Rab Effector Research Articles

Purification and Interaction Analysis of a Plant-Specific RAB5 Effector by In Vitro Pull-Down Assay.

RAB GTPases regulate membrane traffic by interacting with effector proteins in the GTP-bound active form. RAB GTPases are highly conserved in a broad range of eukaryotic organisms, while land plants and some green algal species possess a plant-specific RAB5 group. A plant-specific RAB5 in Arabidopsis called ARA6 was shown to regulate a characteristic trafficking route, and participate in abiotic and biotic stress responses. The identification of ARA6 effectors is a powerful strategy to get insights into the molecular basis of ARA6 functions. Recently, we identified an ARA6 effector, PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2), and characterized its functions by biochemical means. PUF2 was hardly expressed as a recombinant protein in the bacterial system, but we solved this problem by optimizing the codon usage of PUF2 CDS to suite for expression in Escherichia coli. Here, we present the protocol we employed to purify PUF2 protein, and to test its nucleotide state-specific interaction with ARA6 by in vitro pull-down assay. This approach would be extended to analyze the molecular functions of other effector proteins of RAB GTPases.

SYTL4 downregulates microtubule stability and confers paclitaxel resistance in triple-negative breast cancer.

Background: Taxanes are frontline chemotherapeutic drugs for patients with triple-negative breast cancer (TNBC); however, chemoresistance reduces their effectiveness. We hypothesized that the molecular profiling of tumor samples before and after neoadjuvant chemotherapy (NAC) would help identify genes associated with drug resistance.Methods: We sequenced 10 samples by RNA-seq from 8 NAC patients with TNBC: 3 patients with a pathologic complete response (pCR) and the other 5 with non-pCR. Differentially expressed genes that predicted chemotherapy response were selected for in vitro functional screening via a small-scale siRNAs pool. The clinical and functional significance of the gene of interest in TNBC was further investigated in vitro and in vivo, and biochemical assays and imaging analysis were applied to study the mechanisms.Results: Synaptotagmin-like 4 (SYTL4), a Rab effector in vesicle transport, was identified as a leading functional candidate. High SYTL4 expression indicated a poor prognosis in multiple TNBC cohorts, specifically in taxane-treated TNBCs. SYTL4 was identified as a novel chemoresistant gene as validated in TNBC cells, a mouse model and patient-derived organoids. Mechanistically, downregulating SYTL4 stabilized the microtubule network and slowed down microtubule growth rate. Furthermore, SYTL4 colocalized with microtubules and interacted with microtubules through its middle region containing the linker and C2A domain. Finally, we found that SYTL4 was able to bind microtubules and inhibit the in vitro microtubule polymerization.Conclusion: SYTL4 is a novel chemoresistant gene in TNBC and its upregulation indicates poor prognosis in taxane-treated TNBC. Further, SYTL4 directly binds microtubules and decreases microtubule stability.

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway

Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway

The ER-Localized Transmembrane Protein TMEM39A/SUSR2 Regulates Autophagy by Controlling the Trafficking of the PtdIns(4)P Phosphatase SAC1.

TMEM39A, encoding an ER-localized transmembrane protein, is a susceptibility locus for multiple autoimmune diseases. The molecular function of TMEM39A remains completely unknown. Here we demonstrated that TMEM39A, also called SUSR2, modulates autophagy activity by regulating the spatial distribution and levels of PtdIns(4)P. Depletion of SUSR2 elevates late endosomal/lysosomal PtdIns(4)P levels, facilitating recruitment of the HOPS complex to promote assembly of the SNARE complex for autophagosome maturation. SUSR2 knockdown also increases the degradative capability of lysosomes. Mechanistically, SUSR2 interacts with the ER-localized PtdIns(4)P phosphatase SAC1 and also the COPII SEC23/SEC24 subunits to promote the ER-to-Golgi transport of SAC1. Retention of SAC1 on the ER in SUSR2 knockdown cells increases the level of PtdIns(3)P produced by the VPS34 complex, promoting autophagosome formation. Our study reveals that TMEM39A/SUSR2 acts as an adaptor protein for efficient export of SAC1from the ER and provides insights into the pathogenesis of diseases associated with TMEM39A mutations.

Spatiotemporal organization and protein dynamics involved in regulated exocytosis of MMP-9 in breast cancer cells.

