RESUMO
Neurotensin (NT) acts as a primary neurotransmitter and neuromodulator in the CNS and has been involved in a number of CNS pathologies including epilepsy. NT mediates its central and peripheral effects by interacting with the NTSR1, NTSR2, and Sort1/NTSR3 receptor subtypes. To date, little is known about the precise expression of the NT receptors in brain neural cells and their regulation in pathology. In the present work, we studied the cellular distribution of the NTSR2 protein in the rat hippocampus and questioned whether its expression was modulated in conditions of neuroinflammation using a model of temporal lobe epilepsy induced by pilocarpine. This model is characterized by a rapid and intense inflammatory reaction with reactive gliosis in the hippocampus. We show that NTSR2 protein is expressed in hippocampal astrocytes and its expression increases together with astrocyte reactivity following induction of status epilepticus. NTSR2 immunoreactivity is also increased in astrocytes proximal to blood vessels and their end-feet, and in endothelial cells. Proinflammatory factors such as IL1ß and LPS induced NTSR2 mRNA and protein in cultured astroglial cells. Antagonizing NTSR2 with SR142948A decreased NTSR2 expression as well as astroglial reactivity. Together, our results suggest that NTSR2 is implicated in astroglial and gliovascular inflammation and that targeting the NTSR2 receptor may open new avenues in the regulation of neuroinflammation in CNS diseases.
Assuntos
Astrócitos , Pilocarpina , Animais , Astrócitos/metabolismo , Células Endoteliais/metabolismo , Hipocampo/metabolismo , Doenças Neuroinflamatórias , Pilocarpina/metabolismo , Pilocarpina/toxicidade , Ratos , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Convulsões/metabolismoRESUMO
The blood-brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor-mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from wild-type and LDLR-knockout animals (ldlr-/- ) were used to validate the specific LDLR-dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR-targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor-mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.-Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier.
Assuntos
Anticorpos/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Endocitose/fisiologia , Receptores de LDL/metabolismo , Animais , Transporte Biológico/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Humanos , Camundongos , Ratos , Receptores de LDL/deficiênciaRESUMO
Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., J. Med. Chem. 2012, 55 (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL[CMPRLRGC]cDPR) and structure-activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC]c) which specifically binds hLDLR with a KD of 76 nM and has an in vitro blood half-life of â¼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-"Pen"M"Thz"RLRGC]c), which showed the highest KD value of 9 nM. However, this latter analogue displayed the lowest in vitro blood half-life (â¼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 106 s-1.M-1 range, and off-rates varying from the low 10-2 s-1 to the 10-1 s-1 range. Furthermore, all these new analogues showed increased blood half-lives in vitro, reaching â¼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM"Thz"RLRG"Pen"]c), showing a KD of 18 nM and a blood half-life of â¼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled 3H-VH4127 in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.
Assuntos
Vetores Genéticos/normas , Fragmentos de Peptídeos/farmacologia , Receptores de LDL/antagonistas & inibidores , Animais , Células CHO , Cricetulus , Sistemas de Liberação de Medicamentos , Endocitose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de Peptídeos/farmacocinética , Receptores de LDL/fisiologia , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
In recent decades, subcutaneous (SC) administration of monoclonal antibodies (mAbs) has emerged as a promising alternative to intravenous delivery in oncology, offering comparable therapeutic efficacy while addressing patient preferences. This perspective article provides an in-depth analysis of the technological landscape surrounding SC mAb administration in oncology. It outlines various technologies under evaluation across developmental stages, spanning from preclinical investigations to the integration of established methodologies in clinical practice. Additionally, this perspective article explores emerging trends and prospective trajectories, shedding light on the evolving landscape of SC mAb administration. Furthermore, it emphasizes key checkpoints related to quality attributes essential for optimizing mAb delivery via the SC route. This review serves as a valuable resource for researchers, clinicians, and healthcare policymakers, offering insights into the advancement of SC mAb administration in oncology and its implications for patient care.
RESUMO
Small RNA molecules such as microRNA and small interfering RNA (siRNA) have become promising therapeutic agents because of their specificity and their potential to modulate gene expression. Any gene of interest can be potentially up- or down-regulated, making RNA-based technology the healthcare breakthrough of our era. However, the functional and specific delivery of siRNAs into tissues of interest and into the cytosol of target cells remains highly challenging, mainly due to the lack of efficient and selective delivery systems. Among the variety of carriers for siRNA delivery, peptides have become essential candidates because of their high selectivity, stability, and conjugation versatility. Here, we describe the development of molecules encompassing siRNAs against SOD1, conjugated to peptides that target the low-density lipoprotein receptor (LDLR), and their biological evaluation both in vitro and in vivo.
