scholarly journals Signaling-dependent immobilization of acylated proteins in the inner monolayer of the plasma membrane

2006 ◽  
Vol 174 (2) ◽  
pp. 255-265 ◽  
Author(s):  
Elaine F. Corbett-Nelson ◽  
David Mason ◽  
John G. Marshall ◽  
Yves Collette ◽  
Sergio Grinstein
Keyword(s):  
Tyrosine Kinases ◽  
Fluorescent Proteins ◽  
Critical Role ◽  
Lipid Binding ◽  
Binding Domains ◽  
Associated Proteins ◽  
Target Particle ◽  
Restricted Mobility ◽  
Acylated Proteins ◽  
Reduced Mobility

Phospholipids play a critical role in the recruitment and activation of several adaptors and effectors during phagocytosis. Changes in lipid metabolism during phagocytosis are restricted to the phagocytic cup, the area of the plasmalemma lining the target particle. It is unclear how specific lipids and lipid-associated molecules are prevented from diffusing away from the cup during the course of phagocytosis, a process that often requires several minutes. We studied the mobility of lipid-associated proteins at the phagocytic cup by measuring fluorescence recovery after photobleaching. Lipid-anchored (diacylated) fluorescent proteins were freely mobile in the unstimulated membrane, but their mobility was severely restricted at sites of phagocytosis. Only probes anchored to the inner monolayer displayed reduced mobility, whereas those attached to the outer monolayer were unaffected. The immobilization persisted after depletion of plasmalemmal cholesterol, ruling out a role of conventional “rafts.” Corralling of the probes by the actin cytoskeleton was similarly discounted. Instead, the change in mobility required activation of tyrosine kinases. We suggest that signaling-dependent recruitment of adaptors and effectors with lipid binding domains generates an annulus of lipids with restricted mobility.

scholarly journals The Annexin A2/S100A10 System in Health and Disease: Emerging Paradigms

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Nadia Hedhli ◽  
Domenick J. Falcone ◽  
Bihui Huang ◽  
Gabriela Cesarman-Maus ◽  
Rosemary Kraemer ◽  
...  
Keyword(s):  
Serotonin Receptor ◽  
Human Cancer ◽  
High Titer ◽  
Critical Role ◽  
Annexin A2 ◽  
Avascular Osteonecrosis ◽  
Kinase Substrate ◽  
New Findings ◽  
Human Disorders ◽  
Associated Proteins

Since its discovery as a src kinase substrate more than three decades ago, appreciation for the physiologic functions of annexin A2 and its associated proteins has increased dramatically. With its binding partner S100A10 (p11), A2 forms a cell surface complex that regulates generation of the primary fibrinolytic protease, plasmin, and is dynamically regulated in settings of hemostasis and thrombosis. In addition, the complex is transcriptionally upregulated in hypoxia and promotes pathologic neoangiogenesis in the tissues such as the retina. Dysregulation of both A2 and p11 has been reported in examples of rodent and human cancer. Intracellularly, A2 plays a critical role in endosomal repair in postarthroplastic osteolysis, and intracellular p11 regulates serotonin receptor activity in psychiatric mood disorders. In human studies, the A2 system contributes to the coagulopathy of acute promyelocytic leukemia, and is a target of high-titer autoantibodies in patients with antiphospholipid syndrome, cerebral thrombosis, and possibly preeclampsia. Polymorphisms in the humanANXA2gene have been associated with stroke and avascular osteonecrosis of bone, two severe complications of sickle cell disease. Together, these new findings suggest that manipulation of the annexin A2/S100A10 system may offer promising new avenues for treatment of a spectrum of human disorders.

scholarly journals HSD17B13: A Potential Therapeutic Target for NAFLD

2022 ◽  
Vol 8 ◽  
Author(s):  
Hai-bo Zhang ◽  
Wen Su ◽  
Hu Xu ◽  
Xiao-yan Zhang ◽  
You-fei Guan
Keyword(s):  
Liver Disease ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Critical Role ◽  
Chronic Liver ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Nonalcoholic Fatty Liver ◽  
Promising Target ◽  
Associated Proteins