Altered regulation of exocytosis is an important mechanism controlling many diseases, including cancer. Defects in exocytosis have been implicated in many cancer cell types and are generally attributed to mutations in cellular transport, trafficking, and assembly of machinery necessary for exocytosis of secretory vesicle cargo. In these cancers, up-regulation of trafficking and secretion of matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme, is responsible for degrading the extracellular matrix, a necessary step in tumor progression. Using TIRF microscopy, we identified proteins associated with secretory vesicles containing MMP-9 and imaged the local dynamics of these proteins at fusion sites during regulated exocytosis of MMP-9 from MCF-7 breast cancer cells. We found that many regulators of exocytosis, including several Rab GTPases, Rab effector proteins, and SNARE/SNARE modulator proteins, are stably assembled on docked secretory vesicles before exocytosis. At the moment of fusion, many of these components are quickly lost from the vesicle, while several endocytic proteins and lipids are simultaneously recruited to exocytic sites at precisely that moment. Our findings provide insight into the dynamic behavior of key core exocytic proteins, accessory proteins, lipids, and some endocytic proteins at single sites of secretory vesicle fusion in breast cancer cells.

Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling.

Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.

Hexa-Longin domain scaffolds for inter-Rab signalling.

SummaryCPLANE is a protein complex required for assembly and maintenance of primary cilia. It contains several proteins, such as INTU, FUZ, WDPCP, JBTS17 and RSG1 (REM2- and RAB-like small GTPase 1), whose genes are mutated in ciliopathies. Using two contrasting evolutionary analyses, coevolution-based contact prediction and sequence conservation, we first identified the INTU/FUZ heterodimer as a novel member of homologous HerMon (Hermansky-Pudlak syndrome and MON1-CCZ1) complexes. Subsequently, we identified homologous Longin domains that are triplicated in each of these six proteins (MON1A, CCZ1, HPS1, HPS4, INTU and FUZ). HerMon complexes are known to be Rab effectors and Rab GEFs (Guanine nucleotide Exchange Factors) that regulate vesicular trafficking. Consequently, INTU/FUZ, their homologous complex, is likely to act as a GEF during activation of Rab GTPases involved in ciliogenesis. Supplementary information Supplementary data are available at Bioinformatics online.

Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis.

Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little information is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab-A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP-bound) versus wild-type or constitutively active (GTP-bound) RAB-A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyzes of Rab-A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB-A2a, allowing us to propose that TRAPPII is likely to behave as a guanine nucleotide exchange factor (GEF) for the RAB-A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP-bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF-Rab interactions will be crucial to unravel the co-ordination of plant membrane traffic.

WDR91-WDR81 complex-dependent endolysosomal trafficking and neural development

WD40-repeat proteins participate in diverse cellular processes, such as intracellular trafficking, signal transduction, apoptosis, and transcriptional regulation. From genetic screens of the model organism Caenorhabditis elegans , we identified two novel genes that encode homologs of mammalian WDR91 and WDR81, two WD40-repeat proteins with previously unknown functions. Our study revealed that WDR91 and WDR81 form a complex that functions as an effector of the late endosome-specific Rab GTPase Rab7. The WDR91-WDR81 complex is recruited to the endosomes, where these proteins interact with Beclin 1, a subunit of class III PI3K complex, thereby inhibiting the endosomal synthesis of early endosome-specific PtdIns3P and allowing the conversion of early endosomes to late endosomes, ensuring the delivery of the endosomal cargos to the lysosomes. WDR91 or WDR81 loss impairs neurogenesis and development, possibly by affecting endosome-dependent signaling. Furthermore, WDR81 plays a critical role in aggrephagy by interacting with p62 and LC3C, distinct from its role in endosomal trafficking. Taken together, these findings provide important mechanistic insights into WDR91- and WDR81-related neurodevelopmental disorders.

Interaction networks of Weibel-Palade body regulators syntaxin-3 and syntaxin binding protein 5 in endothelial cells