RESUMO
Nanoparticle (NP) surface functionalization with proteins, including monoclonal antibodies (mAbs), mAb fragments, and various peptides, has emerged as a promising strategy to enhance tumor targeting specificity and immune cell interaction. However, these methods often rely on complex chemistry and suffer from batch-dependent outcomes, primarily due to limited control over the protein orientation and quantity on NP surfaces. To address these challenges, a novel approach based on the supramolecular assembly of two peptides is presented to create a heterotetramer displaying VHHs on NP surfaces. This approach effectively targets both tumor-associated antigens (TAAs) and immune cell-associated antigens. In vitro experiments showcase its versatility, as various NP types are biofunctionalized, including liposomes, PLGA NPs, and ultrasmall silica-based NPs, and the VHHs targeting of known TAAs (HER2 for breast cancer, CD38 for multiple myeloma), and an immune cell antigen (NKG2D for natural killer (NK) cells) is evaluated. In in vivo studies using a HER2+ breast cancer mouse model, the approach demonstrates enhanced tumor uptake, retention, and penetration compared to the behavior of nontargeted analogs, affirming its potential for diverse applications.
Assuntos
Nanopartículas , Peptídeos , Nanopartículas/química , Animais , Humanos , Camundongos , Peptídeos/química , Linhagem Celular Tumoral , Feminino , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Neoplasias da Mama/metabolismoRESUMO
Subcutaneous (SC) administration of monoclonal antibodies (mAbs) is a proven strategy for improving therapeutic outcomes and patient compliance. The current FDA-/EMA-approved enzymatic approach, utilizing recombinant human hyaluronidase (rHuPH20) to enhance mAbs SC delivery, involves degrading the extracellular matrix's hyaluronate to increase tissue permeability. However, this method lacks tunable release properties, requiring individual optimization for each mAb. Seeking alternatives, physical polysaccharide hydrogels emerge as promising candidates due to their tunable physicochemical and biodegradability features. Unfortunately, none have demonstrated simultaneous biocompatibility, biodegradability, and controlled release properties for large proteins (≥150 kDa) after SC delivery in clinical settings. Here, a novel two-component hydrogel comprising chitosan and chitosan@DOTAGA is introduced that can be seamlessly mixed with sterile mAbs formulations initially designed for intravenous (IV) administration, repurposing them as novel tunable SC formulations. Validated in mice and nonhuman primates (NHPs) with various mAbs, including trastuzumab and rituximab, the hydrogel exhibited biodegradability and biocompatibility features. Pharmacokinetic studies in both species demonstrated tunable controlled release, surpassing the capabilities of rHuPH20, with comparable parameters to the rHuPH20+mAbs formulation. These findings signify the potential for rapid translation to human applications, opening avenues for the clinical development of this novel SC biosimilar formulation.
Assuntos
Anticorpos Monoclonais , Quitosana , Humanos , Camundongos , Animais , Anticorpos Monoclonais/farmacocinética , Hidrogéis , Preparações de Ação Retardada , Injeções SubcutâneasRESUMO
Here we report the coupling of a cyclic peptide (VH4127) targeting the low density lipoprotein (LDL) receptor (LDLR) noncompetitively to cucurbit[7]uril (CB[7]) to develop a new kind of drug delivery system (DDS), namely, CB[7]-VH4127, with maintained binding affinity to the LDLR. To evaluate the uptake potential of this bismacrocyclic compound, another conjugate was prepared comprising a high-affinity group for CB[7] (adamantyl(Ada)-amine) coupled to the fluorescent tracker Alexa680 (A680). The resulting A680-Ada·CB[7]-VH4127 supramolecular complex demonstrated conserved LDLR-binding potential and improved LDLR-mediated endocytosis and intracellular accumulation potential in LDLR-expressing cells. The combination of two technologies, namely, monofunctionalized CB[7] and the VH4127 LDLR-targeting peptide, opens new avenues in terms of targeting and intracellular delivery to LDLR-expressing tissues or tumors. The versatile transport capacity of CB[7], known to bind a large spectrum of bioactive or functional compounds, makes this new DDS suitable for a wide range of therapeutic or imaging applications.