Nonalcoholic fatty liver disease (NAFLD), especially in its inflammatory form (steatohepatitis, NASH), is closely related to the pathogenesis of chronic liver disease. Despite substantial advances in the management of NAFLD/NASH in recent years, there are currently no efficacious therapies for its treatment. The biogenesis and expansion of lipid droplets (LDs) are critical pathophysiological processes in the development of NAFLD/NASH. In the past decade, increasing evidence has demonstrated that lipid droplet-associated proteins may represent potential therapeutic targets for the treatment of NAFLD/NASH given the critical role they play in regulating the biogenesis and metabolism of lipid droplets. Recently, HSD17B13, a newly identified liver-enriched, hepatocyte-specific, lipid droplet-associated protein, has been reported to be strongly associated with the development and progression of NAFLD/NASH in both mice and humans. Notably, human genetic studies have repeatedly reported a robust association of HSD17B13 single nucleotide polymorphisms (SNPs) with the occurrence and severity of NAFLD/NASH and other chronic liver diseases (CLDs). Here we briefly overview the discovery, tissue distribution, and subcellular localization of HSD17B13 and highlight its important role in promoting the pathogenesis of NAFLD/NASH in both experimental animal models and patients. We also discuss the potential of HSD17B13 as a promising target for the development of novel therapeutic agents for NAFLD/NASH.

scholarly journals Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs

2018 ◽  
Author(s):  
David M. Kern ◽  
SeCheol Oh ◽  
Richard K. Hite ◽  
Stephen G. Brohawn
Keyword(s):  
Lipid Bilayers ◽  
Critical Role ◽  
Anion Channel ◽  
Lipid Binding ◽  
Channel Structure ◽  
Anion Channels ◽  
Cryo Electron Microscopy ◽  
Lipid Nanodiscs ◽  
Insight Into

AbstractHypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here we present single-particle cryo-electron microscopy structures of LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

scholarly journals UBA1 and UBA2, Two Proteins That Interact with UBP1, a Multifunctional Effector of Pre-mRNA Maturation in Plants

2002 ◽  
Vol 22 (12) ◽  
pp. 4346-4357 ◽  
Author(s):  
Mark H. L. Lambermon ◽  
Yu Fu ◽  
Dominika A. Wieczorek Kirk ◽  
Marcel Dupasquier ◽  
Witold Filipowicz ◽  
...  
Keyword(s):  
Steady State ◽  
Rna Binding ◽  
Rna Recognition Motif ◽  
Dependent Manner ◽  
Binding Domains ◽  
Mrna Maturation ◽  
Associated Proteins ◽  
Steady State Levels ◽  
Transient Overexpression

ABSTRACT Nicotiana plumbaginifolia UBP1 is an hnRNP-like protein associated with the poly(A)+ RNA in the cell nucleus. Consistent with a role in pre-mRNA processing, overexpression of UBP1 in N. plumabaginifolia protoplasts enhances the splicing of suboptimal introns and increases the steady-state levels of reporter mRNAs, even intronless ones. The latter effect of UBP1 is promoter specific and appears to be due to UBP1 binding to the 3′ untranslated region (3′-UTR) and protecting the mRNA from exonucleolytic degradation (M. H. L. Lambermon, G. G. Simpson, D. A. Kirk, M. Hemmings-Mieszczak, U. Klahre, and W. Filipowicz, EMBO J. 19:1638-1649, 2000). To gain more insight into UBP1 function in pre-mRNA maturation, we characterized proteins interacting with N. plumbaginifolia UBP1 and one of its Arabidopsis thaliana counterparts, AtUBP1b, by using yeast two-hybrid screens and in vitro pull-down assays. Two proteins, UBP1-associated proteins 1a and 2a (UBA1a and UBA2a, respectively), were identified in A. thaliana. They are members of two novel families of plant-specific proteins containing RNA recognition motif-type RNA-binding domains. UBA1a and UBA2a are nuclear proteins, and their recombinant forms bind RNA with a specificity for oligouridylates in vitro. As with UBP1, transient overexpression of UBA1a in protoplasts increases the steady-state levels of reporter mRNAs in a promoter-dependent manner. Similarly, overexpression of UBA2a increases the levels of reporter mRNAs, but this effect is promoter independent. Unlike UBP1, neither UBA1a nor UBA2a stimulates pre-mRNA splicing. These and other data suggest that UBP1, UBA1a, and UBA2a may act as components of a complex recognizing U-rich sequences in plant 3′-UTRs and contributing to the stabilization of mRNAs in the nucleus.

