A tamoxifen-inducible Cre knock-in mouse for lens-specific gene manipulation.

Mouse models are valuable tools in studying lens biology and biochemistry, and the Cre-loxP system is the most used technology for gene targeting in the lens. However, numerous genes are indispensable in lens development. The conventional knockout method either prevents lens formation or causes simultaneous cataract formation, hindering the studies of their roles in lens structure, growth, metabolism, and cataractogenesis during lens aging. An inducible Cre-loxP mouse line is an excellent way to achieve such a purpose. We established a lens-specific Cre ERT2 knock-in mouse (LCEK), an inducible mouse model for lens-specific gene targeting in a spatiotemporal manner. LCEK mice were created by in-frame infusion of a P2A-CreERT2 at the C-terminus of the last coding exon of the gene alpha A crystallin (Cryaa). LCEK mice express tamoxifen-inducible Cre recombinase uniquely in the lens. Through ROSAmT/mG and two endogenous genes (Gclc and Rbpj) targeting, we found no Cre recombinase leakage in the lens epithelium, but 50-80% leakage was observed in the lens cortex and nucleus. Administration of tamoxifen almost completely abolished target gene expression in both lens epithelium and cortex but only mildly enhanced gene deletion in the lens nucleus. Notably, no overt leakage of Cre activity was detected in developing LCEK lens when bred with mice carrying loxP floxed genes that are essential for lens development. This newly generated LCEK line will be a powerful tool to target genes in the lens for gene functions study in lens aging, posterior capsule opacification (PCO), and other areas requiring precision gene targeting.

Aged Lens Epithelial Cells Suppress Proliferation and Epithelial-Mesenchymal Transition-Relevance for Posterior Capsule Opacification.

Posterior capsule opacification (PCO) is a frequent complication after cataract surgery, and advanced PCO requires YAG laser (Nd: YAG) capsulotomy, which often gives rise to more complications. Lens epithelial cell (LEC) proliferation and transformation (i.e., epithelial-mesenchymal transition (EMT)) are two critical elements in PCO initiation and progression pathogenesis. While PCO marginally impacts aged cataract surgery patients, PCO incidences are exceptionally high in infants and children undergoing cataract surgery. The gene expression of lens epithelial cell aging and its role in the discrepancy of PCO prevalence between young and older people have not been fully studied. Here, we conducted a comprehensive differentially expressed gene (DEG) analysis of a cell aging model by comparing the early and late passage FHL124 lens epithelial cells (LECs). In vitro, TGFß2, cell treatment, and in vivo mouse cataract surgical models were used to validate our findings. We found that aged LECs decelerated rates of cell proliferation accompanied by dysregulation of cellular immune response and cell stress response. Surprisingly, we found that LECs systematically downregulated epithelial-mesenchymal transition (EMT)-promoting genes. The protein expression of several EMT hallmark genes, e.g., fibronectin, αSMA, and cadherin 11, were gradually decreased during LECs aging. We then confirmed these findings in vitro and found that aged LECs markedly alleviated TGFß2-mediated EMT. Importantly, we explicitly confirmed the in vitro findings from the in vivo mouse cataract surgery studies. We propose that both the high proliferation rate and EMT-enriched young LECs phenotypic characteristics contribute to unusually high PCO incidence in infants and children.

Mitotic Activation Around Wound Edges and Epithelialization Repair in UVB-Induced Capsular Cataracts.

Purpose: Ultraviolet B (UVB) has been well documented to induce capsular cataracts; however, the mechanism of the lens epithelial cell-mediated repair process after UVB irradiation is not fully understood. The purpose of this study was to better understand lens epithelial cell repair after UVB-induced epithelium damage. Method: C57BL/6J mice were irradiated by various doses of UVB. Lens morphology and lens capsule opacity were monitored by slit lamp, darkfield microscopy, and phase-contrast microscopy. Lens epithelial cell mitotic activation and cell apoptosis were measured by immunohistochemistry. Lens epithelial ultrastructure was analyzed by transmission electron microscopy. Results: UVB irradiation above a dose of 2.87 kJ/m2 triggered lens epithelial cell apoptosis and subcapsular cataract formation, with a ring-shaped structure composed of multilayered epithelial cell clusters manifesting a dense ring-shaped capsular cataract. The epithelial cells immediately outside the edge of the ring-shaped aggregates transitioned to mitotically active cells and performed wound healing through the epithelialization process. However, repairs ceased when lens epithelial cells made direct contact, and scar-like tissue in the center of the anterior capsule remained even by 6 months after UVB irradiation. Conclusions: Our present study demonstrates that normally quiescent lens epithelial cells can be reactivated for epithelialization repair in response to UV-induced damage.

