-membrane Autophagosome Autophagosome formation & maturation Fusion of membrane bound vesicles with lytic component (Lysosomes) Cargo Degradation End products: Release of degradation products or Cell death Action: Bulk lysosomal degradation Targets Larger cytoplasmic proteins: Folded or unfolded Organelles: Mitochondria; Fractured endoplasmic reticulum; Peroxisomes Mechanisms: Formation of double-membrane, non-Lysosomal vesicle (Autophagosome) Several phases: All ATP dependent Initiation Execution Maturation Initiation Phagophore (Crescent-shaped isolation membrane) generation Membrane is formed de novo, not by budding Sequestering membrane Double layer Separate from Lysosome Contains: LC3-II (or another ATG8 ortholog) Phosphatidylethanolamine-conjugated Bound to inner & outer membranes Phagophore transport From: Perinuclear area Where Nucleation occurs To: Cell periphery Cargo is sequestered Autophagosomes formed Kinesin- & FYCO1-mediated centrifugal transport Phagophore Sequestration Sequestered Contents: Cytoplasm & Organelles Mechanism: Binding to LC3-II or Autophagy receptor (p62) Expansion & Closing of Phagophore Gives rise to Autophagosome Autophagosome Double-walled vesicle Maturation External proteins LC3: Released & Reused Binding of proteins mediating Autophagosome–Lysosome fusion Transport Binding to dynein–dynactin molecular motor Transports autophagosome centripetally: Toward Center of cell Lysosomes at minus end of microtubules Amphisome-formation May fuse with Late endosome: Prior to fusion with lysosome Actions Induction Upregulation of HDAC6 Rapamycin Starvation (Low amino acids) Autophagy: Proteins required General Function: Most at step of vesicle formation Localization: At phagophore assembly site/preautophagosomal structure Autophagy-related proteins Ubiquitin-like General Activated by ATG7 ATG12 Conjugated to ATG5 during active autophagy Microtubule-associated protein 1, light chain 3, alpha (MAP1LC3A) Conjugated to phosphatidylethanolamine Cleaved by ATG4 Associated with completed autophagosome Function: Unknown ATG7 Ubiquitin-activating enzyme E1-like protein Essential for ATG12 conjugation system Associated with membrane fusion in autophagy Disease: SCAR31 ATG5 Essential for autophagy Conjugated to ATG12 Binds Atg16: Part of tetrameric complex of unknown function Conditional knock-out in brain Develops: Aggregates & Ubiquitinated inclusions Disease: SCAR25 ATG3 E2-like protein-conjugating enzyme Covalently attaches ATG/LC3 to phosphatidylethanolamine ATG4 Cysteine protease Cleaves C-terminus of Atg8/LC3 Exposes glycine residue for subsequent conjugation Beclin 1 (BECN1; ATG6) Component of complex Other component Phosphatidylinositol 3-Kinase, Class 3 (PIK3C3; Vps34) Produces PtdIns(3)-phosphate Stimulatory for autophagy Haploinsufficient & Mutated in some cancer lines Especially breast tumours Transmembrane proteins ATG9 Involved in: Autophagosome formation ? Marks site(s) of donor membrane used for phagophore expansion Initial nucleating membrane: Develops into autophagosome May be involved in: Membrane delivery to forming autophagosome ATG27 Complex with ATG23 ATG10 Function: Ubiquitin-conjugating-like enzyme Covalently attaches ATG12 to ATG5 ATG16L1 Binds ATG5 Homo-oligomerizes to form a tetrameric complex Mutations: Missense; Related to Crohn's disease susceptibility ATG32 : May play role in mitophagy Recruits autophagic machinery to mitochondria Regulates selective degradation of mitochondria ULK1 (ATG1 homologue) Serine/threonine protein kinase May be involved in regulation & vesicle formation Involved in axon growth LAMP2: Danon disease Phosphatidylinositol 3-kinase, Class I Controls activation of Kinases: Akt1 & Mammalian target of rapamycin (mTOR) Composed of Catalytic p110 subunits p85 adaptor Main product: PtdIns(3,4,5)P3 Inhibitory for autophagy Autophagy: End pathway results Autophagosome vesicles: Fuse with Lysosomes Requirement: Microtubular system Structure Formed: Autophagolysosome Degradation Contents exposed to acid hydrolases Contents Degraded Sequesterd Cargo molecules Inner membrane with LC3-II Autophagy receptors Pathways after degradation Macromolecules released back into the cytosol Through permeases For reuse in metabolic processes (during starvation) Cargo inactivated or killed When macroautophagy acts in immune response to eliminate microbial pathogens Removal of cytoplasm may result in cell death Major catabolic pathway for Energy generation Breakdown of macromolecules & damaged organelles into their essential constituents Most prominent: During periods of stress or nutrient deprivation

