Extracellular Nef Protein Targets CD4+ T Cells for Apoptosis by Interacting with CXCR4 Surface Receptors (original) (raw)
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Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction
Journal of Virology, 2004
The HIV-1 Nef protein was analyzed for apoptotic structural motifs that interact with the CXCR4 receptor and induce apoptosis in CD4+ lymphocytes. Two apoptotic motifs were identified. One centered on Nef amino acids (aa) 50 to 60, with the overlapping 20-mer peptides retaining about 82% of the activity of the full Nef protein. The second centered on aa 170 to 180, with the overlapping 20-mer peptides retaining about 30% of the activity of the full protein. Significant apoptotic abilities were observed for 11-mer motif peptides spanning aa 50 to 60 and aa 170 to 180, with a scrambled version of the 11-mer motif peptide corresponding to aa 50 to 60 showing no apoptotic ability. Hallmarks of apoptosis, such as the formation of DNA ladders and caspase activation, that were observed with the full-length protein were equally evident upon exposure of cells to these motif peptides. A CXCR4 antibody and the endogenous ligand SDF-1α were effective in blocking Nef peptide-induced apoptosis as...
Cell, 1999
highly conserved. ‡ Department of Physiology CD4 is a type I integral membrane glycoprotein ex-Faculty of Medicine pressed on the surface of thymocytes, T helper lympho-§ Department of Biochemistry cytes, and cells of the monocyte/macrophage lineage Faculty of Science (Maddon et al., 1986). Required for both the maturation University of Geneva and activation of T helper lymphocytes, CD4 stabilizes CH-1211 Geneva 4 the interaction between the T cell receptor (TCR) on the Switzerland surface of lymphocytes and the class II major histocompatibility complex (MHC II) on antigen-presenting cells. Furthermore, it recruits the p56 lck protein tyrosine kinase, Summary bound to its cytoplasmic domain, to the vicinity of the TCR. As such, CD4 functions as a coactivator, because The Nef protein of primate lentiviruses downregulates Lck is a major proximal effector of the T cell activation the cell surface expression of CD4 through a two-step cascade (reviewed in Weiss and Littman, 1994). process. First, Nef connects the cytoplasmic tail of CD4 also serves as the primary receptor for both HIV CD4 with adaptor protein complexes (AP), thereby inand SIV and is dramatically downregulated following ducing the formation of CD4-specific clathrin-coated infection. One important role of this downmodulation pits that rapidly endocytose the viral receptor. Second, is to permit the subsequent release of fully infectious Nef targets internalized CD4 molecules for degradavirions, as excessive levels of CD4 on the surface of tion. Here we show that Nef accomplishes this second virus-producing cells result in blocking the virion incortask by acting as a connector between CD4 and the  poration and function of the viral envelope (Lama et al., subunit of COPI coatomers in endosomes. A sequence submitted). Two HIV-1 proteins, Nef and Vpu, counteract encompassing a critical acidic dipeptide, located nearby this inhibition by downmodulating the cell surface exbut distinct from the AP-binding determinant of HIV-1 pression of CD4. Of the two, Nef exerts the earliest and Nef, is responsible for -COP recruitment and for routmost robust influence (Chen et al., 1996; Lama et al., ing to lysosomes. A novel class of endosomal sorting submitted). motif, based on acidic residues, is thus revealed, and The study of Nef-induced CD4 downregulation has -COP is identified as its downstream partner. provided exciting insights into the mechanisms by which cells can modulate the expression of a surface molecule.
