Herve Durand - Academia.edu (original) (raw)

Papers by Herve Durand

Research in Virology, 1997

We have cloned the nef, vif, vpr and vpu genes of HIV1 in the pGEX system to produce auxiliary pr... more We have cloned the nef, vif, vpr and vpu genes of HIV1 in the pGEX system to produce auxiliary proteins of HIV1 as N-terminal fusions with glutathione S-transferase (GST}. Some GST proteins are difficult to obtain under standard conditions. The synthesis and solubility varied considerably from one protein to another. We investigated the reasons for the poor production of GST-Vpr, GST-Vpu and GST-Vif. Interestingly, using this GST prokaryotic model, we demonstrated that Vpr, which is known to block the cell cycle of mammalian and yeast cells at the G2 phase, is also bacteriostatic for Escherichia coil The effect on E. coil was specific to Vpr, and was not linked to the expression of the other HIV1 proteins. This suggests that Vpr interferes with components of cell replication that are conserved from prokaryotes to eukaryotes. Thus, E. coil appears to be a convenient model system for studies on the function of Vpr.

Journal of Biological Chemistry, 1999

Activation of NF-B transcription factors requires phosphorylation and ubiquitin-proteasome-depend... more Activation of NF-B transcription factors requires phosphorylation and ubiquitin-proteasome-dependent degradation of IB proteins. We provide evidence that a human F-box protein, h-␤TrCP, a component of Skp1-Cullin-F-box protein (SCF) complexes, a new class of E3 ubiquitin ligases, is essential for inducible degradation of IB␣. ␤TrCP associates with Ser 32-Ser 36 phosphorylated, but not with unmodified IB␣ or Ser 32-Ser 36 phosphorylation-deficient mutants. Expression of a F-box-deleted ␤TrCP inhibits IB␣ degradation, promotes accumulation of phosphorylated Ser 32-Ser 36 IB␣, and prevents NF-B-dependent transcription. Our findings indicate that ␤TrCP is the adaptor protein required for IB␣ recognition by the SCF ␤TrCP E3 complex that ubiquitinates IB␣ and makes it a substrate for the proteasome.

European Journal of Biochemistry, 1994

The activity of the Src family protein-tyrosine kinase ~5 6 "~ is regulated by phosphorylation an... more The activity of the Src family protein-tyrosine kinase ~5 6 "~ is regulated by phosphorylation and dephosphorylation of two critical tyrosine residues Tyr394 and Tyr505. Tyr394 is autophosphorylated after p56Ick activation, whereas phosphorylation of Tyr505 is believed to be due to p50Ak which negatively modulates ~5 6 "~ activity. To determine whether Tyr505 could be autophosphorylated, we used the prokaryotic glutathione S-transferase expression system to express wild-type Lck, the mutants [Y394F]Lck and [Y505F]Lck, a kinase-deficient ~5 6 "~ with a mutation of the ATP-binding site [K273E]Lck and a double mutant [Y394F, Y505FILck. We studied the kinase activities and the patterns of autophosphorylation for tyrosine residues in these mutants and wild-type Lck both in vivo and in vitro. Wild-type Lck, [Y505F]Lck and [Y394F]Lck were phosphorylated on tyrosine. Both the kinasedeficient mutant[K273E]Lck and the double mutant [Y394F, Y505FILck did not react with monoclonal anti-phosphotyrosine antibody [anti-Y(P) mAb], thus providing evidence that (a) the bacterial strains used lacked intrinsic protein-tyrosine kinase activities, and therefore tyrosine phosphorylations of wild-type Lck, [Y505F]Lck and [Y394F]Lck are due to autophosphorylation occurring in vivo in bacteria, and (b) that p56"* can only be autophosphorylated on two tyrosine residues, namely Tyr394 and Tyr505. Phosphopeptide mapping analysis confirmed that ~5 6 ' "~ can undergo autophosphorylation on these two tyrosine residues. We propose that autophosphorylation at Tyr505 of ~5 6 "~ may represent an accessory mechanism for the down-regulation of the tyrosine kinase activity of ~56''~. Ten distinct members of the src gene family have been cloned and the encoded protein-tyrosine kinases have been characterized (Mustelin, 1994). These include Src, Yes, Fgr, Fyn, Lyn, Lck, Blk, Hck, Yrk and Rak. The protein-tyrosine kinase ~56"' has been found to be exclusively expressed in cells of the lymphoid lineage, predominantly in T lymphocytes (