The endothelium stores the hemostatic protein Von Willebrand factor (VWF) in endothelial storage organelles called Weibel-Palade bodies (WPBs). During maturation, WPBs recruit a complex of Rab GTPases and effectors that associate with components of the SNARE machinery that control WPB exocytosis. Recent genome wide association studies have found links between genetic variations in the SNAREs syntaxin-2 (STX2) and syntaxin binding protein 5 (STXBP5) and VWF plasma levels, suggesting a role for SNARE proteins in regulating VWF release. Moreover, we have previously identified the SNARE proteins syntaxin-3 and STXBP1 as regulators of WPB release. In this study we used an unbiased iterative interactomic approach to identify new components of the WPB exocytotic machinery. An interactome screen of syntaxin-3 identifies a number of SNAREs and SNARE associated proteins (STXBP2, STXBP5, SNAP23, NAPA and NSF). We show that the VAMP-like domain (VLD) of STXBP5 is indispensable for the interaction with SNARE proteins and this capacity of the VLD could be exploited to identify an extended set of novel endothelial SNARE interactors of STXBP5. In addition, an STXBP5 variant with an N436S substitution, which is linked to lower VWF plasma levels, does not show a difference in interactome when compared with WT STXBP5. SignificanceThe hemostatic protein Von Willebrand factor plays a pivotal role in vascular health: quantitative or qualitative deficiencies of VWF can lead to bleeding, while elevated levels of VWF are associated with increased risk of thrombosis. Tight regulation of VWF secretion from WPBs is therefore essential to maintain vascular homeostasis. We used an unbiased proteomic screen to identify new components of the regulatory machinery that controls WPB exocytosis. Our data expand the endothelial SNARE protein network and provide a set of novel candidate WPB regulators that may contribute to regulation of VWF plasma levels and vascular health.

ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-\u03baB via phosphoinosotide remodeling of endosomes

Complement promotes vascular inflammation in transplant organ rejection and connective tissue diseases. Here we identify ZFYVE21 as a complement-induced Rab5 effector that induces non-canonical NF-κB in endothelial cells (EC). In response to membrane attack complexes (MAC), ZFYVE21 is post-translationally stabilized on MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of cellular phosphoinositide content with miltefosine reduces ZFYVE21 induction, EC activation, and allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC and is detected in human renal and synovial tissues. Our data identifies ZFYVE21 as a Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway in complement-mediated conditions.

Molecular cloning of the Rab7 effector RILP (Rab-interacting lysosomal protein) in Litopenaeus vannamei and preliminary analysis of its role in white spot syndrome virus infection

To investigate the role of the Rab7 effector RILP (Rab-interacting lysosomal protein) in white spot syndrome virus (WSSV) infection, the full-length cDNA of RILP (LvRILP) was cloned in Litopenaeus vannamei, which consists of 1595 bp and encodes a polypeptide of 411 amino acids. Sequence analysis and multiple sequence alignment displayed that LvRILP contained a conserved RILP region from 277 amino acid to 325 amino acid. Both the LvRILP and Rab7 mRNA were most highly expressed in stomach and most lowly expressed in hemocyte, which were significantly up-regulated and exhibited similar kinetics post WSSV infection. The interaction of Rab7 with LvRILP was verified by both GST Pull-down and ELISA. Meanwhile, the results of Pull-down assays showed that the GST-tagged VP28 (GST-VP28), His-tagged Rab7 (His-Rab7) and His-RILP formed a tripartite complex. After silencing by specific LvRILP dsRNA, the LvRILP mRNA level exhibited a significant reduction, and the expression levels of three WSSV genes ie1, wsv477 and vp28 all exhibited decreases at 24, 36 and 48 h post WSSV infection. These results suggested that the Rab7 effector RILP was involved in WSSV infection.

ZFYVE21 is a Novel Complement-Induced Rab5 Effector Protein Mediating Cardiac Allograft Vasculopathy

Purpose Complement promotes vascular inflammation in transplant organ rejection, a process leading to cardiac allograft vasculopathy. In this study, we identify a new Rab5 effector protein, ZFYVE21, that mediates this process. We elucidate a ZFYVE21-SMURF2-pAkt signaling axis which activates non-canonical NF-kB, and we define the clinical relevance of this sequence. Methods We developed FACS-assisted protocols enabling proteomic analysis of MAC+Rab5+ endosomes and semi-quantitative analyses of the phosphoinositide content of these structures. A humanized mouse model incorporating human coronary artery segments and human lymphoid cells was employed to define a role for ZFYVE21-associated signaling in cardiac allograft vasculopathy. Software-assisted analyses of human transplant biopsies analyzing ZFYVE21 colocalization was performed using a computer algorithm we helped to developed in-house. Results In response to deposition of membrane attack complexes (MAC) on endothelial cells (EC), ZFYVE21 is post-translationally stabilized via recruitment to MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of the phosphoinositide content of MAC+Rab5+ endosomes with miltefosine reduces ZFYVE21 induction, EC activation, and MAC-induced allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC- but not ligand-induced non-canonical NF-κB and is a biomarker for complement-mediated endothelial signaling in transplant biopsies with antibody-mediated rejection. Conclusion Our data identifies ZFYVE21 as a novel Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway as a drug target and biomarker for allograft vasculopathy.

