Credit seminar on

ESCRT proteins in physiology and disease

Speaker: Sandeep Kumar Guide : Dr. S. K. Sood

Animal Biochemistry

Contents• Introduction• Roles in physiology and Diseases Multivesicular bodies formation Cytokinesis HIV budding

Development Signal attenuation and Cancer Autophagy and Neurodegeneration

Introduction• ESCRT: Endosomal sorting complex required for transport

• ESCRT – first discovered in yeast that were defective in formation of MVB and called as Vps E complex

• Originally identified as regulators of protein targeting to lysosomes.

• ESCRTs work by mediating cargo recruitment and the formation of intraluminal vesicles during the maturation of MVBs

• ESCRTs are actually four distinct complexes: ESCRT-0, -I, -II, -III.

Multivesicular Bodies Formation

(Daniel P. Nickerson et.al.,2009)

GLUE

• Multivesicular bodies (MVBs): The endocytic organelle, mediates either the lysosomal degradation or recycling of proteins.

•The formation and scission of these intraluminal vesicles are mediated by the ESCRT complexes.

Multivesicular Bodies Formation

(Oliver Schmidt et.al.,2008)

ESCRT in Cytokinesis

(Bethan McDonald et.al.,2009)

• 3 Steps in cytokinesis Assembly of central spindles

Formation of cleavage FurrowFinal abscission at mid body

ESCRT in Cytokinesis

(Anna Caballe et.al.,2011)

HIV Budding

(Bo Meng et.al.,2013)

HIV Budding

(Carlton et.al.,2009)

ESCRT Proteins in Development• bicoid mRNA is coupled to the microtubule-dependent transport pathway

by binding Staufen, which forms a complex with three ESCRT-II proteins, Vps22, Vps25 and Vps36.

• A mutant form of any of these ESCRT-II proteins abolishes the bicoid mRNA gradient.

Bico

idN

anog

Anterior Posterior

Bicoid protein Gradient

Nanog protein Gradient

(Irion U & St Johnston D. ,2007)

Drosophila egg

ESCRT in Cell Signalling

(Wegner et.al.,2009)

Cancer• Signaling Endosomes: Early endosomes containing activated

receptors are in the ‘on’ state in terms of signaling.

• EGFR: best studied receptor tyrosine kinases

• excessive signaling Cancer

• Tsg101: Tumor Susceptibility gene Mdm2

DNA damageATM serine kinase

p53 p

Arrest cell Cycle in G1 phase

p53 Ub

Degradation of p53

Tsg101

Cancer

(Nobuyuki Tanaka et.al.,2008)

Mdm2

p53

ESCRT in Autophagy• Phagophore closure require ESCRT as in cytokinesis & Budding.• Formation of autolysosomes is inhibited in cells depleted of Tsg101 or

Vps24(Fig E)• ESCRT-III dysfunction caused by loss of mSnf7-2 or CHMP2BIntron5

expression leads to the accumulation of autophagosomes in neurons (Fig F)

(Lee et.al.,2007) (Filimonenko et. al.,2008)

• Jin-A Lee, Anne Beigneux, S. Tariq Ahmad, Stephen G. Young,2,4 and Fen-Biao GaoGladstone Institute of Neurological Disease and Department of NeurologyGladstone Institute of Cardiovascular Disease and Department of MedicineUniversity of California,San Francisco, California 94158Summary Defect in the endosomal-lysosomal pathway have been implicated in a number of neurodegenerative disorders[1]. A key step in the endocytic regulation of transmembrane proteins occurs in a subset of late endosomal compartments known as multivesicular bodies (MVBs), whose formation is controlled by endosomal sorting complex required for transport (ESCRT) [2, 3]. The roles of ESCRT in dendritic maintenance and neurodegeneration remain unknown. Here, we show that mSnf7-2, a key component of ESCRT-III, is highly expressed in most mammalian neurons. Loss of mSnf7-2 in mature cortical neurons caused retraction of dendrites and neuronal cell loss. mSnf7-2 binds to CHMP2B, another ESCRT-III subunit, in which a rare dominant mutation is associated with frontotemporal dementia linked to chromosome 3 (FTD3). Ectopic expression of the mutant protein CHMP2BIntron5 also caused dendritic retraction prior to neurodegeneration. CHMP2BIntron5 was associated more avidly than with mSnf7-2, resulting in sequestration CHMP2BWT of mSnf7-2 in ubiquitin-positive late-endosomal vesicles in cortical neurons. Moreover, loss of mSnf7-2 or CHMP2BIntron5 expression caused the accumulation of autophagosomes in cortical neurons and flies. These findings indicate that ESCRT-III dysfunction is associated with the autophagy pathway, suggesting a novel neurodegeneration mechanism that may have important implications for understanding FTD and other age-dependent neurodegenerative diseases.

Neurodegeneration• mSnf7-2 and CHMP2B is Required for Dendritic Integrity.

• CHMP2BIntron5 bind more stronger with mSnf7-2 than CHMP2BWT so

CHMP2BIntron5 fails to dissociate from ESCRT-III properly and aberrant MVB formation occur

• Ubiqitinated P62 and Htt PolyQ aggeregate- FTD And Huntington (Filimonenko et.

al.,2007)

mSnf7-2 CHMP2B(Lee et.al.,2007)

Summary ESCRT: necessity of eukaryotes for viability • The ESCRT machinery has evolved as a conserved device for abscission of

narrow membrane tubes filled with cytosol, mediating inward budding of vesicles into MVEs as well as cell separation during cytokinesis.

• Enveloped RNA viruses have acquired the ability to hijack this machinery to facilitate their budding from the plasma membrane.

• The functions of ESCRT subunits as tumour suppressors in Drosophila models can be correlated to their ability to mediate receptor downregulation and cancer.