Journal of Cancer Research and Clinical Oncology, 1991

Addition of purified plasmin or plasminogen (0.1 gM) to serum-free culture media elevated cellula... more Addition of purified plasmin or plasminogen (0.1 gM) to serum-free culture media elevated cellular D-myo-inositol 1,4,5-trisphosphate (InsP3) levels in human colorectal carcinoma cells within 1 h to double those of control cells. This was accompanied by decreases in cellular phosphatidylinositol bisphosphate by 40% in cells exposed to fibrinolytic ligands for up to 1 h. The effect was not due to opening of Ca 2 + channels of the type blocked by 5 gM nifedipine, and 100 gM EGTA, a Ca 2+ chelator, did not suppress plasmin's ability to elevate InsP 3. Binding assays at 4 ~ C with 125I-labelled plasmin indicated maximum binding within 1 h suggesting that the effects ofplasmin may be associated with its cell-binding function. These cells could convert exogenous plasminogen to plasmin with endogenous activation and this was accompanied by a decrease in radioactive phosphatidylinositol well below control levels (13% of control). Our results contribute to evidence for the association of plasmin-binding sites with a signalling system. A cell signalling system indirectly or directly associated with plasmin binding, would permit carcinoma cells to coordinate extracellular fibrinolysis with cell migration and motility through second messengers.

Journal of Biological Chemistry

Nef is a 27-kDa myristylated protein conserved in most human immunodeficiency virus (HW-1, HIV-2,... more Nef is a 27-kDa myristylated protein conserved in most human immunodeficiency virus (HW-1, HIV-2, and simian immunodeficiency virus isolates. Simian immunodeficiency virus Nef is required in macaques for both high viral load and full pathological effects. Nef downregulates the cell surface expression of CD4 by a posttranslational mechanism that is not yet fully elucidated. W e have used the yeast two-hybrid system to identify cellular proteins that interact with Nef.

Atherosclerosis, 2015

Objective: We have previously reported that SASH1 expression is increased in circulating human mo... more Objective: We have previously reported that SASH1 expression is increased in circulating human monocytes from smokers and was positively correlated with the number of carotid atherosclerotic plaques. The aim of this study was to further validate the link between smoking, SASH1 and atherosclerosis within the vascular wall and to assess the impact of SASH1 expression on endothelial cell functions. Method: Human carotids with atherosclerotic plaques were obtained from 58 patients (45 of them with known smoking status: smoker, non-smoker, ex-smokers), and were processed for gene expression analyses and immunostaining. To investigate its function, SASH1 was silenced in human aortic endothelial cells (HAECs) using two different siRNA and subcellular localization of SASH1 was determined by immunostaining and subcellular fractionation. Subsequently the transcriptomic analyses and functional experiments (wound healing, WST-1 proliferation or Matrigel assays) were performed to characterize SASH1 function. Results: SASH1 was expressed in human vascular cells (HAECs, smooth muscle cells) and in monocytes/ macrophages. Its tissue expression was significantly higher in the atherosclerotic carotids of smokers compared to non-smokers (p < 0.01). In HAECs, SASH1 was expressed mostly in the cytoplasm and SASH1 knockdown resulted in an increased cell migration, proliferation and angiogenesis. Transcriptomic and pathway analyses showed that SASH1 silencing results in a decreased CYP1A1 expression possibly through the inhibition of TP53 activity. Conclusion: We showed that SASH1 expression is increased in atherosclerotic carotids in smokers and its silencing affects endothelial angiogenic functions; therefore we provide a potential link between smoking and atherosclerosis through SASH1 expression.