Retrograde transport of Akt by a neuronal Rab5-APPL1 endosome

Long-distance axonal trafficking plays a critical role in neuronal function and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.

The integral function of the endocytic recycling compartment is regulated by RFFL-mediated ubiquitylation of Rab11 effectors

Endocytic trafficking is regulated by ubiquitylation (also known as ubiquitination) of cargoes and endocytic machineries. The role of ubiquitylation in lysosomal delivery has been well documented, but its role in the recycling pathway is largely unknown. Here, we report that the ubiquitin (Ub) ligase RFFL regulates ubiquitylation of endocytic recycling regulators. An RFFL dominant-negative (DN) mutant induced clustering of endocytic recycling compartments (ERCs) and delayed endocytic cargo recycling without affecting lysosomal traffic. A BioID RFFL interactome analysis revealed that RFFL interacts with the Rab11 effectors EHD1, MICALL1 and class I Rab11-FIPs. The RFFL DN mutant strongly captured these Rab11 effectors and inhibited their ubiquitylation. The prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype and to delay cargo recycling. RFFL directly ubiquitylates these Rab11 effectors in vitro, but RFFL knockout (KO) only reduced the ubiquitylation of Rab11-FIP1. RFFL KO had a minimal effect on the ubiquitylation of EHD1, MICALL1, and Rab11-FIP2, and failed to delay transferrin recycling. These results suggest that multiple Ub ligases including RFFL regulate the ubiquitylation of Rab11 effectors, determining the integral function of the ERC.

Membrane Binding by Synaptotagmin-Like Protein 4: Site Directed Mutagenesis of the Lipid Interaction Surface

Membrane-binding proteins play crucial roles in many biological process such as vesicle trafficking and exocytosis. Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector protein whose overexpression is known to suppress insulin secretion despite increasing the number of secretory vesicles docked to the plasma membrane. Therefore, its structural mechanism may hold clues for understanding the regulation of efficiency in exocytosis signaling. Slp-4 binds to vesicular Rab effector proteins through its N-terminal Slp homology domain, to SNARE complexes via its central linker region, and contains C-terminal tandem C2A and C2B domains that interact with plasma membrane lipids. We have previously shown that the Slp-4 C2A domain binds with high affinity to liposomes approximating the lipid composition of the plasma membrane interior leaflet. The C2A domain has a lysine cluster that binds to phosphatidylinositol-(4, 5)-bisphosphate (PIP2) and soluble inositol phosphates with micromolar affinity, but much more strongly to membranes containing both PIP2 and anionic background lipids such as phosphatidylserine (PS). We are combining computational and experimental approaches to reveal which residues of the Slp-4 C2A domain participate in binding PS and PIP2. Using mutational analysis and protein binding assays, here we show that mutation of specific individual amino acid residues tends to have modest effects on the domain's membrane affinity; however, combined mutations at multiple predicted binding sites have greater effects. This finding indicates that the C2A domain interacts with plasma membrane lipids through multiple binding sites that act in a concerted manner to produce its high membrane affinity.

To Pinpoint the Location and the Orientation of Proteins Associated with Dense-Core Vesicles (DCVs) using CLEM

Exocytosis of catecholamine (adrenaline and noradrenaline) from adrenal chromaffin cells is important for stress responses and insulin release from pancreatic beta cells is critical in regulating blood glucose levels. These exocytic processes are highly regulated by a dozens of key proteins in eukaryotic cells. While the origin, transport, and delivery of cargo-loaded vesicles (dense-core vesicles) and the proteins involved is well studied, how these proteins are organized with respect to dense-core vesicles (DCVs) at the nanoscale remains to be explored. Here, we directly image several proteins that are key regulators of the transport, docking, and priming of DCVs at the plasma membrane. Specifically, correlative super-resolution (dSTORM) light and platinum replica transmission electron microscopy (CLEM) imaging was performed on PC12 cells (a rat adrenal chromaffin-derived neuroendocrine cell line) that were transfected with proteins of interest. We aligned super-resolution dSTORM images of Rab proteins (Rab27a and Rab3a) and Rab effector proteins (granuphilin and Rabphilin) with corresponding TEM images of the plasma membrane and observed the precise correlation between proteins and vesicles at the plasma membrane. The nano-scale molecular architecture of DCVs generated from this correlative imaging will help us understand how key proteins guide the docking, priming, and fusion of DCVs in endocrine cells.

