2006 — 2007 |
Snapp, Erik L. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Polycystic Liver Disease and Er Quality Control @ Albert Einstein Col of Med Yeshiva Univ
[unreadable] DESCRIPTION (provided by applicant): The products of the autosomal-dominant polycystic liver disease (PCLD) genes affect the folding and quality control of membrane and secretory proteins. The PCLD genes, PRKCSH and SEC63, encode the endoplasmic reticulum (ER) proteins Sec63, a cofactor for the lumenal ER chaperone BiP, and glucosidase II b, which modulates glycoprotein interactions with the lectin chaperones calnexin and calreticulin. How these mutations promote cyst formation in hepatic biliary duct cells (cholangiocytes) while remaining effectively silent in most other tissues remains unclear. To dissect the molecular mechanisms of PCLD, we will develop a tissue culture model for PCLD and will investigate the cellular consequences of the PCLD mutations. Specifically, we will 1) Establish and characterize PCLD cholangiocyte cell lines, and 2) Use the PCLD cell lines to identify downstream targets of the PCLD mutants. These studies will provide some of the first insights into the cell biology of PCLD and will identify substrates that require the activities of Sec63 and glucosidase IIb. [unreadable] [unreadable] [unreadable]
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0.934 |
2008 — 2009 |
Schmidt, Marion Snapp, Erik L. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Detecting and Responding to Misfolded Protein Burdens in Aging Cells @ Albert Einstein Col of Med Yeshiva Univ
[unreadable] DESCRIPTION (provided by applicant): An increase in cellular protein misfolding and a decrease in degradation of proteins are characteristics of cellular and organismal aging. Prevention of misfolded protein accumulation is normally mediated by the cellular quality control (QC) machinery, including chaperones and the proteasome. Loss of QC machinery function can occur at multiple regulatory levels. However, many of the QC components are especially long lived proteins. Acute loss of a cell's QC capacity predisposes the cells to rapid death. In contrast, aging appears to be a gradual process that occurs over the course of several days to years, not a catastrophic process. We hypothesize that discrete components of the cellular protein quality control and homeostasis machinery are selectively and progressively inactivated during aging, potentially due to oxidative damage. As a consequence, the ability of cells to respond to misfolded protein stress or maintain homeostasis is likely to be impaired. While studies have addressed changes in mRNA levels, protein levels, and oxidative damage to QC proteins, little is known regarding the functionality of the QC proteins in aging cells. In this proposal, we will directly measure QC protein function at the levels of individual QC proteins, their substrates, and global misfolded protein stress. These problems will be investigated by 1) developing new methods for detecting misfolded protein stress and QC machinery function in living cells, 2) determining which QC components lose functionality in aging cells and in an aging model of oxidatively stressed cells, and 3) using genetic, pharmacologic, and biophysical fluorescence methods to define how crosstalk between cytoplasmic and secretory protein QC systems modulate misfolded protein levels and stress in aging cells. Project Narrative: Several protein misfolding diseases increase in incidence and severity as people age. We are studying, at the cellular level, the mechanisms that normally prevent misfolded proteins from accumulating in cells and how these mechanisms are altered during aging. [unreadable] [unreadable] [unreadable]
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0.934 |
2013 — 2015 |
Ahn, Natalie G [⬀] Palmer, Amy E (co-PI) [⬀] Palmer, Amy E (co-PI) [⬀] Snapp, Erik L. Verkhusha, Vladislav |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Technologies to Define and Map Novel Interorganelle Macromolecular Interactions
DESCRIPTION (provided by applicant): Interorganelle interactions are key processes controlling eukaryotic cell function, and dysregulation of these interactions has been implicated in many human diseases. However, relatively little is known about macromolecular complexes that mediate organelle interactions, due to obstacles that have been difficult to overcome. First, many relevant proteins are integral membrane proteins, which are hard to purify and maintain weak but physiologically important binding interactions. Capturing such interactions by conventional biochemical and genetic approaches is technically difficult. Second, simultaneously tracking transient organelle populations and interactions requires the ability to follow real time dynamics in living cells using multi-color fluorescent probes. However, many fluorescent proteins (FPs) used for live imaging are compromised by the oxidizing environment of many organelles, including ER, Golgi, and secretory vesicles. The goal of this proposal is to define protein complexes that define and modulate novel organelle subpopulations, using a combination of new technologies in mass spectrometry and fluorescent protein based probes for live cell imaging. Our Specific Aims are: (1) Identify candidate protein markers of novel organelles and interorganellar protein complexes. We will develop a proteomics strategy to profile proteins within organelle subpopulations that are dynamic and transient, as well as macromolecular complexes that bridge organelles. (2) Develop novel biosensors to track these protein markers in living cells, by time resolved imaging and high resolution microscopy. We will maximize the available colors of the fluorescent protein spectrum for use in multi-color live cell imaging studies, by solving key problems in fluorescent protein reporters caused by organellar environments that restrict their folding and function. (3) Apply these methods to cutting edge problems in cell biology, addressing mechanisms underlying (i) ER stress and Ca2+-mediated organelle remodeling, (ii) Zn2+ homeostasis, and (iii) cell polarity. We will combine technologies developed in Aims 1 and 2 to create a new experimental workflow which integrates mass spectrometry/proteomics, biosensor design, and high resolution fluorescence microscopy, and apply this to relevant problems in collaborator labs in Aim 3. Our proposal establishes a unique, multidisciplinary collaboration between a team of four investigators, who are leading experts in technologies of proteomics/mass spectrometry, protein engineering and biosensor design, and cutting edge methods for high resolution cell imaging. The combined expertise from these investigators gives us a unique opportunity to discover novel organelles and macromolecular complexes involved in interorganelle contacts, and define their cell biology.
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0.957 |