Research interests: Protein aggregation and molecular chaperones in cancer

The Cashikar Laboratory

                                                              research opportunities

Education
BSc         1989        Bangalore University, Bangalore, India
MSc         1991       Madurai Kamaraj University, Madurai, India
PhD         1997       Centre for Cellular and Molecular Biology, Hyderabad, India
Postdoc   1997-01   Dr. Susan Lindquist, HHMI/University of Chicago, Chicago, Ill.
                2001-02   Dr. Susan Lindquist, Whitehead Institute/MIT, Cambridge, Mass.

Current Research

The term molecular chaperone encompasses several families of highly conserved proteins that mediate the folding and assembly of other proteins though they are not components of the final functional structure. Many chaperones are highly overproduced during stress conditions, such as heat, oxidative stress, viral infection, anoxia and even normal aging – most likely due to a reduced availability or functional capacity of the molecular chaperones themselves. Consequently, they are also called heat shock or stress proteins (Hsps). When molecular chaperones go awry, proteins misfold. Inevitably, the misfolded proteins clump together into protein aggregates. Many diseases are characterized by the accumulation of intracellular or extracellular protein aggregates. Our overall goal is to understand the cross-talk between molecular chaperones and protein aggregation in relation to neurodegenerative diseases and cancer.

Mechanisms that clear protein aggregates.

That some cells do not accumulate aggregates indicates their ability to clear protein aggregates normally. In baker’s yeast (Saccharomyces cerevisiae), aggregated proteins are reactivated by the protein disaggregation machinery, which includes molecular chaperones of the Hsp100 family together with Hsp70/Hsp40. However, these mechanisms are not well understood in mammalian cells. We are interested in identifying such mechanisms using yeast as a model system.

There are many advantages of using yeast as an experimental system. Yeast is currently the single best-characterized eukaryote. We have previously demonstrated its utility as an excellent model for biomedical research in cancer and neurodegenerative diseases. As a unicellular organism, it is simple to work with, yet it extensively shares the molecular complexities of metazoans. It provides an invaluable option in working with either haploid or diploid cells. Its genome was the first eukaryotic genome to be sequenced, paving the way to generate a knockout strain for every gene in the genome; to compile microarray data on a vast array of conditions; and to clarify important genetic, proteomic and metabolite networks.

In an effort to utilize this unprecedented level of detail in any biological system, we have undertaken the genome-wide screening approach and identified novel components (genes) that influence aggregate clearance. We are interested in deciphering the intricacies of their molecular mechanisms.

Key References:

Mir S, Fiedler D, Cashikar AG. Ssd1 is required for thermotolerance and Hsp104-mediated protein disaggregation in Saccharomyces cerevisiae. Mol Cell Biol. 2009 Jan;29(1):187-200.

Xia L, Jaafar L, Cashikar AG, Flores-Rozas H. Identification of Genes Required for Protection to Doxorubicin by a Genome-Wide Screen in Saccharomyces cerevisiae. Cancer Res. 2007 Dec 1;67(23):11411-8.

Cooper AA,* Gitler AD,* Cashikar AG, Haynes CM, Hill KJ, BhullarB, Liu K, Xu K, Strathern KE, Liu F, Cao S, Caldwell KA, Caldwell GA, Kolodner RD, LaBaer J, Rochet JC, Bonini NM, Lindquist SL. Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science. 2006 Jul 21;313(5785):324-8. (*Equal contribution.)

Yeger-Lotem E,* Riva L,* Su LJ,# Gitler AD,# Cashikar AG, King OD, Auluck PK, Geddie ML, Valastyan JS, Karger DR, Lindquist S, Fraenkel E. Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity. Nat Genet. 2009 Mar;41(3):316-23.

Wendler P, Shorter J, Plisson C, Cashikar AG, Lindquist SL, Saibil HR. Novel AAA+ subunit packing creates an expanded cavity for protein disaggregation by the protein-remodeling factor Hsp104. Cell. 2007;131:1366-77.

Cashikar AG, Schirmer EC, Hattendorf DA, Glover JR, Ramakrishnan MS, Ware DM, Lindquist SL. Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein. Mol Cell. 2002 Apr;9(4):751-60.

Molecular chaperones in human disease.

Members of the sHsp family of molecular chaperones protect cells from a variety of environmental conditions, such as heat and oxidative stress, by antagonizing protein aggregation. In vivo, a number of functions have been proposed for sHsps – enhancing stress resistance, regulating cytoskeletal dynamics, inhibiting apoptosis, etc. sHsps share a conserved α-crystallin domain of 80 to 100 amino acids at their C terminus whereas their N-terminal regions are highly variable in sequence and length. It has been proposed that sHsps aid in refolding of denatured proteins by holding them in a reactivation-competent state. The sHsps form dynamic oligomeric structures, ranging from 9 to 50 subunits and resembling a hollow soccer ball.

The human genome codes for 10 genes for sHsps, differing from 45 to 85 percent in sequence. Mutations in human sHsps lead to several aggregation diseases – for example, mutations in αA-crystallin leads to cataracts, mutations in αB-crystallin leads to desmin-related myopathy and missense mutations in Hsp27 are associated with Charcot-Marie-Tooth disease. Both Hsp27 and αB-crystallin have been found in proteinaceous inclusions of Alzheimer’s and Parkinson’s diseases. These diseases are characterized by the accumulation of extracellular or intracellular protein aggregates. The aggregates may consist of fibrillar structures of misfolded protein, termed amyloids. The sHsps are highly protective against toxicity induced by Parkinson’s (α-synuclein) or Huntington’s (polyglutamine) disease model systems. Our goal is to understand the mechanism by which the sHsps protect cells from protein aggregates.

Key References:

Cashikar AG, Duennwald M, Lindquist SL. A Chaperone Pathway in Protein Disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104. J Biol Chem. 2005 Jun 24;280(25):23869-75. Erratum in: J Biol Chem. 2006 Mar 31;281(13):8996.

Serio TR,* Cashikar AG,* Kowal AS, Sawicki GJ, Moslehi JJ, Serpell L, Arnsdorf MF, Lindquist SL. Nucleated conformational conversion and the replication of conformational information by a yeast prion determinant. Science. 2000 Aug 25;289(5483):1317-21. (*Equal contribution.)

Serio TR, Cashikar AG, Moslehi JJ, Kowal AS, Lindquist SL. Yeast prion [psi +] and its determinant, Sup35p. Methods Enzymol. 1999;309:649-73.

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