Zhaohui Xu - US grants

Protein translocation is one of the fundamental aspects of cell biology. Understanding how proteins move from their sites of synthesis to their sites of action is relevant since almost half of the proteins of a cell are transported into or across a membrane. In fact, the amounts and locations of particular proteins are controlled during development, during the cell cycle, and for maintenance of healthy cells, failure in secretion results in not only activity deficiency at the indeed subcellular location but also toxic levels of molecules in the wrong place. The long term objectives of the following proposal are to understand the molecular mechanisms by which cellular machinery translocates proteins across membrane. The current focus is on the early events that occurs in the E. coli Sec translocation system, with emphasis on mechanisms regarding (1) the binding of a polypeptide by a translocation dedicated chaperone SecB (2) the general recognition motif within the translocating polypeptide, and (3) the interaction of SecB with its membrane receptor SecA and the effect of polypeptide binding by SecB. Our approach will be to use high resolution X-ray crystallography to establish the three- dimensional structures of SecB and its relevant complexes with peptides and/or SecA. Mutational and biochemical experiments will then be used to complement structural studies. The combination of these approaches will help us to understand the physical chemistry that govern protein translocation by the Sec system.

DESCRIPTION (provided by applicant): Diabetes and impaired glucose tolerance is a major cause of morbidity and mortality worldwide. Normal glucose homeostasis requires that insulin is properly synthesized and secreted from the beta cell upon periodic increases in blood glucose. Disturbances in beta cell function could result in the loss of glucose-stimulated insulin secretion, a major cause for type II diabetes. Recent studies demonstrated an association between beta cell function, proliferation, and/or survival with an intracellular signaling pathway termed the unfolded protein response (UPR). Upon accumulation of unfolded proteins in the lumen of the endoplasmic reticulum, signal transduction pathways are activated to increase the protein folding capacity and the protein degradative machinery. In addition, protein synthesis is also transiently attenuated. These responses collectively enable cells to tolerate and survive conditions that disrupt normal protein folding and protein secretion processes in the ER. The long-term goal of this proposal is to understand the molecular mechanism of the UPR through structure/function studies of the molecules involved. A class of novel ER trans-membrane receptors including IRE1, PERK, and ATF6 mediate activation of the UPR. Of these, IRE1 and PERK are more closely related and appear to be activated through a similar mechanism. Therefore, our current proposal will focus on the structure and function of these two ER trans-membrane receptors. Under normal conditions, both IRE1 and PERK are kept in an inactive, monomeric form by binding to ER chaperone BiP. Upon receiving stress signals, BiP is released from the receptors, which dimerize to activate downstream signaling events. We will use high resolution X-ray crystallography to determine the structures of the lumenal activation/dimerization domains of IRE1 and PERK as well as their complexes with BiP. In collaboration with Dr. Randy Kaufman at the University of Michigan, we will use molecular biology and biochemistry tools to further probe the structure and function relationship of these proteins. The results we obtained from these studies have the potential for providing novel insights into human disease and may also lead to new therapeutic strategies.

DESCRIPTION (provided by applicant): Diabetes and impaired glucose tolerance is a major cause of morbidity and mortality worldwide. Normal glucose homeostasis requires that insulin is properly synthesized and secreted from the beta cell upon periodic increases in blood glucose. Disturbances in beta cell function could result in the loss of glucose-stimulated insulin secretion, a major cause for type II diabetes. Recent studies demonstrated an association between beta cell function, proliferation, and/or survival with an intracellular signaling pathway termed the unfolded protein response (UPR). Upon accumulation of unfolded proteins in the lumen of the endoplasmic reticulum, signal transduction pathways are activated to increase the protein folding capacity and the protein degradative machinery. In addition, protein synthesis is also transiently attenuated. These responses collectively enable cells to tolerate and survive conditions that disrupt normal protein folding and protein secretion processes in the ER. The long-term goal of this proposal is to understand the molecular mechanism of the UPR through structure/function studies of the molecules involved. A class of novel ER trans-membrane receptors including IRE1, PERK, and ATF6 mediate activation of the UPR. Of these, IRE1 and PERK are more closely related and appear to be activated through a similar mechanism. Therefore, our current proposal will focus on the structure and function of these two ER trans-membrane receptors. Under normal conditions, both IRE1 and PERK are kept in an inactive, monomeric form by binding to ER chaperone BiP. Upon receiving stress signals, BiP is released from the receptors, which dimerize to activate downstream signaling events. We will use high resolution X-ray crystallography to determine the structures of the lumenal activation/dimerization domains of IRE1 and PERK as well as their complexes with BiP. In collaboration with Dr. Randy Kaufman at the University of Michigan, we will use molecular biology and biochemistry tools to further probe the structure and function relationship of these proteins. The results we obtained from these studies have the potential for providing novel insights into human disease and may also lead to new therapeutic strategies.