Assuntos
Compostos Macrocíclicos , Peptídeos , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Sistemas de Liberação de Medicamentos , Peptídeos/química , Receptores de LDL/metabolismoRESUMO
p50/dynamitin (DM) is a major subunit of the microtubule-associated dynactin complex that is required for stabilization and attachment of its two distinct structural domains, namely the Arp1 rod and the shoulder/sidearm. Here, we define the determinants of p50/DM required for self-oligomerization of the protein and for interactions with other subunits of the dynactin complex. Whereas the N-terminal 1-91-amino acid region of the protein is required and sufficient for binding to the Arp1 rod, additional determinants contained within the first half of the protein are required for optimal recruitment of the p150(Glued) subunit of the shoulder/sidearm. Overexpression experiments confirmed that the N-terminal 1-91-amino acid region of p50/DM is critical for dynactin functionality, because this fragment acts as a dominant negative to inhibit both dynein-dependent and -independent functions of the complex. Furthermore, the first two predicted coiled-coil motifs of p50/DM contain determinants that mediate self-association of the protein. Interestingly, p50/DM self-association does not contribute to p50/DM-induced disruption of the dynactin complex, but most likely participates in the stabilization of the complex.
Assuntos
Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Fator II de Transcrição COUP/metabolismo , Galinhas , Chlorocebus aethiops , Sequência Conservada , Complexo Dinactina , Células HeLa , Humanos , Dados de Sequência Molecular , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de ProteínaRESUMO
Despite clinical advances in diagnosis and treatment, pancreatic ductal adenocarcinoma (PDAC) remains the third leading cause of cancer death, and is still associated with poor prognosis and dismal survival rates. Identifying novel PDAC-targeted tools to tackle these unmet clinical needs is thus an urgent requirement. Here we use a peptide conjugate that specifically targets PDAC through low-density lipoprotein receptor (LDLR). We demonstrate by using near-infrared fluorescence imaging the potential of this conjugate to specifically detect and discriminate primary PDAC from healthy organs including pancreas and from benign mass-forming chronic pancreatitis, as well as detect metastatic pancreatic cancer cells in healthy liver. This work paves the way towards clinical applications in which safe LDLR-targeting peptide conjugate promotes tumor-specific delivery of imaging and/or therapeutic agents, thereby leading to substantial improvements of the PDAC patient's outcome.
Assuntos
Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Receptores de LDL/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Receptores de LDL/metabolismoRESUMO
It was recently reported that human immunodeficiency virus type 1 (HIV-1) Vpr induced the proteasomal degradation of the nuclear UNG2 enzyme for efficient virus replication. We confirm here that HIV-1 infection and Vpr expression reduce the level of endogenous UNG2, but this effect is not reverted by treatment with the proteasome inhibitor MG132. Moreover, this reduction is not mediated by Vpr binding to UNG2 and is independent of the Vpr-induced G(2) arrest. Finally, we show that Vpr influences the UNG2 promoter without affecting UNG1 gene expression. These data indicate that the Vpr-induced decrease of UNG2 level is mainly related to a transcriptional effect.
Assuntos
DNA Glicosilases/biossíntese , Regulação Viral da Expressão Gênica , Transcrição Gênica , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Fase G2 , HIV-1/genética , Células HeLa , Humanos , Leupeptinas/farmacologia , Microscopia de Fluorescência/métodos , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Replicação ViralRESUMO
The HIV-1 accessory protein viral protein R (Vpr) causes G2 arrest and apoptosis in infected cells. We previously identified the DNA damage-signaling protein ATR as the cellular factor that mediates Vpr-induced G2 arrest and apoptosis. Here, we examine the mechanism of induction of apoptosis by Vpr and how it relates to induction of G2 arrest. We find that entry into G2 is a requirement for Vpr to induce apoptosis. We investigated the role of the mitochondrial permeability transition pore by knockdown of its essential component, the adenine nucleotide translocator. We found that Vpr-induced apoptosis was unaffected by knockdown of ANT. Instead, apoptosis is triggered through a different mitochondrial pore protein, Bax. In support of the idea that checkpoint activation and apoptosis induction are functionally linked, we show that Bax activation by Vpr was ablated when ATR or GADD45alpha was knocked down. Certain mutants of Vpr, such as R77Q and I74A, identified in long-term nonprogressors, have been proposed to inefficiently induce apoptosis while activating the G2 checkpoint in a normal manner. We tested the in vitro phenotypes of these mutants and found that their abilities to induce apoptosis and G2 arrest are indistinguishable from those of HIV-1NL4-3 vpr, providing additional support to the idea that G2 arrest and apoptosis induction are mechanistically linked.
Assuntos
Translocador 1 do Nucleotídeo Adenina/fisiologia , Apoptose/fisiologia , Ciclo Celular/fisiologia , Fase G2/fisiologia , Produtos do Gene vpr/fisiologia , Proteína X Associada a bcl-2/fisiologia , Translocador 1 do Nucleotídeo Adenina/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos T CD4-Positivos/citologia , Caspases/fisiologia , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Regulação para Baixo , Regulação Viral da Expressão Gênica , Produtos do Gene vpr/genética , HIV-1/genética , HIV-1/patogenicidade , HIV-1/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutagênicos/farmacologia , Mutação/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteína X Associada a bcl-2/genética , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells-or organs that express the LDLR.