scholarly journals PAH- and PCB-induced Alterations of Protein Tyrosine Kinase and Cytokine Gene Transcription in Harbor Seal (Phoca Vitulina) PBMC

2005 ◽  
Vol 12 (2) ◽  
pp. 91-97 ◽  
Author(s):  
Jennifer C. C. Neale ◽  
Thomas P. Kenny ◽  
Ronald S. Tjeerdema ◽  
M. Eric Gershwin
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Critical Role ◽  
Harbor Seal ◽  
Lymphocyte Function ◽  
Altered Expression ◽  
Protein Tyrosine

Mechanisms underlyingin vitroimmunomodulatory effects of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were investigated in harbor seal peripheral leukocytes, via real-time PCR. We examined the relative genetic expression of the protein tyrosine kinases (PTKs)FynandItk, which play a critical role in T cell activation, and IL-2, a cytokine of central importance in initiating adaptive immune responses. IL-1, the macrophage-derived pro-inflammatory cytokine of innate immunity, was also included as a measure of macrophage function. Harbor seal PBMC were exposed to the prototypic immunotoxic PAH benzo[a]pyrene (BaP), 3,3',4,4',5,5'-hexachlorobiphenyl (CB-169), a model immunotoxic PCB, or DMSO (vehicle control). Exposure of Con A-stimulated harbor seal PBMC to both BaP and CB-169 produced significantly altered expression in all four targets relative to vehicle controls. The PTKsFynandItkwere both up-regulated following exposure to BaP and CB-169. In contrast, transcripts for IL-2 and IL-1 were decreased relative to controls by both treatments. Our findings are consistent with those of previous researchers working with human and rodent systems and support a hypothesis of contaminant-altered lymphocyte function mediated (at least in part) by disruption of T cell receptor (TCR) signaling and cytokine production.

scholarly journals Binding Dynamics of Hepatitis C Virus' NS5A Amphipathic Peptide to Cell and Model Membranes

2007 ◽  
Vol 81 (12) ◽  
pp. 6682-6689 ◽  
Author(s):  
Nam-Joon Cho ◽  
Kwang Ho Cheong ◽  
ChoongHo Lee ◽  
Curtis W. Frank ◽  
Jeffrey S. Glenn
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Critical Role ◽  
Membrane Binding ◽  
Lipid Binding ◽  
Model Membranes ◽  
Cellular Membranes ◽  
Kinetic Measurements ◽  
Amphipathic Peptide ◽  
Target Membrane

ABSTRACT Membrane association of the hepatitis C virus NS5A protein is required for viral replication. This association is dependent on an N-terminal amphipathic helix (AH) within NS5A and is restricted to a subset of host cell intracellular membranes. The mechanism underlying this specificity is not known, but it may suggest a novel strategy for developing specific antiviral therapy. Here we have probed the mechanistic details of NS5A AH-mediated binding to both cell-derived and model membranes by use of biochemical membrane flotation and quartz crystal microbalance (QCM) with dissipation. With both assays, we observed AH-mediated binding to model lipid bilayers. When cell-derived membranes were coated on the quartz nanosensor, however, significantly more binding was detected, and the QCM-derived kinetic measurements suggested the existence of an interacting receptor in the target membranes. Biochemical flotation assays performed with trypsin-treated cell-derived membranes exhibited reduced AH-mediated membrane binding, while membrane binding of control cytochrome b5 remained unaffected. Similarly, trypsin treatment of the nanosensor coated with cellular membranes abolished AH peptide binding to the cellular membranes but did not affect the binding of a control lipid-binding peptide. These results therefore suggest that a protein plays a critical role in mediating and stabilizing the binding of NS5A's AH to its target membrane. These results also demonstrate the successful development of a new nanosensor technology ideal both for studying the interaction between a protein and its target membrane and for developing inhibitors of that interaction.