Structure of HIV-1 Vpr in complex with the human nucleotide excision repair protein hHR23A.

HIV-1 Vpr is a prototypic member of a large family of structurally related lentiviral virulence factors that antagonize various aspects of innate antiviral immunity. It subverts host cell DNA repair and protein degradation machineries by binding and inhibiting specific post-replication repair enzymes, linking them via the DCAF1 substrate adaptor to the Cullin 4 RING E3 ligase (CRL4DCAF1). HIV-1 Vpr also binds to the multi-domain protein hHR23A, which interacts with the nucleotide excision repair protein XPC and shuttles ubiquitinated proteins to the proteasome. Here, we report the atomic resolution structure of Vpr in complex with the C-terminal half of hHR23A, containing the XPC-binding (XPCB) and ubiquitin-associated (UBA2) domains. The XPCB and UBA2 domains bind to different sides of Vpr's 3-helix-bundle structure, with UBA2 interacting with the α2 and α3 helices of Vpr, while the XPCB domain contacts the opposite side of Vpr's α3 helix. The structure as well as biochemical results reveal that hHR23A and DCAF1 use overlapping binding surfaces on Vpr, even though the two proteins exhibit entirely different three-dimensional structures. Our findings show that Vpr independently targets hHR23A- and DCAF1- dependent pathways and highlight HIV-1 Vpr as a versatile module that interferes with DNA repair and protein degradation pathways.

Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells.

The present study aims to understand the mechanism of the lens epithelial cell's strong anti-apoptotic capacity and survival in the mature human lens that, on the one hand, maintains lens transparency over several decades, while on the other hand, increases the risk of posterior capsule opacification (PCO). Here we compared FHL124 cells and HeLa cells, spontaneously immortalized epithelial cell lines derived from the human lens and cervical cancer cells, respectively, of their resistance to TNFα-mediated cell death. TNFα plus cycloheximide (CHX) triggered almost all of HeLa cell death. FHL124 cells, however, were unaffected and able to block caspase-8 activation as well as prevent caspase-3 and PARP-1 cleavage. Interestingly, despite spontaneous NFκB and AP-1 activation and upregulation of multiple cell survival/anti-apoptotic genes in both cell types, only FHL124 cells were able to survive the TNFα challenge. After screening and comparing the cell survival genes, cFLIP was found to be highly expressed in FHL124 cells and substantially upregulated by TNFα stimulation. FHL124 cells with a mild cFLIP knockdown manifested a profound apoptotic response to TNFα stimulus similar to HeLa cells. Most importantly, we confirmed these findings in an ex vivo lens capsular bag culture system. In conclusion, our results show that cFLIP is a critical gene that is regulating lens epithelial cell survival.

Aging lens epithelium is susceptible to ferroptosis.

Age-related cataracts (ARC) are the primary cause of blindness worldwide, and oxidative stress is considered the central pathogenesis of age-related cataractogenesis. Interestingly, ample evidence suggests that there is no remarkable apoptosis present in aged and cataractous human lenses despite the profound disruption of redox homeostasis, raising an essential question regarding the existence of other cell death mechanisms. Here we sought to explore the lens epithelial cell's (LEC) susceptibility to ferroptosis after documentation has concluded that aged and cataractous human lenses manifest with increased reactive oxygen species (ROS) formation, elevated lipid peroxidation, and accumulative intracellular redox-active iron, constituting the three hallmarks of ferroptosis during aging and cataractogenesis. Here we show that very low concentrations of system Xc- inhibitor Erastin (0.5 µM) and glutathione peroxidase 4 (GPX4) inhibitor RSL3 (0.1 µM) can drastically induce human LEC (FHL124) ferroptosis in vitro and mouse lens epithelium ferroptosis ex vivo. Depletion of intracellular glutathione (GSH) in human LECs and mouse lens epithelium significantly sensitizes ferroptosis, particularly under RSL3 challenge. Intriguingly, both human LECs and the mouse lens epithelium demonstrate an age-related sensitization of ferroptosis. Transcriptome analysis indicates that clusters of genes are up-or down-regulated in aged LECs, impacting cellular redox and iron homeostases, such as downregulation of both cystine/glutamate antiporter subunits SLC7A11 and SLC3A2 and iron exporter ferroportin (SLC40A1). Here, for the first time, we are suggesting that LECs are highly susceptible to ferroptosis. Moreover, aged and cataractous human lenses may possess more pro-ferroptotic criteria than any other organ in the human body.

