Chaperone –mediated autophagy: Molecular mechanism and role in cellular homeostasis

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Chaperone mediated autophagy Molecular
mechanism and role in cellular homeostasis
CREDIT SEMINAR

• Lukram Kenedy Singh

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INTRODUCTION

• Autophagy is an evolutionary conserved and
  strictly regulated lysosomal pathway that
  degrades cytoplasmic materials and organelles.
• Autophagy is activated during stress conditions
  such as amino acid starvation, unfolded protein
  responses and viral infections.
• Depending on the delivery routes of
  cytoplasmic material to the lysosomal lumen three
  different autophagic routes are known
  1)Macroautophagy
• 2)Microautophagy
• 3)Chaperone-mediated autophagy

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Types of Autophagy
Tocris Bioscience Scientific Review series
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Chaperone Mediated Autophagy
Proceedings of the American thoracic society 2010
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How does CMA work?

• CMA is multi- step process that involves
  
• (1) substrate recognition and lysosomal
  targeting
• (2) substrate binding and unfolding
• (3) substrate translocation and
• (4) substrate degradation .
• Substrate recognition
• Recognition of substrate proteins in the
  cytoplasm by binding of constitutive chaperone ,
  the heat shock cognate protein of 70 KDa (hsc70)
  to a pentapeptide motif(KFERQ) present in the
  substrate.
• This motif consists of an amino acid
  glutamine(Q) residue at the beginning, one of the
  two positively charged amino acid arginine (R)or
  lysine (K), hydrophobic amino acids phenylalanine
  (F) and negatively charged glutamic acid (E).

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• Substrate binding
• Once bound to the chaperone, the substrate is
  targeted to the surface of the lysosomes .
• The substrate interacts with the cytosolic tail
  of single-span membrane protein
  lysosome-associated protein 2A(LAMP-2A).
• LAMP-2A is present at the lysosomal membrane as
  monomers
• and in association with other proteins to
  form a multi- proteins complex required for
  substrate translocation.
• Transition of LAMP-2A monomer to form multimer
  (700KDa) and the stability of LAMP-2A is
  maintained by hsp90 located at the luminal side
  of the lysosome.

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• Substrate translocation
• Translocation of the substrate protein
  across the lysosomal membrane requires the
  presence of hsc70(lys-hsc70).
• Translocation function actively by pulling
  the substrate proteins in a ratchet-like manner
  or alternatively hold onto the substrate
  passively to prevent its return to the cytosol.
• A pair of protein GFAP and EF1a specifically
  modulate LAMP-2A assembly/disassembly in a GTP-
  dependent manner.
• Association of the GFAP to the
  translocation complex contributes to its
  stabilization.

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• Substrate degradation
• Once the substrate has passed through the
  translocation complex disassembly occurs by the
  mobilization of GFAP from the complex to bind
  phosphorylated form of GFAP resident in the
  membrane.
• The disintegrate LAMP-2A monomers are partially
  cleaved by cathepsin A and a membrane associated
  metalloprotease.

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Mechanism of Chaperone mediated autophagy
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Regulation of CMA

• The main target of CMA regulation is LAMP-2A,
  whose levels in the lysosomal membrane directly
  correlate with CMA activity.
• Under stress condition (oxidative stress ),
  lysosomal LAMP-2A levels increase through
  transcriptional upregulation.
• In most cases, changes in the lysosomal levels of
  LAMP-2A are regulated directly at the membrane
  and do not require de novo synthesis of LAMP-2A.
• LAMP-2A is also subjected to tightly regulated
  degradation at the lysosomal membrane through
  sequential cleavage by cathepsin A and memebrane
  associated metalloprotease.

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Local regulation of CMA activity in the lysosome

• In low CMA activity, LAMP-2A is recruited to
  lysosomal membrane(L.Mb) microdomains.
• Partial cleavage of LAMP-2A by cathepsinA
  followed by rapid degradation in the lumen.
• When the CMA is activated, association of LAMP-2A
  with membrane microdomains decreases.
• Substrate binding to the cytosolic tail of
  LAMP-2A promotes multimerization of LAMP-2A to
  form translocation complex.

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• Intermediate filament protein glial fibrilillary
  acidic protein (GFAP) and elongation factor 1a
  (EF1-a) also participates in modulation of
  LAMP-2A dynamics.
• These two proteins modify the stability of the
  multimeric LAMP-2A complex and association of
  LAMP-2A with the lipid microdomains in a
  GTP-dependent manner.
• Lysosomal GFAP partitions into two
  subpopulations unphosphorylated GFAP that binds
  to the multimer of LAMP-2A and phosphorylated
  GFAP.
• EF1a in the presence GTP is released from the
  lysosomal membrane allowing dissociation of GFAP
  from the translocation complex and its binding to
  GFAP-P.
• Dissociation of GFAP favours the rapid
  disassembly of LAMP-2A multimeric complex and
  mobilization to lipid microdomain.

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Physiological functions of CMA
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Physiological functions of CMA.

• CMA contribute to amino acid recycling during
  prolong starvation, a condition where CMA is
  maximally activated.
• In quality control of cells where CMA pathway
  could selectively removed single proteins from
  the cytosol and cytosolic- assembled protien
  complexes.
• CMA also contributes to cell type- specific
  functions 1)Degradation of transcription factor
  Pax2 and 2) selective degradation of neuronal
  survival factor.
• Degradation of transcription factor Pax2 by
  CMA in kidney is important to control tubular
  cell growth.
• Selective degradation of a neuronal
  survival factor (MEF2D) by is essential for
  proper neuronal responses to injury.