PLOS ONE, 2015

Bariatric surgery is associated to improvements in obesity-associated comorbidities thought to be... more Bariatric surgery is associated to improvements in obesity-associated comorbidities thought to be mediated by a decrease of adipose inflammation. However, the molecular mechanisms behind these beneficial effects are poorly understood. We analyzed RNA-seq expression profiles in adipose tissue from 22 obese women before and 3 months after surgery. Of 15,972 detected genes, 1214 were differentially expressed after surgery at a 5% false discovery rate. Upregulated genes were mostly involved in the basal cellular machinery. Downregulated genes were enriched in metabolic functions of adipose tissue. At baseline, 26 modules of coexpressed genes were identified. The four most stable modules reflected the innate and adaptive immune responses of adipose tissue. A first module reflecting a non-specific signature of innate immune cells, mainly macrophages, was highly conserved after surgery with the exception of DUSP2 and CD300C. A second module reflected the adaptive immune response elicited by T lymphocytes; after surgery, a disconnection was observed between genes involved in T-cell signaling and mediators of the signal transduction such as CXCR1, CXCR2, GPR97, CCR7 and IL7R. A third module reflected neutrophil-mediated inflammation; after surgery, several genes were dissociated from the module, including S100A8, S100A12, CD300E, VNN2, TUBB1 and FAM65B. We also identified a dense network of 19 genes involved in the interferon-signaling pathway which was strongly preserved after surgery, with the exception of DDX60, an antiviral factor involved in RIG-I-mediated interferon signaling. A similar loss of connection was observed in lean mice compared to their obese counterparts. These results suggest that improvements of the inflammatory state following surgery might be explained by a disruption of immuno-inflammatory cascades involving a few crucial molecules which could serve as potential therapeutic targets.

Molecular cell, 1998

HIV-1 Vpu interacts with CD4 in the endoplasmic reticulum and triggers CD4 degradation, presumabl... more HIV-1 Vpu interacts with CD4 in the endoplasmic reticulum and triggers CD4 degradation, presumably by proteasomes. Human beta TrCP identified by interaction with Vpu connects CD4 to this proteolytic machinery, and CD4-Vpu-beta TrCP ternary complexes have been detected by coimmunoprecipitation. beta TrCP binding to Vpu and its recruitment to membranes require two phosphoserine residues in Vpu essential for CD4 degradation. In beta TrCP, WD repeats at the C terminus mediate binding to Vpu, and an F box near the N terminus is involved in interaction with Skp1p, a targeting factor for ubiquitin-mediated proteolysis. An F-box deletion mutant of beta TrCP had a dominant-negative effect on Vpu-mediated CD4 degradation. These data suggest that beta TrCP and Skp1p represent components of a novel ER-associated protein degradation pathway that mediates CD4 proteolysis.

Research in virology

The humoral immune response of 34 macaques experimentally infected with SIVmac-251 was studied us... more The humoral immune response of 34 macaques experimentally infected with SIVmac-251 was studied using a combination of an epitope library and synthetic peptides. The course of the immune response was checked for up to 9 months postinfection with a panel of clones expressing SIV fragments. A systematic study was performed with synthetic peptides covering the whole transmembrane (TM) and external (SU) envelope proteins. Seven major immunodominant epitopes were characterized. Four are localized in the SU protein: one in the V1 region (111-130), one in the Cys loop of the V3 region (311-330) and two in the C-terminal end (501-520 and 511-530). Three are localized in the TM protein: one in the extracellular domain (601-619), one in the anchor domain (731-750) and one in the intracytoplasmic domain (861-881). Among these epitopes, only one, 601-619, was found to be reactive with all sera and can be defined as the principal immunodominant epitope.

Virology, 1996

The Vpu and CD4 cytoplasmic domains were found, by using a two-hybrid assay in yeast, to interact... more The Vpu and CD4 cytoplasmic domains were found, by using a two-hybrid assay in yeast, to interact in the absence of their membrane anchor domains. Studies on several deletion and point mutants revealed that the overall structure of the Vpu cytoplasmic domain is required for this interaction. The Vpu amino acid residues involved in the interaction with CD4 were identified. Deletion of the C-terminal residues of Vpu, required for CD4 degradation, as well as the double mutation on the casein kinase II phosphorylation sites S52N -S56N, also involved in CD4 degradation, resulted in the loss of interaction with CD4 and in the inability to induce CD4 degradation. These results suggest that the ability of Vpu to mediate the degradation of CD4 is linked to its capacity to physically interact with CD4. However, additional mutagenesis on the S52 site revealed that the interaction between the cytoplasmic domains of Vpu and CD4 is not sufficient for in vitro Vpu-mediated CD4 degradation. ᭧