Vitamin C is a source of oxoaldehyde and glycative stress in age-related cataract and neurodegenerative diseases.

Oxoaldehyde stress has recently emerged as a major source of tissue damage in aging and age-related diseases. The prevailing mechanism involves methylglyoxal production during glycolysis and modification of arginine residues through the formation of methylglyoxal hydroimidazolones (MG-H1). We now tested the hypothesis that oxidation of vitamin C (ascorbic acid or ASA) contributes to this damage when the homeostatic redox balance is disrupted especially in ASA-rich tissues such as the eye lens and brain. MG-H1 measured by liquid chromatography mass spectrometry is several fold increased in the lens and brain from transgenic mice expressing human vitamin C transporter 2 (hSVCT2). Similarly, MG-H1 levels are increased two- to fourfold in hippocampus extracts from individuals with Alzheimer's disease (AD), and significantly higher levels are present in sarkosyl-insoluble tissue fractions from AD brain proteins than in the soluble fractions. Moreover, immunostaining with antibodies against methylglyoxal hydroimidazolones reveals similar increase in substantia nigra neurons from individuals with Parkinson's disease. Results from an in vitro incubation experiment suggest that accumulated catalytic metal ions in the hippocampus during aging could readily accelerate ASA oxidation and such acceleration was significantly enhanced in AD. Modeling studies and intraventricular injection of 13 C-labeled ASA revealed that ASA backbone carbons 4-6 are incorporated into MG-H1 both in vitro and in vivo, likely via a glyceraldehyde precursor. We propose that drugs that prevent oxoaldehyde stress or excessive ASA oxidation may protect against age-related cataract and neurodegenerative diseases.

HIV-1 Vpr counteracts HLTF-mediated restriction of HIV-1 infection in T cells.

Lentiviruses, including HIV-1, possess the ability to enter the nucleus through nuclear pore complexes and can infect interphase cells, including those actively replicating chromosomal DNA. Viral accessory proteins hijack host cell E3 enzymes to antagonize intrinsic defenses, and thereby provide a more permissive environment for virus replication. The HIV-1 Vpr accessory protein reprograms CRL4DCAF1 E3 to antagonize select postreplication DNA repair enzymes and activates the DNA damage checkpoint in the G2 cell cycle phase. However, little is known about the roles played by these Vpr targets in HIV-1 replication. Here, using a sensitive pairwise replication competition assay, we show that Vpr endows HIV-1 with a strong replication advantage in activated primary CD4+ T cells and established T cell lines. This effect is disabled by a Vpr mutation that abolishes binding to CRL4DCAF1 E3, thereby disrupting Vpr antagonism of helicase-like transcription factor (HLTF) DNA helicase and other DNA repair pathway targets, and by another mutation that prevents induction of the G2 DNA damage checkpoint. Consistent with these findings, we also show that HLTF restricts HIV-1 replication, and that this restriction is antagonized by HIV-1 Vpr. Furthermore, our data imply that HIV-1 Vpr uses additional, yet to be identified mechanisms to facilitate HIV-1 replication in T cells. Overall, we demonstrate that multiple aspects of the cellular DNA repair machinery restrict HIV-1 replication in dividing T cells, the primary target of HIV-1 infection, and describe newly developed approaches to dissect key components.

HIV-1 Vpr Reprograms CLR4DCAF1 E3 Ubiquitin Ligase to Antagonize Exonuclease 1-Mediated Restriction of HIV-1 Infection.

