Degradation

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PROTEIN DEGRADATION^{^1,2}

General principles
Protease classes

Autophagy
Chaperones
Cysteine
Lysosomal
Metalloproteinases
Mitochondrial
Muscle
Serine
Ubiquitin-Proteasome

Also see
Apoptosis
Caspases
External link: Prolysis

GENERAL PRINCIPLES

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
ê Proteolytic activation of enzyme	Factor IX Chapel Hill
ê Proteolytic inactivation of enzyme	Factor V Leiden
é Protein expression 2° stabilized mRNA	Hyperprothrombinemia
ê Enzyme activity; Disulfide bond Δ	Factor VII
ê Enzyme activity; Loss of highly conserved arginine	Limb Girdle MD 2A
ê 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 ê serine protease activity
Response to inflammation	é matrilysin & gelatinase B in GBS nerves

SERINE PROTEASES

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
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 ® é Dissolution of blood clots (fibrinolysis) ® Excessive bleeding

CYSTEINE PROTEASES

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
  - Diseases: Cathepsin C : Papillon-Lefevre ; Haim-Munk
- 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-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

LYSOSOMAL PROTEASES

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
Lysosomal formation: From phagocytosis, endocytosis, autophagy
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
- 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
  - Sialic acid storage diseases: Sialin (SLC17A5)
    - Infantile sialic acid storage disorder
    - Salla disease
- HMSN 2B
- Storage disorders
  - Glycogen: Acid maltase; Lamp-2
  - Toxic: Amiodarone; Chloroquine; Perhexiline
  - Inclusions: Cockayne
  - Aging: Lipofuscin accumulation
  - Other: XMEA

METALLOPROTEINASE

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 : Required for maturation of iron-containing proteins
- 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	ê Tolerance
Proteolysis of basement membrane	Blood-brain barrier damage
Proteolysis of extracellular matrix	é Cell migration
é Cytokine activation from precursors	é Inflammation
Proteolytic activation of zymogens	é 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

Degradation Pathway

End products
Large 26S proteasome
Overall process
Ubiquitin
Ubiquitin binding process

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
- 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

Ubiquitination regulated cellular processes

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 disorders⁹
  - Ubiquitin protein ligase (E3)
    - Angelman syndrome: UBE3A
    - LGMD 2H : TRIM32
    - X-linked Mental Retardation Syndrome: CUL4B
    - Mental retadation: HUWE1 duplication (with (HSD17B10)
  - 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; PGP9.5)
    - Protein function: Deubiquitinating enzyme
    - Disorders
      - Parkinson's disease
      - 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)
  - 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 : Huntington's disease
    - PrP : Prion disease
    - CFTR : Cystic fibrosis
    - p53 : Cancer
  - Ubiquitin-proteasome pathway cofactor
    - Ubiquitin : Alzheimer's, late onset

MITOCHONDRIAL PROTEASES

Paraplegin
- Hereditary spastic paraparesis 7 (recessive)
- Metalloproteinase
Elastase 2
- Granulocytes & erythroblasts
- Serine protease

PROTEIN FOLDING & CHAPERONES⁸

Protein folding propensity is intrinsic property of peptide
- Proteins in "non-functionally" (Unfolded/Partially) folded configurations more likely to be degraded
- Mutant proteins: Exposed for longer time in "non-functionally" folded configurations
  - Abnormal folding
  - Increased likelihood of spontaneous unfolding
- Disorders
  - Endoplasmic reticulum: Cystic fibrosis (δ508) ; α-1-antitrypsin deficiency (E342K)
  - Mitochondria: MCAD deficiency (K304E) ; SCAD deficiency (R22W)
  - Cytosol: Phenyketonuria (A104D)
Molecular chaperones
- Function
  - Minimize abnormal folding & aggregation of proteins
  - High chaperone levels enhance rescue of floding defects
- Mechanism: Recognize amino acid domains with hydrophobic side chains
  - Normally sequestered inside normally folded protein
- Types of chaperones
  - Folding helper
    - Mechanism: Cycle between high & low affinity forms by conformational changes
    - Cycling mediated by ATP & other mechanisms
    - Families: Hsp70; Hsp90; Chaperonins; Lectin chaperones; Clp/Hsp100; AAA
    - Diseases
      - Hereditary spastic paraplegia (SPG7): Paraplegin
      - Peroxisome biogenesis disorder
  - Holding
    - Mechanism: Hydrophobic surfaces mediate reversible binding of unfolded peptides
    - Role
      - Protect partilly folded/misfolded proteins from aggregation & degradation
      - Protection needed until folding helper chaperones become available
    - Family: Small Hsp (sHsp)
    - Diseases
      - Cataract: α-crystallin
      - Desmin-related myopathy: ab-crystallin
Other protein folding disorders
- Abnormally folded protein not secreted in enough quantity for proper function: Cystic fibrosis
- Abnormally folded protein becomes insoluble & deposits in tissues: Amyloid
- Charlevoix-Saguenay - Spastic Ataxia: Sacsin protein