Diabetes and impaired glucose tolerance is a major cause of morbidity and mortality worldwide. Normal glucose homeostasis requires that insulin is properly synthesized and secreted from pancreatic [unreadable]-cells upon periodic increases in blood glucose level. Disturbances in [unreadable]-cell function could result in the loss of glucose-stimulated insulin secretion, a major cause for type II diabetes. Recent studies demonstrated an association between [unreadable]-cell function, proliferation, and survival with an intracellular signaling pathway termed the Unfolded Protein Response (UPR). Upon accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER), signal transduction pathways are activated to increase the rate of protein clearance from the ER and to decrease the rate of overall protein synthesis and translocation into the ER. These responses collectively enable cells to tolerate and survive conditions that disrupt the normal protein folding and protein secretion processes in the ER. The longterm goal of this proposal is to understand the molecular mechanism of the UPR and ultimately design new therapeutic approaches to treat diabetes and other related diseases. A class of novel transmembrane ER stress sensors including IRE1, PERK, and ATF6 mediate activation of the UPR. Our current study will focus on the structure and function of IRE1 and PERK. Under normal conditions, both IRE1 and PERK are maintained in an inactive, monomeric form by binding to ER chaperone BiP. Upon ER stress, BiP is released from these two proteins, both of which homodimerizes to activate downstream signaling events. Three specific aims will be pursued in this proposal. First, crystal structure of the PERK luminal domain will be determined to identify molecular interactions that mediate PERK-specific UPR activation in the ER lumen. Second, crystal structure of IRE1 luminal domain in complex with BiP will be studied to understand the molecular mechanism underlying BiP-dependent regulation of the UPR signaling. Third, crystal structure of IRE1 cytosolic domain will be characterized to examine molecular interactions that mediate phosphorylation-dependent IRE1 activation on the cytoplasmic side of the ER membrane. Results from these studies will significantly broaden and deepen our understanding towards to the molecular mechanism underlying the UPR activation.

DESCRIPTION (provided by applicant): The Endosomal Sorting Complex Required for Transport (ESCRT) is a membrane scission machine that functions to direct membrane budding away from the cytoplasm. Its role has been documented in several biological processes that are important to cellular homeostasis and defense against aging, including multi-vesicular body (MVB) biogenesis, membrane abscission during cytokinesis and autophagosome formation in autophagy. In addition, viruses such as HIV-1 and Ebola virus have hijacked the ESCRT machinery for their own usage in budding from the plasma membrane of infected human cells. Therefore, a detailed understanding of the molecular mechanism of the ESCRT function can provide insights into the pathophysiology of a range of human diseases from cancer, viral infection to neurodegeneration. The ESCRT machinery consists of four core ESCRT complexes (-0, -I, -II, and -III) and the Vps4 ATPase complex. It has been shown that the central reaction that drives membrane scission is executed by the ESCRT-III complex and fueled by Vps4. As the only energy-consuming enzyme in the ESCRT machinery, Vps4 is an excellent target for pharmacological intervention of the ESCRT function. A number of proteins including Vta1, Vps60, Did2 and Ist1 are regulators of Vps4 activity in vivo. The long-term goal of the proposal is to delineate the molecular mechanism that underlies the regulation of the Vps4 ATPase complex so that novel strategies can be devised for preventive and therapeutic discovery. Using a combined approach of structural biology, biochemistry and cell biology, the current project will pursue three specific aims: (1) to determine the structural basis of regulation of Vps4 oligomerization by Vta1;(2) to elucidate the molecular mechanism of action by Vps60;(3) to investigate the structural basis and biological role of Ist1 and Did2 interactions. These studies will produce high-resolution structural information describing the molecular interactions involving Vps4 and its associated regulators. Combined with insights gained from the concurrent structure/function relationship study, these results will significantly advance our understanding of the molecular mechanism underlying the function and regulation of Vps4. PUBLIC HEALTH RELEVANCE: The Vps4 ATPase complex is involved in a myriad of physiological and pathological processes that have implications in a number of human diseases including cancer, viral infection and neurodegeneration. Understanding the molecular mechanism underlying the function and regulation of Vps4 will enable us to target the protein complex for preventive and therapeutic discovery, which may lead to novel treatments for these diseases that have affected millions of lives.