Apoptosis Enhancement by the HIV-1 Nef Protein
The Journal of Immunology, 2001
The HIV-1 nef gene, essential for AIDS pathogenesis, encodes a 27-kDa protein (Nef) whose biochemical and biological functions are unclear. It has been suggested that Nef expression contributes to the T cell depletion observed during the disease by promoting their apoptosis. We report that in CD4(+) human lymphoblastoid cell lines transfected with the nef cDNA obtained from three different HIV-1 strains, expression of the Nef protein enhances and accelerates the response to four unrelated apoptotic agents (staurosporine, anisomycin, camptothecin, and etoposide) but not to an anti-Fas agonist Ab. Nef reduces the expression of the anti-apoptotic proteins Bcl-2 and Bcl-X(L) and induces a striking enhancement of apoptotic hallmarks, including mitochondrial depolarization, exposure of phosphatidylserine on the cell surface, activation of caspase-3, and cleavage of the caspase target poly(ADP-ribose) polymerase. Interestingly, the peptide Z-Val-Ala-DL-Asp-fluoromethylketone (a broad-spectrum caspase inhibitor) reduces, but does not abolish, phosphatidylserine exposure, suggesting that Nef also activates a caspase-independent apoptotic pathway. Surprisingly, Nef expression increases DNA degradation but without causing oligonucleosomal fragmentation. An increased apoptotic response and down-modulation of Bcl-2/Bcl-X(L) following Nef expression are observed also in NIH-3T3 fibroblasts. These data show that Nef enhances programmed cell death in different cell types by affecting multiple critical components of the apoptotic machinery independently from the Fas pathway.
Proceedings of the National Academy of Sciences, 1996
Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death. Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA. Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif. Apoptosis mediated by interleukin 13 converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB. As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAFI and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.
ARF1 Regulates Nef-Induced CD4 Degradation
Current Biology, 2004
and Molecular [13] revealed the importance of residues located in a C-terminal flexible loop of HIV-1 Nef in CD4 late-endoso-Medicine 3 Department of Cell Physiology and Metabolism mal migration, they also indicated that the Nef-COP interaction strongly depends on a then-unidentified co-4 Frontiers in Genetics Research Program University of Geneva factor found in a COP-depleted fraction of the cytosol [13]. Because endosomal COP functions depend on 1211 Geneva Switzerland ARF1 in vitro [21], we reasoned that ARF1 might be the necessary cofactor. The results presented here indicate that this is the case. Summary Results Background: The HIV Nef protein downregulates CD4 through sequential connection with clathrin-coated pits Nef Late-Endosomal Targeting and Interactions and the COP1 coatomer, resulting in accelerated endowith ARF1 cytosis and lysosomal targeting. We previously reported that a Nef molecule mutated in Results: Here we report that the small GTPase ARF1 two distal acidic residues (Nef EE 155 QQ) inefficiently tarcontrols the Nef-induced, COP-mediated late-endosogeted CD4 to lysosomes [13]. To extend this observamal targeting of CD4. We find that Nef binds ARF1 dition, we expressed wild-type Nef, Nef EE 155 QQ , or the nonrectly and can recruit the GTPase onto endosomal memmyristoylated Nef G 2 A mutant with a Myc-tagged form of branes. Furthermore, a complex comprising Nef, ARF1, ARF1 in BHK cells, prepared early-and late-endosomal and COP can be immunoprecipitated from cells exfractions on a flotation gradient [22], and analyzed the pressing the viral protein. Residues in a C-terminal loop resulting material by Western blotting (Figure 1). Wildof the viral protein facilitate both these interactions and type Nef and Nef EE 155 QQ were both found in early-endothe targeting of Nef and CD4 to acidic late endosomes, somal fractions (lanes 3 and 7). However, only the wildwhereas other residues primarily involved in mediating type protein was detected in late-endosomal fractions CD4 endocytosis are dispensable for this process. Fi-(lanes 2 and 6). As expected from its failure to bind nally, a dominant-negative ARF1 mutant blocks the mimembranes, Nef G 2 A did not copurify with endosomes gration of the Nef-CD4 complex to lysosomes. (lanes 10 and 11). ARF1 can interact with several target Conclusions: Our results support a model in which membranes, including endosomes, and-not surpris-ARF1 is the immediate downstream partner of Nef for ingly-was found associated with early-and late-endo-CD4 lysosomal targeting. somal fractions under all conditions. However, ARF1 appeared somewhat more enriched in late endosomes
Journal of Biological …, 2001
Tumor necrosis factor (TNF)-␣-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-␣-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-␣ induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-␣ signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-␣-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-␣, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death. TNF-␣ 1 is a cytokine produced by the immune system in response to virus infection (1). Signaling by TNF-␣ through the TNF R1 induces a variety of cellular responses including the recruitment and activation of caspase-8 in the receptor complex, which initiates a pathway to implement cell death by apoptosis. Caspase-8, which is a cysteine protease (2, 3), then cleaves cellular substrates including the Bcl-2 family member Bid to truncated Bid (tBid), which interacts with proapoptotic Bax and Bak (4-8). Bax and Bak are monomeric in healthy cells, however, the tBid-Bax and tBid-Bak interactions cause conformational
Regulation of Cell-Death and Immune Defense by receptors of the TNF/NGF family
2006
Receptors of the TNF/NGF family control all aspects of immune defense and also play important roles in the regulation of embryonic developmental processes. Our studies are aimed toward elucidating the mechanisms that regulate the induction of two cardinal activities of these receptors: programmed cell death, an effect mediated by members of the caspase cysteine-protease family, which are activated by the receptors in a proteinsynthesis independent manner, and the induction of immune defense mechanisms through gene activation, which to a large extent relies on stimulation of transcription factors of the NF-kappa B family. We are applying genetic screens and proteomic approaches to identify the signaling proteins that participate in the induction of cell death and immune defense, and complement these in vitro studies by transgenic approaches for elucidating the in vivo role of these proteins.