Autophagy: Steps & Molecules From: Chris Weihl ULK complex ULK1 ATG13 ATG101 FIP200 Association: Class III PI3K complex   VPS34, VPS15, Beclin1, RUBCN   ATG14, WIPI2, DFCP1 Membrane expansion ATG9: Phospholipid delivery Conjugation System ATG12-ATG5-ATG16L1 complex LC3: Cleaved by ATG4 Receptors: p62 Other: ATG3; ATG7 Fusion Machinery UVRAG STX7 RAB7 SNAP29 VAMP7/8 LAMP2 HOPS PLEKHM1 Autolysosome-related Molecules SNX4/5/17 complex TEFB/TEF13 INPP5K Alternate Pathway Secretory Autophagy

• Organelle pathway
  • Lysosome damage
  • IL1B sequestration membrane
  • Autophagosome
  • Secretion across: Plasma membrane
• Related Structures & Molecules
  • Secretary autophagy receptor: TRIM16
  • Autophagosome: IL1B; Sec22B
  • Plasma membrane: STX 3/4; SNAP 23/29
  • No involvement: Golgi apparatus
• Cargo
  • Unconventionally secreted proteins: Proteins with no secretory tag
  • Presecretion locations: Autophagosome
  • Localization after secretion
    • Extracellular space: Common
    • Surface membrane: Galectins; Annexins; Tissue transglutaminase (TTG)
• Secretory Autophagy: Functions
  • Cytosolic proteins with extracellular functions: IL-1β; IL-18; HMGB1; Galectin-3
  • Removal of Aggregate-forming proteins: α-Synuclein; Amyloid-β
  • Extracellular release of Cytoplasmic organelles: Mitochondria; Autophagic organelles; Phagolysosomes; Endoplasmic reticulum
  • Synaptic remodeling
  • Microbial release & Transmission: Poliovirus; M. marinum; B. abortus; Influenza A; Coxsackie; Morbillivirus
  • Regulates conventional secretory pathway
• Functions of secreted proteins
  • Inflammation: Cytokines; Regulation
• Associated disorders
  • XMEA
  • Colchicine toxicity
  • Dermatomyositis with Vascular Pathology
  • Congenital MG: GFPT1; DPAGT1
  • Acid Maltase deficiency

From: R Schmidt

From: R Schmidt

From: R Schmidt

From: R Schmidt

• Histology: Vacuoles & Aggregates
  • Autophagic
  • Sarcolemmal features
  • LC3 & p62 stain
• Autophagy-associated proteins & disorders
  • Muscle
    • Autophagosome-Lysosome fusion
      • Danon: LAMP2
    • Cargo degradation
      • XMEA: VMA21
      • Acid maltase deficiency
      • CLN3
      • Chloroquine-Hydroxychloroquine myopathy
    • Autophagosome maturation
      • Colchicine myopathy
    • Cargo recognition (Granulophagy) defects
      • MSP1: VCP
    • Chaperone-Assisted Selective Autophagy
      • Myofibrillar myopathies
    • Autophagy increased: PI3K/AKT/mTOR pathway
      • Atrophy: Cachexia; Disuse
    • Autophagy induction: Possible disorders
      • SCAR31: ATG7
      • Collagen VI
      • Centronuclear myopathies
      • LGMDR8: TRIM32
    • Other possible autophagy disorders
      • HIBM2: GNE myopathy
      • Sporadic IBM (IM-VAMP)
      • LC3 aggregates: Targets; DM-VP
  • Neuropathy
    • HSAN2B: FAM134B
  • Motor neuron syndromes
    • ALS1: SOD1; Mutants induce autophagy
    • ALS2: Alsin
    • ALS4: SETX
    • ALS5: SPG11
    • ALS10: TDP43; Related to autophagy initiation
    • ALS11: FIG4
    • ALS12: OPTN; Autophagy receptor
    • ALS14: VCP; Mutants induce autophagy
    • FTD-ALS1: c9orf72
    • FTD-ALS3 & NADGP: SQSTM1; Autophagy receptor
    • FTD-ALS4: TBK1; Autophagy initiation
    • FTD-ALS6: VCP
    • FTD-ALS7: CHMP2B
    • SMAX2: UBA1
  • CNS
    • Niemann-Pick, Type C: NPC1
    • Ataxia
      • SCAR20: SNX14
      • SCAR25: ATG5
      • SCAR31: ATG7
    • SENDA (NBIA5): WDR45
    • Spastic paraparesis
      • SPG11: Spatacsin
      • SPG15: ZFYVE26
      • SPG49: TECPR2
      • Parkinson-Spasticity: ATP6AP2
    • CLN3
    • Vici syndrome: EPG5
    • Tuberous sclerosis: TSC1 ; TSC2
  • Inflammatory bowel disease associations
    • Autophagy 16-like 1 (ATG16L1)
    • LRG47
  • 'lurcher' mouse
    • Glutamate receptor, ionotropic, delta 2 (GRID2) : Mutations
    • Activation of autophagy
  • 3-methyladenine: Blocks autophagy