• The ESCRT machinery is important for neuronal function. Even though the neurodegeneration in CHMP2B mutant cells has been attributed to impaired autophagy

Unanswered Questions• How are MVBs that fuse with the lysosome different from MVBs

that fuse with the plasma membrane to release exosomes? • Why only ESCRT subunit CHMP2B and Snf7-2 found to be

mutated in neurodegenerative diseases. Why.?• It is possible that one gene copy of the ESCRT subunits is

sufficient for normal development, but that both alleles are required under certain conditions of physiologic stress. Why.?

• There is no role of ESCRT II in cytokinesis but if there is mutation in ESCRT II it block cytokinesis. Why.?

• Which tumors over-express ESCRT and reduce.?• why depletion of the vpsE genes is lethal in multicellular

organisms but not in yeast.

…..?????

The yeast and human ESCRT proteins

AbbreviationsAAA ATPases associated with diverse cellular activitiesAIP1 ALG-2 interacting protein 1, synonym for AlixBro BCK1-like resistance to osmotic shockCC Coiled-coilCHMP Chromatin modifying protein (human orthologs of ESCRT-III subunits)Doa Degradation of alpha2DUB De-ubiquitinating enzymeEAP ELL associated protein (human orthologs of ESCRT-II subunits)EEA1 Early endosomal antigen 1EGF Epidermal growth factor receptorEGFR EGF receptor ESCRT Endosomal sorting complex required for transportFYVE Fab1, YOTB, Vac1, EEA1GAT GGA and TOMGLUE GRAM-like ubiquitin binding in EAP45GRAM Glucosyltransferases, Rab-like GTPase activators and myotubularinsHIV Human immunodeficiency virus

Hrs Hepatocyte growth factor receptor substrateHse Has symptoms of class E mutants; resembles Hrs, STAM, EastMIT Microtubule interacting and traffickingMVB Multivesicular bodyNZF Npl4 zinc fingerPI(3)P phosphatidylinositol 3-phosphateSnf Sucrose non-fermentingSTAM Signal transducing adaptor moleculeTsg101 Tumor susceptibility gene 101, the human ortholog of vps23Vps Vacuolar protein sortingUb UbiquitinUEV Unusual E2 variantUIM Ubiquitin interacting motifVHS Vps27, Hrs, STAMWW Tryptophan-TryptophanFTD3 frontotemporal dementia linked to chromosome 3

Abbreviations

References • Henne,W.M., Buchkovich,N.J. & Emr,E. The ESCRT Pathway. Developmental Cell 21, 71-91 (2011)• Williams,R.L. & Urbe,S. The emerging shape of the ESCRT machinery. Nat. Rev. Mol.

Cell Biol. 8, 355–368 (2007)• Schiel,J.A., Childs,C. & Prekeris,R. Endocytic transport and cytokinesis: From regulation of the

cytoskeleton to midbody inheritance. Trends in Cell Biology 23:7 319-327 (2013)• Hanson,P.I., Shim,S. & Merrill,S.A. Cell biology of the ESCRT machinery. Current Opinion in Cell

Biology 21,568–574 (2009)• Nickerson,D.P., Russell,R.G. & Odorizzi,G. A concentric circle model of multivesicular

body cargo sorting. EMBO reports 8, 644–650 (2007) • Rusten,T.E. & Stenmark,H. How do ESCRT proteins control autophagy? J. Cell Sci. 122, 2179-2183 (2009)• Lee,J.A., Beigneux, A., Ahmad,S.T., Young,S.G. & Gao,F.B. ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration. Curr. Biol.

17, 1561-1567 (2007)• Schmidt,O. & Teis,D. The ESCRT machinery. Current Biology 22 No 4 R116 • Michelet,X., Djeddi,A. & Legouis,R. Developmental and cellular functions of the ESCRT

machinery in pluricellular organisms. Biol. Cell 102, 191–202 (2010)• Roxrud,I., Stenmark,H. & Malerød,L. ESCRT & Co. Biol. Cell 102, 293–318 (2010)

References • McCullough,J., Colf,L.A. & Sundquist W.L. Membrane Fission Reactions of the Mammalian

ESCRT Pathway Annu. Rev. Biochem. 82, 663–92 (2013)• McDonald,B. & Serrano, J.M. No strings attached: the ESCRT machinery in viral budding and

cytokinesis. J.Cell Sci. 122, 2167-2177 (2009)• Rusten,T.E., Vaccari,T. & Stenmark, H. Shaping development with ESCRTs. Nat. Cell Bio. 14:1 38-

45 (2012)• Rodahl,L.M., Stuffers,S., Lobert,H. & Stenmark,H. The role of ESCRT proteins in attenuation of

cell signalling. Biochem. Soc. Trans. 37, 137–142 (2009)• Carlton,J. The ESCRT machinery: a cellular apparatus for sorting and scission Biochem. Soc.

Trans. 38, 1397–1412 (2010)• Meng,B.O. & Lever,A. Wrapping up the bad news – HIV assembly and release. Retrovirology

10:5 (2013)• Weiss,E.R. & Göttlinger,H. The Role of Cellular Factors in Promoting HIV Budding. J. Mol. Biol.

410, 525–533 (2011)• Pincetic,A. & Leis,J. TheMechanism of Budding of Retroviruses fromCellMembrane. Advances

in Virology 2009, Pg. 9 (2008)• Carlton,J.C. & Serrano,J.M. The ESCRT machinery: new functions in viral and cellular biology.

Biochem. Soc. Trans. 37, 195–199 (2009) • Caballe,A. & Serrano,J.M. ESCRT Machinery and Cytokinesis: the Road to Daughter Cell

Separation Traffic 12, 1318–1326 (2011)

THANK YOU

THAN

K YO

U

THANK YOU