Resistance of the target islet tissue to autoimmune destruction contributes to genetic susceptibility in Type 1 diabetes

Type 1 diabetes occurs when self-reactive T lymphocytes destroy the insulin-producing islet β cells of the pancreas. The defects causing this disease have often been assumed to occur exclusively in the immune system. We present evidence that genetic variation at the Idd9 diabetes susceptibility locus determines the resilience of the targets of autoimmunity, the islets, to destruction. Susceptible islets exhibit hyper-responsiveness to inflammatory cytokines resulting in enhanced cell death and increased expression of the death receptor Fas. Fas upregulation in β cells is mediated by TNFR2, and colocalization of TNFR2 with the adaptor TRAF2 in NOD β cells is altered. TNFR2 lies within the candidate Idd9 interval and the diabetes-associated variant contains a mutation adjacent to the TRAF2 binding site. A component of diabetes susceptibility may therefore be determined by the target of the autoimmune response, and protective TNFR2 signaling in islets inhibit early cytokine-induced damage required for the development of destructive autoimmunity. This article was reviewed by Matthiasvon Herrath, HaraldVon Boehmer, and Ciriaco Piccirillo (nominated by Ethan Shevach)

Evolution of TNF-Induced Apoptosis Reveals 550 My of Functional Conservation

The Precambrian explosion led to the rapid appearance of most major animal phyla alive today. It has been argued that the complexity of life has steadily increased since that event. Here we challenge this hypothesis through the characterization of apoptosis in reef-building corals, representatives of some of the earliest animals. Bioinformatic analysis reveals that all of the major components of the death receptor pathway are present in coral with high-predicted structural conservation with Homo sapiens. The TNF receptor-ligand superfamilies (TNFRSF/TNFSF) are central mediators of the death receptor pathway, and the predicted proteome of Acropora digitifera contains more putative coral TNFRSF members than any organism described thus far, including humans. This high abundance of TNFRSF members, as well as the predicted structural conservation of other death receptor signaling proteins, led us to wonder what would happen if corals were exposed to a member of the human TNFSF (HuTNFα). HuTNFα was found to bind directly to coral cells, increase caspase activity, cause apoptotic blebbing and cell death, and finally induce coral bleaching. Next, immortalized human T cells (Jurkats) expressing a functional death receptor pathway (WT) and a corresponding Fas-associated death domain protein (FADD) KO cell line were exposed to a coral TNFSF member (AdTNF1) identified and purified here. AdTNF1 treatment resulted in significantly higher cell death (P \u3c 0.0001) in WT Jurkats compared with the corresponding FADD KO, demonstrating that coral AdTNF1 activates the H. sapiens death receptor pathway. Taken together, these data show remarkable conservation of the TNF-induced apoptotic response representing 550 My of functional conservation

The stromal cell–derived factor-1α/CXCR4 ligand–receptor axis is critical for progenitor survival and migration in the pancreas

The SDF-1α/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1α and CXCR4 expression in fetal pancreas. We have found that SDF-1α and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) γ–nonobese diabetic mouse. We show that SDF-1α stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1α on ductal cell migration. Importantly, blocking the SDF-1α/CXCR4 axis in IFNγ-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1α–CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration

The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas.

The SDF-1alpha/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1alpha and CXCR4 expression in fetal pancreas. We have found that SDF-1alpha and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) gamma-nonobese diabetic mouse. We show that SDF-1alpha stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1alpha on ductal cell migration. Importantly, blocking the SDF-1alpha/CXCR4 axis in IFNgamma-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1alpha-CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration

DIAPH1-MFN2 Interaction Regulates Mitochondria-SR/ER Contact and Modulates Ischemic/Hypoxic Stress

Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages. Solution structure studies affirm the interaction between the Diaphanous Inhibitory Domain and the cytosolic GTPase domain of MFN2. In male rodent and human cardiomyocytes, DIAPH1-MFN2 interaction regulates mitochondrial turnover, mitophagy, and oxidative stress. Introduction of synthetic linker construct, which shorten the mitochondria-SR/ER distance, mitigated the molecular and functional benefits of DIAPH1 silencing in ischemia. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of mitochondria-SR/ER contact networks. We propose that targeting pathways that regulate DIAPH1-MFN2 interactions may facilitate recovery from tissue ischemia

Use of retroviral peptide libraries for the identification of novel cellular targets of HIV-1 and the discovery of novel inhibitors

Great strides have been made toward understanding and fighting the Human Immunodeficiency Virus-1 in the thirty years since its discovery (HIV-1). Unfortunately, a true cure remains elusive due to the fact that all of the current pharmaceutical approaches are rendered ineffective by HIV's rapid mutation rate. Furthermore these drugs have severe side effects and, while effective at delaying the onset of Acquired Immunodeficiency Syndrome (AIDS) and extending the lifespan of the infected individuals, are very expensive and ultimately fail to stop the virus. Researchers may spend years using rational design approaches to model a drug that would block the active site of an HIV enzyme, to realize the virus develops resistance within several months of treatment. It is therefore imperative to develop novel approaches to finding direct and indirect means of blocking infection. This dissertation describes the adaptation of retroviral technology in order to discover novel peptide inhibitors of HIV-1 or HIV-1-interacting proteins. It describes the development of high-complexity libraries of random peptides, targeted to specific cellular compartments. The aim of these libraries is to block HIV-1 during various discrete steps in its lifecycle : specifically post-entry, pre-integration early events in the cytoplasm or nucleus, and late events such as processing of the viral polypeptide by its Protease enzyme. It also describes the discovery of a potential new binding partner: the COP9 Signalosome complex. The COP9 Signalosome complex, identified through a screen utilizing one of the libraries, could become a potential new target for HIV-1 inhibition. The complex as well as the interacting 14-3-3 Zeta/Delta protein, which was shown to bind only in the presence of HIV-1 proteins, was purified via a novel approach that allows for a rapid, efficient and specific isolation of large protein complexes and their binding partner