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Alterations in CMA contribute to disease
Trends in cell biology 2012
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Reduced CMA and neurodegerative diseases

• In neurodegenerative pathologies there is
  failure of the proteolytic systems to
  adequately dispose deleterious protein.
• Mishandling of aberrant proteins alters
  proteostasis and leads to the precipitation of
  protein aggregates .
• Parkinsons disease (PD)
• Impairment of CMA is linked to the
  pathogenesis of parkinsons disease (PD).
• Dysfunction in CMA has been observed in both
  familial and sporadic PD.
• In familial two most commonly mutated
  proteins a-synuclein and leucine rich repeat
  kinase 2(LRRK2) undergo degradation via CMA.

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Mechanism of CMA Failure in parkinsons disease
Cell research 2014
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CMA activity in aging

• Functional decline in CMA also occurs with
  physiological aging.
• Age-dependent decay in CMA appears to be
  caused by age-related changes in lipid
  constituents of lysosomal membranes that alter
  the dynamics and stability of LAMP-2A
• Genetic manipulation to preserve CMA
  activity in old rodents by expressing exogenous
  copy of LAMP-2A in mouse has proven effective
  improving the healthspan of aged animals.( Zhang
  C et al.)
• Restored CMA functions in the transgenic
  animals results in improved cellular homeostasis.

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Cross talk between different proteolytic system.

• CMA activity is tightly coordinated with
  macroautophagy and UPS.
• Crosstalk between the CMA and macroautophagy is
  observed by constitutive activation of CMA in
  cells deficient in macroautophagy.
• Compromised CMA perturbs functioning of the UPS
  during the early stage of CMA blockage by
  affecting the turnover of specifc proteosome
  subunits.
• In HD dual failure of macroautophagy and UPS is
  compensates by constitutive upregulation of CMA.

Cell research 2014
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Case study

• Cell metab. (2014)
• Jaime L. Schneider, Yousin suh and Anna Maria
  Cuervo.

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Introduction

• Chaperone-mediated autophagy (CMA) is a
  catabolic pathway for selective degradation of
  cytosolic proteins in lysosome.
• CMA activity is decreases with age ,but the
  consequences of this functional decline in vivo
  remain unknown.
• Blockage of CMA causes hepatic glycogen depletion
  and hepatosteatosis.
• In this study they have generated a mouse with a
  conditional knockout for LAMP-2A and performed
  physiological and proteomic analysis to study
  effect of CMA in liver and the consequences of
  the failure of this pathway. .

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Experimental procedures

• Animals
• Male C57BL/6 mice ( wild-type or transgenic
  for Albumin-Cref/f) 3-5 months of age.
• L2AKO mice were generated using LoxP
  insertion to delete the exon region in LAMP2 gene
  encodes for LAMP-2A variant

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• Subcellular fractionation and isolation of
  lysosomes
• Mouse liver lysosomes were isolated from a
  light mitochondrial-lysosomal fration in a
  discontinous metrizamide density gradeint.
• Histological procedures
• Livers were fixed in 10 neutral buffer
  formalin and stained with HE and periodic
  stain(PAS)
• For oil-red-O staining ,liver tissue was
  frozen in OCT, sectioned and stained.

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Liver specific L2AKO mice decrease CMA activity
and display signs of liver damage and reduced
liver function.
Overall of all these findings suggested that
suppression of hepatic CMA in vivo leads to liver
damage and a decline in liver function.
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Altered lipid metabolism and hepatostasis in
liver-specifc L2AKO
Overall of these findings support that loss of
hepatic CMA leads to the alterations in fat
metabolism that render livers more vulnerable to
lipid challenges
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Increase energy expenditure and reduced
peripheral adiposity in liver L2AKOmice
RD
HFD
These finding shows that failure of hepatic CMA
activity leads to changes in metabolism that
compromise the ability to adapt to the energetic
requirements in response to different nutritional
challenges
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CMA regulates hepatic levels of carbohydrate
metabolim enzymes in response to starvation
Overall of these findings support that
compromised degradation of glycolytic enzymes and
increase levels in intracellular levels are
responsible for the alterations in carbohydrate
metabolism.
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Liver enzymes related to lipid metabolism undergo
regulated degradation by CMA
Overall findings supported that Hepatocyte CMA
blockage alters liver lipid metabolism
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Conclusion

• In this work through generation of a mouse model
  with ablated CMA activity it was identified CMA
  as a regulator of hepatic metabolism.
• The metabolic dysfunction observed upon blockage
  of hepatic CMA suggests that age- dependent
  decline in CMA activity may contribute to
  energetic deficiencies.
• They found that by blockage of CMA in liver,
  peripheral tissues such as WAT and BAT were also
  affected.
• The pronounced metabolic changes in L2AKO mice
  could be in part explained by tight
  interconnection between carbohydrate and lipid
  metabolism pathways.
• They identified the consequences of defective
  hepatic CMA activity go beyond the mere
  disruption of protein quality control to include
  to compromised ability to maintain metabolic
  homeostasis.

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Conclusion

• CMA identify and degrade single protein
  selectively in the lysosome.
• 30 cytosolic proteins are CMA substrate.
• CMA is at the forefront of cellular function in
  cellular stress
• Interventions to prevent blockage in old age may
  have therapeutic value in age related disease.
• The better molecular characterization of the
  components that participate in CMA allowed the
  identification of pathogenic conditions.

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