Blood, 2003
constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis binding domain peptide accelerates − B by a TAT-NEMO κ Inhibition of NFhttp://bloodjournal.hematologylibrary.org/content/102/6/2259.full.html Updated information and services can be found at: (1930 articles) Signal Transduction (973 articles) Phagocytes (1086 articles) Gene Expression (746 articles) Apoptosis Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub\_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: Delivery of biologically active peptides into human polymorphonuclear neutrophils (PMNs) has implications for studying cellular functions and may be therapeutically relevant. The transcription factor nuclear factor-B (NF-B) regulates the expression of multiple genes controlling inflammation, proliferation, and cell survival. PMNs play a crucial role in first-line defense. Targeting NF-B in these cells may promote apoptosis and therefore facilitate resolution of inflammation. We used an 11-amino acid sequence NEMO-binding domain (NBD) that selec-tively inhibits the IKK␥ (NEMO)/IKK interaction, preventing NF-B activation. An HIV-TAT sequence served as a highly effective transducing shuttle. We show that lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), and dexamethasone (DEX) significantly reduced apoptosis after 20 hours. LPS, but not GM-CSF or DEX, activated NF-B as shown by IB␣ degradation, NF-B DNA binding, and transcriptional activity. The TAT-NBD blocked LPSinduced NF-B activation and NF-Bdependent gene expression. TAT-NBD accelerated constitutive PMN apoptosis dose dependently and abrogated LPSdelayed apoptosis. These results provide a proof of principle for peptide delivery by TAT-derived protein transduction domains to specifically inhibit NF-B activity in PMNs. This strategy may help in controlling various cellular functions even in short-lived, transfection-resistant primary human cells. (Blood. 2003;102:2259-2267)
Cell, 1997
family, while the length and sequence of the cytoplasmic segments differ significantly. Department of Genetics Proteolysis of membrane-associated TNF produces Osaka University Medical School soluble TNF. The proteolysis is mediated by a membrane 2-2 Yamada-oka, Suita metalloproteinase (Gearing et al., 1994). Similarly, mem-Osaka 565 brane-bound FasL undergoes metalloproteinase-medi-Japan ated proteolytic cleavage to generate soluble cytokine Osaka Bioscience Institute (Tanaka et al., 1996). A specific metalloproteinase inhibi-6-2-4 Furuedai, Suita tor blocks the processing of TNF as well as FasL, sug-Osaka 565 gesting that a similar enzyme cleaves TNF and FasL. Japan Since the CD40 ligand is also cleaved off from the membrane to become soluble, it is likely that all TNF family members are processed to a soluble form. The soluble Introduction form of human FasL is functional, but mouse FasL loses its activity when it is cleaved from the membrane. Fur-There is an old Japanese saying that "Once we are in thermore, membrane-bound TNF is more active than the land of the living, we will eventually die." This is true, soluble TNF in activating the type II TNF receptor (Grell not only for human beings, but also for the cells that et al., 1995). These results may indicate that FasL and constitute our bodies. By repeated cell division (mitosis) TNF work locally via cell-cell interactions under physioand differentiation, a fertilized egg produces billions of logical conditions and that the purpose of shedding TNF cells to create our bodies. During this process, many or FasL is to attenuate the process. surplus or harmful cells are generated, and they must The functional, soluble forms of TNFs as well as hube removed or killed (Jacobson et al., 1997 [this issue man FasL exist as trimers (Tanaka et al., 1997). It has