The FASEB Journal, 2006

Increasing evidence suggests that secreted phospholipases A2 (sPLA2s) play an important role in t... more Increasing evidence suggests that secreted phospholipases A2 (sPLA2s) play an important role in the pathophysiology of atherosclerosis. Among sPLA2s, the human group X (hGX) enzyme has the highest catalytic activity toward phosphatidylcholine, one of the major phospholipid species of cell membranes and low-density lipoprotein (LDL). Our study examined the presence of hGX sPLA2 in human atherosclerotic lesions and investigated the ability of hGX modified LDL to alter human endothelial cell (HUVEC) function. Our results show that hGX sPLA2 is present in human atherosclerotic lesions and that the hydrolysis of LDL by hGX sPLA2 results in a modified particle that induces lipid accumulation in human monocyte-derived macrophages. Acting on endothelial cells, hGX-modified LDL activates the MAP kinase pathway, which leads to increased arachidonic acid release, increased expression of adhesion molecules on the surface of HUVEC, and increased adhesion of monocytes to HUVEC monolayers. Together, our data suggest that LDL modified by hGX, rather than hGX itself may have strong proinflammatory and proatherogenic properties, which could play an important role in the propagation of atherosclerosis.-Karabina, S. A., Brocheriou, I., Le Naour, G., Agrapart, M., Durand, H., Gelb, M., Lambeau, G., Ninio, E. Atherogenic properties of LDL particles modified by human group X secreted phospholipase A2 on human endothelial cell function. FASEB J. 20, E1890 -E1900 (2006)

Molecular Cell, 1998

Molecular and Cellular Biology, 2001

The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we s... more The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein ␤TrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IB␣ and ␤-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation and on ATF4 serine residue 219 present in the context of DSGXXXS, which is similar but not identical to the motif found in other substrates of ␤TrCP. ATF4 ubiquitination in HeLa cells is enhanced in the presence of ␤TrCP. The F-box-deleted ␤TrCP protein behaves as a negative transdominant mutant that inhibits ATF4 ubiquitination and degradation and, subsequently, enhances its activity in cyclic AMP-mediated transcription. ATF4 represents a novel substrate for the SCF ␤TrCP complex, which is the first mammalian E3 ubiquitin ligase identified so far for the control of the degradation of a bZIP transcription factor.

Journal of Receptors and Signal Transduction, 1990

The pre-incubation of human colorectal carcinoma cells SW 1116 with 25 to 100 uM purified ganglio... more The pre-incubation of human colorectal carcinoma cells SW 1116 with 25 to 100 uM purified gangliosides resulted in 35-60% inhibition of specific (125I)-plasmin binding to the cell surface. After 5 to 6 days in culture, tumor cells were pre-incubated at 4 degrees for 1 to 4 h followed by post-incubation with (125I)-plasmin by techniques previously described. At 25 uM the capacity for inhibition of plasmin binding was GT1b greater than GQ1b greater than or equal to GD1a greater than GM1 less than or equal to GgOse 4Cer. Thus a terminal sialyl moiety appears to be necessary (p less than 0.05) although exogenous N-acetyl neuraminic acid was ineffective (p greater than 0.05), indicating a role for the lipid portion of the ganglioside. Other (glyco)lipids such as sphingosine, fucolipid H-1 and sulfatide were without significant effect. The inhibition could not be reversed by the presence of 10 mM Ca+2, EDTA, pre-treatment of the cell with carboxypeptidase or pretreatment of plasmin with neuraminidases. The inhibition was however reversed by post-incubation in control medium without exogenous ganglioside. Cell counts determined prior to, and after ganglioside incubation showed that the effect was not due to cell death or detachment from the culture surface. The dissociation constant for (125I)-plasmin binding was 5.6 x 10(-8) M (700,000 sites/cell), but in the presence of trisialoganglioside (GT1b), Scatchard plots suggested diversification of binding sites with 280,000 sites/cell at Kd 2.6 x 10(-8) M and 820,000 sites/cell at Kd 2.1 x 10(-7) M. Another interpretation of the Scatchard plot in the presence of ganglioside was that the glycolipid imposed negative cooperativity on plasmin binding to the cell surface. These results suggest that certain gangliosides can affect tumor cell invasiveness by altering protease binding to the cell surface.