Cell Delivery and Recirculation

2004 ◽  
Author(s):  
W. Mark Saltzman
Keyword(s):  
Tissue Engineering ◽  
Cell Adhesion ◽  
Cell Fate ◽  
Critical Role ◽  
Cell Movement ◽  
The Body ◽  
Associated Proteins ◽  
Adult Organism ◽  
Adenine Deaminase

Perhaps the simplest realization of tissue engineering involves the direct administration of a suspension of engineered cells—cells that have been isolated, characterized, manipulated, and amplified outside of the body. One can imagine engineering diverse and useful properties into the injected cells: functional enzymes, secretion of drugs, resistance to immune recognition, and growth control. We are most familiar with methods for manipulating the cell internal chemistry by introduction or removal of genes; for example, the first gene therapy experiments involved cells that were engineered to produce a deficient enzyme, adenine deaminase (see Chapter 2). But genes also encode systems that enable cell movement, cell mechanics, and cell adhesion. Conceivably, these systems can be modified to direct the interactions of an administered cell with its new host. For example, cell adhesion signals could be introduced to provide tissue targeting, cytoskeleton-associated proteins could be added to alter viscosity and deformability (in order to prolong circulation time), and motor proteins could be added to facilitate cell migration. Ideally, cell fate would also be engineered, so that the cell would move to the appropriate location in the body, no matter how it was administered; for example, transfused liver cells would circulate in the blood and, eventually, crawl into the liver parenchyma. Cells find their place in developing organisms by a variety of chemotactic and adhesive signals, but can these same signaling mechanisms be engaged to target cells administered to an adult organism? We have already considered the critical role of cell movement in development in Chapter 3. In this chapter, the utility of cell trafficking in tissue engineering is approached by first considering the normal role of cell recirculation and trafficking within the adult organism. Most cells can be easily introduced into the body by intravenous injection or infusion. This procedure is particularly appropriate for cells that function within the circulation; for example, red blood cells (RBCs) and lymphocytes. The first blood transfusions into humans were performed by Jean-Baptiste Denis, a French physician, in 1667. This early appearance of transfusion is startling, since the circulatory system was described by William Harvey only a few decades earlier, in 1628.

scholarly journals Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities

2016 ◽  
Vol 213 (5) ◽  
pp. 513-524 ◽  
Author(s):  
Holly A. Ping ◽  
Lauren M. Kraft ◽  
WeiTing Chen ◽  
Amy E. Nilles ◽  
Laura L. Lackner
Keyword(s):  
Plasma Membrane ◽  
Specific Binding ◽  
Coiled Coil ◽  
Lipid Binding ◽  
General Mechanism ◽  
Cell Cortex ◽  
Pleckstrin Homology Domain ◽  
Binding Domains ◽  
Coiled Coil Domain ◽  
Mitochondrial Distribution

The mitochondria–ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering.

scholarly journals Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function

Blood ◽  
2009 ◽  
Vol 113 (2) ◽  
pp. 338-346 ◽  
Author(s):  
Ramon Urrea Moreno ◽  
Juana Gil ◽  
Carmen Rodriguez-Sainz ◽  
Elena Cela ◽  
Victor LaFay ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Critical Role ◽  
Lipid Binding ◽  
Inflammatory Disorder ◽  
C2 Domain ◽  
Biophysical Modeling ◽  
Binding Assays ◽  
Calcium Dependent ◽  
Pathogen Elimination ◽  
Cytotoxic Function

Abstract Perforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function.

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