of Cell]. For example, thymocytes that have failed to not yet been demonstrated whether membrane-bound rearrange their T cell-receptor gene, or whose T cell TNF or FasL are trimers. However, lymphotoxin , a receptor may recognize their own tissues, must be elimimember of the TNF family, consists of a heterotrimer nated. The magnitude of the cell death is staggering: of one ␣ (lymphotoxin-␣, or TNF) and two  chains more than 95% of thymocytes die in the thymus during (lymphotoxin-) on the membrane (Androlewicz et al., maturation. Even in adults, senescent cells are removed 1992), suggesting that membrane-bound TNF and FasL and replaced by newly generated cells to maintain hohave the potential to form trimeric structures. X-ray meostasis. The cell death that occurs during emdiffraction analyses of TNF␣ and TNF have indicated bryogenesis, metamorphosis, endocrine-dependent tisthat each monomer forms an elongated, antiparallel sue atrophy, and normal tissue turnover is "programmed -pleated sheet sandwich with a jelly roll topology cell death," mediated by a process termed "apoptosis." (Jones et al., 1989). Amino acids conserved among Here, I focus on apoptosis controlled by cytokines. members of the TNF family are mainly within the  Two death factors, Fas ligand (FasL) or tumor necrosis strands. Computer-assisted modeling of FasL based on factor (TNF), bind to their receptors and induce the amino acid sequence suggests that FasL has a simiapoptosis, killing the cells within hours. In a classic defilar tertiary structure to TNF␣ and TNF. nition of apoptosis, cells die by "suicide;" that is, cells The Fas and TNF Receptor Family programmed to die would do so autonomously. How-Fas (also known as APO-1 or CD95), the receptor for ever, the identification of death factor-receptor pairs FasL, is a type I-membrane protein (Itoh et al., 1991; that regulate apoptosis indicates that apoptosis can Oehm et al., 1992) and a member of the TNF receptor also be controlled by an external killer in some instances. (TNFR) family, which includes two TNFRs (TNFR1 and TNFR2), the receptor for lymphotoxin-, the NGF recep-Death Factor and Receptor tor (p75), CD40, CD27, and CD30 (Nagata and Golstein, Fas Ligand and the TNF Family 1995). This family is still growing, and three new mem-Cytokines are a family of proteins that regulate cellular bers have recently been identified. They are human DR-3 proliferation and differentiation by binding to their spe-(death receptor-3)/Wsl-1 (Chinnaiyan et al., 1996; Kiston cific receptors on target cells. Cytokines are grouped et al., 1996), human HVEM (herpes virus early mediator) into at least three subfamilies based on structure, cyste-(Montogomery et al., 1996), and chicken CAR1 (cytoine-knot growth factors, tumor necrosis factor, and helipathic avian leukosis-sarcoma virus receptor) (Brojatsch cal cytokines. FasL belongs to the TNF family (Suda et et al., 1996). The extracellular region of the TNF receptor al., 1993; Nagata and Golstein, 1995), which includes family members carries 2-6 repeats of a cysteine-rich TNF, lymphotoxin, CD30 ligand, 4-1BB ligand, CD40 subdomain that has about 25% similarity among various ligand, CD27 ligand, and TRAIL (TNF-related apoptosismembers. In contrast, the cytoplasmic regions have little inducing ligand). FasL is synthesized as a type II-memsimilarity among the members, except for Fas, TNFR1, brane protein; that is, its N terminus is in the cytoplasm DR-3/Wsl-1, and CAR1, as discussed below. and its C-terminal region extends into the extracellular TNF induces apoptosis and activates the transcription space. The extracellular region of about 150 amino acids factor NF-B. It can also stimulate the proliferation of thymocytes. Although both TNFR1 and TNFR2 can is well conserved (20-25%