Grab recruitment by Rab27A-Rabphilin3a triggers Rab3A activation in human sperm exocytosis

Sperm must undergo the regulated exocytosis of its dense core granule (the acrosome reaction, AR) to fertilize the egg. We have previously described that Rabs3 and 27 are organized in a RabGEF cascade within the signaling pathway elicited by exocytosis stimuli in human sperm. Here, we report the identity and the role of two molecules that link these secretory Rabs in the RabGEF cascade: Rabphilin3a and GRAB. Like Rab3 and Rab27, GRAB and Rabphilin3a are present, localize to the acrosomal region and are required for calcium-triggered exocytosis in human sperm. Sequestration of either protein with specific antibodies introduced into streptolysin O-permeabilized sperm impairs the activation of Rab3 in the acrosomal region elicited by calcium, but not that of Rab27. Biochemical and functional assays indicate that Rabphilin3a behaves as a Rab27 effector during the AR and that GRAB exhibits GEF activity toward Rab3A. Recombinant, active Rab27A pulls down Rabphilin3a and GRAB from human sperm extracts. Conversely, immobilized Rabphilin3a recruits Rab27 and GRAB; the latter promotes Rab3A activation. The enzymatic activity of GRAB toward Rab3A was also suggested by in silico and in vitro assays with purified proteins. In summary, we describe here a signaling module where Rab27A-GTP interacts with Rabphilin3a, which in turn recruits a guanine nucleotide-exchange activity toward Rab3A. This is the first description of the interaction of Rabphilin3a with a GEF. Because the machinery that drives exocytosis is highly conserved, it is tempting to hypothesize that the RabGEF cascade unveiled here might be part of the molecular mechanisms that drive exocytosis in other secretory systems.

HIV-1 Nef Hijacks Lck and Rac1 Endosomal Traffic To Dually Modulate Signaling-Mediated and Actin Cytoskeleton-Mediated T Cell Functions.

Endosomal traffic of TCR and signaling molecules regulates immunological synapse formation and T cell activation. We recently showed that Rab11 endosomes regulate the subcellular localization of the tyrosine kinase Lck and of the GTPase Rac1 and control their functions in TCR signaling and actin cytoskeleton remodeling. HIV-1 infection of T cells alters their endosomal traffic, activation capacity, and actin cytoskeleton organization. The viral protein Nef is pivotal for these modifications. We hypothesized that HIV-1 Nef could jointly alter Lck and Rac1 endosomal traffic and concomitantly modulate their functions. In this study, we show that HIV-1 infection of human T cells sequesters both Lck and Rac1 in a pericentrosomal compartment in an Nef-dependent manner. Strikingly, the Nef-induced Lck compartment contains signaling-competent forms (phosphorylated on key Tyr residues) of Lck and some of its downstream effectors, TCRζ, ZAP70, SLP76, and Vav1, avoiding the proximal LAT adaptor. Importantly, Nef-induced concentration of signaling molecules was concomitant with the upregulation of several early and late T cell activation genes. Moreover, preventing the concentration of the Nef-induced Lck compartment by depleting the Rab11 effector FIP3 counteracted Nef-induced gene expression upregulation. In addition, Nef extensively sequesters Rac1 and downregulates Rac1-dependent actin cytoskeleton remodeling, thus reducing T cell spreading. Therefore, by modifying their endosomal traffic, Nef hijacks signaling and actin cytoskeleton regulators to dually modulate their functional outputs. Our data shed new light into the molecular mechanisms that modify T cell physiology during HIV-1 infection.

RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses

SummaryHIV-1 broadly neutralizing antibodies (bnAbs) are difficult to induce with vaccines but are generated in ∼50% of HIV-1-infected individuals. Understanding the molecular mechanisms of host control of bnAb induction is critical to vaccine design. Here, we performed a transcriptome analysis of blood mononuclear cells from 47 HIV-1-infected individuals who made bnAbs and 46 HIV-1-infected individuals who did not and identified in bnAb individuals upregulation of RAB11FIP5, encoding a Rab effector protein associated with recycling endosomes. Natural killer (NK) cells had the highest differential expression of RAB11FIP5, which was associated with greater dysregulation of NK cell subsets in bnAb subjects. NK cells from bnAb individuals had a more adaptive/dysfunctional phenotype and exhibited impaired degranulation and cytokine production that correlated with RAB11FIP5 transcript levels. Moreover, RAB11FIP5 overexpression modulated the function of NK cells. These data suggest that NK cells and Rab11 recycling endosomal transport are involved in regulation of HIV-1 bnAb development.