Use of retroviral peptide libraries for the identification of novel cellular targets of HIV-1 and the discovery of novel inhibitors

Great strides have been made toward understanding and fighting the Human Immunodeficiency Virus-1 in the thirty years since its discovery (HIV-1). Unfortunately, a true cure remains elusive due to the fact that all of the current pharmaceutical approaches are rendered ineffective by HIV's rapid mutation rate. Furthermore these drugs have severe side effects and, while effective at delaying the onset of Acquired Immunodeficiency Syndrome (AIDS) and extending the lifespan of the infected individuals, are very expensive and ultimately fail to stop the virus. Researchers may spend years using rational design approaches to model a drug that would block the active site of an HIV enzyme, to realize the virus develops resistance within several months of treatment. It is therefore imperative to develop novel approaches to finding direct and indirect means of blocking infection. This dissertation describes the adaptation of retroviral technology in order to discover novel peptide inhibitors of HIV-1 or HIV-1-interacting proteins. It describes the development of high-complexity libraries of random peptides, targeted to specific cellular compartments. The aim of these libraries is to block HIV-1 during various discrete steps in its lifecycle : specifically post-entry, pre-integration early events in the cytoplasm or nucleus, and late events such as processing of the viral polypeptide by its Protease enzyme. It also describes the discovery of a potential new binding partner: the COP9 Signalosome complex. The COP9 Signalosome complex, identified through a screen utilizing one of the libraries, could become a potential new target for HIV-1 inhibition. The complex as well as the interacting 14-3-3 Zeta/Delta protein, which was shown to bind only in the presence of HIV-1 proteins, was purified via a novel approach that allows for a rapid, efficient and specific isolation of large protein complexes and their binding partner

Use of retroviral peptide libraries for the identification of novel cellular targets of HIV-1 and the discovery of novel inhibitors

Includes bibliographical references.Great strides have been made toward understanding and fighting the Human Immunodeficiency Virus-1 in the thirty years since its discovery (HIV-1). Unfortunately, a true cure remains elusive due to the fact that all of the current pharmaceutical approaches are rendered ineffective by HIV's rapid mutation rate. Furthermore these drugs have severe side effects and, while effective at delaying the onset of Acquired Immunodeficiency Syndrome (AIDS) and extending the lifespan of the infected individuals, are very expensive and ultimately fail to stop the virus. Researchers may spend years using rational design approaches to model a drug that would block the active site of an HIV enzyme, to realize the virus develops resistance within several months of treatment. It is therefore imperative to develop novel approaches to finding direct and indirect means of blocking infection. This dissertation describes the adaptation of retroviral technology in order to discover novel peptide inhibitors of HIV-1 or HIV-1-interacting proteins. It describes the development of high-complexity libraries of random peptides, targeted to specific cellular compartments. The aim of these libraries is to block HIV-1 during various discrete steps in its lifecycle : specifically post-entry, pre-integration early events in the cytoplasm or nucleus, and late events such as processing of the viral polypeptide by its Protease enzyme. It also describes the discovery of a potential new binding partner: the COP9 Signalosome complex. The COP9 Signalosome complex, identified through a screen utilizing one of the libraries, could become a potential new target for HIV-1 inhibition. The complex as well as the interacting 14-3-3 Zeta/Delta protein, which was shown to bind only in the presence of HIV-1 proteins, was purified via a novel approach that allows for a rapid, efficient and specific isolation of large protein complexes and their binding partner

Purification of the COP9 Signalosome Complex and Binding Partners from Human T Cells

Abstract The COP9 Signalosome (CSN) is a highly conserved eight subunit protein complex associated with a wide range of essential biological functions in eukaryotic cells, and directly involved in processes including deneddylation, phosphorylation, and ubiquitination. Despite its significant role, very few studies have been undertaken to reveal the interactions between the CSN and its binding partners, and none in human T cells. Here we present a purification method for the CSN and binding proteins via the Streptavidin-Binding Peptide (SBP) fused to CSN Subunit 1 (CSN1). Using this method, coupled with liquid chromatography-mass spectrometry analysis, we identified all eight subunits of the CSN, as well as expected and putative novel binding partners such as a tumor suppressor under the control of Cullin4a-ligase complex; Neurofibromin 2 (Merlin). This work presents a method for fast, reliable, and specific affinity-based purification of a protein complex from a nonadherent cell line. The purification of the CSN and binding partners from T cells can elucidate the roles of CSN in a cell type where it has never been studied before. This proteomic-based approach can broaden our understanding of the functions of the CSN in contexts such as viral-host interactions or immune activation in their natural milieu