Assuntos
Peptídeos/metabolismo , Receptores de LDL/metabolismo , Sequência de Aminoácidos , Animais , Sistemas de Liberação de Medicamentos , Endocitose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Engenharia de Proteínas , Ratos , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: HIV-1 Vpr is a dynamic protein that primarily localizes in the nucleus, but a significant fraction is concentrated at the nuclear envelope (NE), supporting an interaction between Vpr and components of the nuclear pore complex, including the nucleoporin hCG1. In the present study, we have explored the contribution of Vpr accumulation at the NE to the Vpr functions, including G2-arrest and pro-apoptotic activities, and virus replication in primary macrophages. RESULTS: In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first alpha-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first alpha-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors. CONCLUSION: These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.
Assuntos
Núcleo Celular/virologia , HIV-1/fisiologia , Macrófagos/virologia , Membrana Nuclear/metabolismo , Replicação Viral , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Ciclo Celular , HIV-1/química , HIV-1/metabolismo , Células HeLa , Humanos , Técnicas do Sistema de Duplo-Híbrido , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/biossíntese , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Drug delivery to the central nervous system is hindered by the presence of physiological barriers such as the blood-brain barrier. To accomplish the task of nutrient transport, the brain endothelium is endowed with various transport systems, including receptor-mediated transcytosis (RMT). This system can be used to shuttle therapeutics into the central nervous system (CNS) in a noninvasive manner. Therefore, the low-density lipoprotein receptor (LDLR) is a relevant target for delivering drugs. From an initial phage display biopanning, a series of peptide ligands for the LDLR was optimized leading to size reduction and improved receptor binding affinity with the identification of peptide 22 and its analogues. Further real-time biphoton microscopy experiments on living mice demonstrated the ability of peptide 22 to efficiently and quickly cross CNS physiological barriers. This validation of peptide 22 led us to explore its binding on the extracellular LDLR domain from an NMR-oriented structural study and docking experiments.
Assuntos
Barreira Hematoencefálica/metabolismo , Oligopeptídeos/síntese química , Peptídeos Cíclicos/síntese química , Receptores de LDL/metabolismo , Medula Espinal/metabolismo , Animais , Corantes Fluorescentes , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Relação Estrutura-Atividade , TranscitoseRESUMO
Although HIV-1 Vpr displays several functions in vitro, limited information exists concerning their relevance during infection. Here, we characterized Vpr variants isolated from a rapid and a long-term non-progressor (LTNP). Interestingly, vpr alleles isolated from longitudinal samples of the LTNP revealed a dominant sequence that subsequently led to diversity similar to that observed in the progressor patient. Most of primary Vpr proteins accumulated at the nuclear envelope and interacted with host-cell partners of Vpr. They displayed cytostatic and proapoptotic activities, although a LTNP allele, harboring the Q65R substitution, failed to bind the DCAF1 subunit of the Cul4a/DDB1 E3 ligase and was inactive. This Q65R substitution correlated with impairment of Vpr docking at the nuclear envelope, raising the possibility of a functional link between this property and the Vpr cytostatic activity. In contradiction with published results, the R77Q substitution, found in LTNP alleles, did not influence Vpr proapoptotic activity.
Assuntos
Produtos do Gene vpr/genética , Genes vpr , Sequências Repetidas Terminais , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Alelos , Sequência de Aminoácidos , Apoptose , Variação Genética , Humanos , Leucócitos Mononucleares/citologia , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In addition to its positive signaling function in the antigen presentation process, CD4 acts as the primary receptor for HIV-1. Contact between CD4 and the viral envelope leads to virus entry, but can also trigger apoptosis of uninfected CD4+ T-cells through a mechanism that is poorly understood. We show that Siva-1, a death domain-containing proapoptotic protein, associates with the cytoplasmic domain of CD4. This interaction is mediated by the cysteine-rich region found in the C-terminal part of the Siva-1 protein. Expression of Siva-1 specifically increases the susceptibility of both T-cell lines and unstimulated human primary CD4+ T-lymphocytes to CD4-mediated apoptosis triggered by the HIV-1 envelope, and results in activation of a caspase-dependent mitochondrial pathway. The same susceptibility is observed in T-cells expressing a truncated form of CD4 that is able to recruit Siva-1 but fails to associate with p56Lck, indicating that Siva-1 participates in a pathway independent of the p56Lck kinase activity. Altogether, these results suggest that Siva-1 might participate in the CD4-initiated signaling apoptotic pathway induced by the HIV-1 envelope in T-lymphoid cells.