Autophagy

General

Action: Process of subcellular degradation
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
Substrates
- Proteins: Folded or unfolded
- Protein complexes
- Entire organelles
Associated processes & disorders
- Myopathies
- Cancer
- Neurodegeneration
- Major histocompatibility complex (MHC) class II antigen processing
- Removal of bacterial & viral pathogens

Chaperone-mediated autophagy

General: Direct translocation of cytosolic proteins with pentapeptide motif across lysosomal membrane
- Requires actions of cytosolic & lysosomal chaperones to unfold substrates
- Formation of a protein complex: Between cytosolic "substrate" & "chaperone"
- Complex then interacts with a lysosomal membrane receptor (LAMP 2a) for translocation into the 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
Disease: ? Blocked in some forms of Parkinson's disease

Macroautophagy (Autophagy)

Stages
- Membrane sequestration of cytoplasm: Double-membrane autophagosome
- Fusion of membrane bound vesicles with lytic component (lysosomes)
- Degradation
- Release of degradation products or cell death
Morphological hallmark: Double membrane sequestering vesicle (Autophagosome)
Action: Bulk lysosomal degradation
Targets
- Larger cytoplasmic proteins: Folded or unfolded
- Organelles: Mitochondria; Fractured endoplasmic reticulum; Peroxisomes
Mechanisms: Formation of double-membrane vesicle of nonlysosomal origin (Autophagosome)
- Several phases: All ATP dependent
  - Initiation
  - Execution
  - Maturation
- Initiation
  - Generation of crescent-shaped isolation membrane or phagophore
    - Membrane is formed de novo, not by budding
    - Sequestering membrane: Double layer; Separate from lysosome
  - Phagophore sequesters cytoplasm and organelles
- Expansion & eventual closing of phagophore: Gives rise to autophagosome
- 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
      - ATG8/LC3 (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
    - ATG5
      - Essential for autophagy
      - Conjugated to ATG12
      - Binds Atg16: Part of tetrameric complex of unknown function
      - Conditional knock-out in brain: Develop aggregates & ubiquitinated inclusions
    - ATG3
      - E2-like protein-conjugating enzyme
      - Covalently attaches ATG/LC3 to phosphatidylethanolamine
    - ATG4
      - Cysteine protease
      - Cleaves the C-terminus of Atg8/LC3 to expose a glycine residue for subsequent conjugation
    - Beclin 1 (BECN1; ATG6)
      - Component of complex that also includes: Class III phosphotidylinositol (PtdIns)-3-kinase (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 to allow expansion of phagophore
        
        Initial nucleating membrane that develops into an autophagosome
        
        May be involved in delivering membrane 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
    - ULK1 (ATG1 homologue)
      - Serine/threonine protein kinase
      - May be involved in regulation & vesicle formation
      - Involved in axon growth
    - LAMP2: Danon disease
    - Class I Phosphatidylinositol 3-kinase
      - Control 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
- Induction
  - Upregulation of HDAC6
  - Rapamycin
  - Starvation (Low amino acids)
End results
- Vesicles (Autophagosomes) fuse with lysosomes
  - Requirement: Microtubular system
  - Forms: Autophagolysosome
  - Degradation: Contents exposed to acid hydrolases
- 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 as part of immune response to eliminate microbial pathogens
  - Removal of cytoplasm may result in cell death
- Major catabolic pathway for
  - Energy generation
  - Breakdown of macromolecules and damaged organelles into their essential constituents
  - Most prominent: During periods of stress or nutrient deprivation

Disorders of Autophagy

Autophagic vacuoles
- With sarcolemmal features
- Autophagic vacuoles: Other
Autophagy-associated proteins & disorders
- 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

INCREASED PROTEIN BREAKDOWN

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)

DECREASED PROTEIN BREAKDOWN

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

Return to Neuromuscular Home Page

References
1. NEJM 1996;335:1897-1905
2. Human Mutation 1999;13:87-98
3. Immunol Today 1997;18:189-198
4. FASEB J 1997;11;1067-1075
5. Cell Mol Life Sci 1998;54:253-262
6. J Neuroimmunol 1999;94:1-14, Sem Cancer Biol 2000;10:415-433
7. Annu. Rev. Immunol. 1999;17:739-779
8. Human Mutation 1999;14:199-215
9. Molec Genet Metab 2000:71:261-266

3/4/2008

PROTEIN DEGRADATION1,2

UBIQUITIN-PROTEASOME PROTEIN DEGRADATION 3-5,7

Autophagy

MUSCLE PROTEIN DEGRADATION

PROTEIN DEGRADATION^{^1,2}

UBIQUITIN-PROTEASOME PROTEIN DEGRADATION ^3-5,⁷