Muscle Protein Degradation

• Stimuli
  • Fasting (Early)
  • Acidosis: Diabetes mellitus (Uncontrolled)
  • Metabolic: Uremia; Sepsis; Burns; Hyperthyroid
  • Inactivity: Denervation; Disuse
  • Glucocorticoids: Inhibited by insulin
  • Paraneoplastic
    • ? Tumor effect via tumor factors such as PIF
    • Increased production of proteasomal subunits
• Related molecules
  • Cytokines
    • Interferon-γ (IFN-γ)
      • Induces: Proteasome activator (PA28); 20S core proteasome subunits
      • Protein cleavage
        • Increased: After hydrophobic & basic amino acids
          
        • Reduced: After acidic & small amino acids
          
        • Generates peptides that bind to MHC I molecules
    • Tumor necrosis factor-α (TNF-α)
      • Inhibits skeletal myogenesis: ? via reduction in MyoD
      • Induces muscle fiber atrophy
    • Mediator: TNF-α & IFN-γ activate transcription factor Nuclear Factor-κB (NF-κB)
  • Transcription factors
    • NF-κB
    • Forkhead Box, class O (FOXO): Transcription factors; Reduces protein degradation
      • FOXO-3A : Upregulates atrogin-1; Produces reduced muscle fiber size
      • FOXO-1A : Overexpression produces muscle atrophy
  • Ubiquitin binding protein: ZNF216
    • Binds polyubiquitin chains
    • Associates with 26S proteasome
    • Colocalized with aggresome
    • Increased with denervation & fasting-induced muscle atrophy
    • Upregulated by expression of FOXO
    • ZNF216 deficiency
      • Resistance to denervation-induced atrophy
      • Ubiquitinylated proteins markedly accumulate with denervation
  • Inhibitors
    • Eicosapentaenoic acid
• Pathway: Ubiquitin-proteasome
  • Ubiquitin pathway protein upregulation
    • Some E2 carrier proteins; Ubiquitin; 20S core proteasome
  • Increased ubiquitination of cell proteins
  • Requires ATP
• Resulting amino acids
  • Used for hepatic gluconeogenesis
  • Energy generation in muscle: Leucine; Isoleucine; Valine
  • 3-methylhistidine from myofibrillar proteins (actin & myosin) lost in urine
    • Used as measure of muscle catabolism (when diet is meat free)

• Stimuli
  • Fasting: Prolonged
  • Low protein diet
  • Endocrine: Hypothyroid; Hypopituitary
  • Clenbuterol

Ferroptosis ¹⁴

• General features
  • Non-apoptotic regulated cell death
  • Iron
    • Dependent
    • Accumulation (Free Fe⁺⁺)
  • Caused by: Redox imbalance
  • Leads to Excessive: Lipid reactive oxygen species (ROS); Lipid peroxidation
• Pathology: Mitochondria
  • Size: Small
  • Outer membrane: Damaged
  • Cristae: Reduced or Absent
• Genes involved
  • GPX4, ACSL4, NRF2, TfR1, FTH1, LPCAT3, SLC7A11,SLC39A14, NCOA4, FSP1, COX2, ACSL4, P53, HSPB1
• Associated pathway: Xc-GSH-GPX4
  • Subunits: SLC7A11; SLC3A2
  • Glutathione synthesis
  • Inducer: Erastin interaction with VDAC2/3
• Relations to Skeletal muscle atrophy
  • Sarcopenia: Iron increased in aging muscle
  • Renal disease, chronic
  • Sepsis-Induced
  • Cisplatin
  • Motor neuron disease-related