Viral accessory proteins hijack host cell E3 ubiquitin ligases to antagonize innate/intrinsic defenses and thereby provide a more permissive environment for virus replication. Human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr reprograms CRL4DCAF1 E3 to antagonize select postreplication DNA repair enzymes, but the significance and role of these Vpr interactions are poorly understood. To gain additional insights, we performed a focused screen for substrates of CRL4DCAF1 E3 reprogrammed by HIV-1 Vpr among known postreplication DNA repair proteins and identified exonuclease 1 (Exo1) as a novel direct HIV-1 Vpr target. We show that HIV-1 Vpr recruits Exo1 to the CRL4DCAF1 E3 complex for ubiquitination and subsequent proteasome-dependent degradation and that Exo1 levels are depleted in HIV-1-infected cells in a Vpr-dependent manner. We also show that Exo1 inhibits HIV-1 replication in T cells. Notably, the antagonism of Exo1 is a conserved function of main group HIV-1 and its ancestor Vpr proteins in the simian immunodeficiency virus from chimpanzee (SIVcpz) lineage, further underscoring the relevance of our findings. Overall, our studies (i) reveal that HIV-1 Vpr extensively remodels the cellular postreplication DNA repair machinery by impinging on multiple repair pathways, (ii) support a model in which Vpr promotes HIV-1 replication by antagonizing select DNA repair enzymes, and (iii) highlight the importance of a new class of restrictions placed on HIV-1 replication in T cells by the cellular DNA repair machinery.IMPORTANCE HIV-1 polymerase reverse transcribes the viral RNA genome into imperfectly double-stranded proviral DNA, containing gaps and flaps, for integration into the host cell chromosome. HIV-1 reverse transcripts share characteristics with cellular DNA replication intermediates and are thought to be converted into fully double-stranded DNA by cellular postreplication DNA repair enzymes. Therefore, the finding that the HIV-1 accessory protein Vpr antagonizes select postreplication DNA repair enzymes that can process HIV-1 reverse transcripts has been surprising. Here, we show that one such Vpr-antagonized enzyme, exonuclease 1, inhibits HIV-1 replication in T cells. We identify exonuclease 1 as a member of a new class of HIV-1 restriction factors in T cells and propose that certain modes of DNA "repair" inhibit HIV-1 infection.

HIV-1 Vpr protein directly loads helicase-like transcription factor (HLTF) onto the CRL4-DCAF1 E3 ubiquitin ligase.

The viral protein R (Vpr) is an accessory virulence factor of HIV-1 that facilitates infection in immune cells. Cellular functions of Vpr are tied to its interaction with DCAF1, a substrate receptor component of the CRL4 E3 ubiquitin ligase. Recent proteomic approaches suggested that Vpr degrades helicase-like transcription factor (HLTF) DNA helicase in a proteasome-dependent manner by redirecting the CRL4-DCAF1 E3 ligase. However, the precise molecular mechanism of Vpr-dependent HLTF depletion is not known. Here, using in vitro reconstitution assays, we show that Vpr mediates polyubiquitination of HLTF, by directly loading it onto the C-terminal WD40 domain of DCAF1 in complex with the CRL4 E3 ubiquitin ligase. Mutational analyses suggest that Vpr interacts with DNA-binding residues in the N-terminal HIRAN domain of HLTF in a manner similar to the recruitment of another target, uracil DNA glycosylase (UNG2), to the CRL4-DCAF1 E3 by Vpr. Strikingly, Vpr also engages a second, adjacent region, which connects the HIRAN and ATPase/helicase domains. Thus, our findings reveal that Vpr utilizes common as well as distinctive interfaces to recruit multiple postreplication DNA repair proteins to the CRL4-DCAF1 E3 ligase for ubiquitin-dependent proteasomal degradation.

HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins.

HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus, separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host.

The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes.

HIV-1 favors integration into active genes and gene-enriched regions of host cell chromosomes, thus maximizing the probability of provirus expression immediately after integration. This requires cleavage and polyadenylation specificity factor 6 (CPSF6), a cellular protein involved in pre-mRNA 3' end processing that binds HIV-1 capsid and connects HIV-1 preintegration complexes to intranuclear trafficking pathways that link integration to transcriptionally active chromatin. CPSF6 together with CPSF5 and CPSF7 are known subunits of the cleavage factor I (CFIm) 3' end processing complex; however, CPSF6 could participate in additional protein complexes. The molecular mechanisms underpinning the role of CPSF6 in HIV-1 infection remain to be defined. Here, we show that a majority of cellular CPSF6 is incorporated into the CFIm complex. HIV-1 capsid recruits CFIm in a CPSF6-dependent manner, which suggests that the CFIm complex mediates the known effects of CPSF6 in HIV-1 infection. To dissect the roles of CPSF6 and other CFIm complex subunits in HIV-1 infection, we analyzed virologic and integration site targeting properties of a CPSF6 variant with mutations that prevent its incorporation into CFIm We show, somewhat surprisingly, that CPSF6 incorporation into CFIm is not required for its ability to direct preferential HIV-1 integration into genes. The CPSF5 and CPSF7 subunits appear to have only a minor, if any, role in this process even though they appear to facilitate CPSF6 binding to capsid. Thus, CPSF6 alone controls the key molecular interactions that specify HIV-1 preintegration complex trafficking to active chromatin.

CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain.

SAMHD1 is a nuclear deoxyribonucleoside triphosphate triphosphohydrolase that contributes to the control of cellular deoxyribonucleoside triphosphate (dNTP) pool sizes through dNTP hydrolysis and modulates the innate immune response to viruses. CyclinA2-CDK1/2 phosphorylates SAMHD1 at Thr-592, but how this modification controls SAMHD1 functions in proliferating cells is not known. Here, we show that SAMHD1 levels remain relatively unchanged during the cell division cycle in primary human T lymphocytes and in monocytic cell lines. Inactivation of the bipartite cyclinA2-CDK-binding site in the SAMHD1 C terminus described herein abolished SAMHD1 phosphorylation on Thr-592 during S and G2 phases thus interfering with DNA replication and progression of cells through S phase. The effects exerted by Thr-592 phosphorylation-defective SAMHD1 mutants were associated with activation of DNA damage checkpoint and depletion of dNTP concentrations to levels lower than those seen upon expression of wild type SAMHD1 protein. These disruptive effects were relieved by either mutation of the catalytic residues of the SAMHD1 phosphohydrolase domain or by a Thr-592 phosphomimetic mutation, thus linking the Thr-592 phosphorylation state to the control of SAMHD1 dNTPase activity. Our findings support a model in which phosphorylation of Thr-592 by cyclinA2-CDK down-modulates, but does not inactivate, SAMHD1 dNTPase in S phase, thereby fine-tuning SAMHD1 control of dNTP levels during DNA replication.

Mechanism of allosteric activation of SAMHD1 by dGTP.

SAMHD1, a dNTP triphosphohydrolase (dNTPase), has a key role in human innate immunity. It inhibits infection of blood cells by retroviruses, including HIV, and prevents the development of the autoinflammatory Aicardi-Goutières syndrome (AGS). The inactive apo-SAMHD1 interconverts between monomers and dimers, and in the presence of dGTP the protein assembles into catalytically active tetramers. Here, we present the crystal structure of the human tetrameric SAMHD1-dGTP complex. The structure reveals an elegant allosteric mechanism of activation through dGTP-induced tetramerization of two inactive dimers. Binding of dGTP to four allosteric sites promotes tetramerization and induces a conformational change in the substrate-binding pocket to yield the catalytically active enzyme. Structure-based biochemical and cell-based biological assays confirmed the proposed mechanism. The SAMHD1 tetramer structure provides the basis for a mechanistic understanding of its function in HIV restriction and the pathogenesis of AGS.

Tetramerization of SAMHD1 is required for biological activity and inhibition of HIV infection.

SAMHD1 is a dGTP-activated dNTPase that has been implicated as a modulator of the innate immune response. In monocytes and their differentiated derivatives, as well as in quiescent cells, SAMHD1 strongly inhibits HIV-1 infection and, to a lesser extent, HIV-2 and simian immunodeficiency virus (SIV) because of their virion-associated virulence factor Vpx, which directs SAMHD1 for proteasomal degradation. Here, we used a combination of biochemical and virologic approaches to gain insights into the functional organization of human SAMHD1. We found that the catalytically active recombinant dNTPase is a dGTP-induced tetramer. Chemical cross-linking studies revealed SAMHD1 tetramers in human monocytic cells, in which it strongly restricts HIV-1 infection. The propensity of SAMHD1 to maintain the tetrameric state in vitro is regulated by its C terminus, located outside of the catalytic domain. Accordingly, we show that the C terminus is required for the full ability of SAMHD1 to deplete dNTP pools and to inhibit HIV-1 infection in U937 monocytes. Interestingly, the human SAMHD1 C terminus contains a docking site for HIV-2/SIVmac Vpx and is known to have evolved under positive selection. This evidence indicates that Vpx targets a functionally important element in SAMHD1. Together, our findings imply that SAMHD1 tetramers are the biologically active form of this dNTPase and provide new insights into the functional organization of SAMHD1.

HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1.

The sterile alpha motif and HD domain-containing protein-1 (SAMHD1) inhibits infection of myeloid cells by human and related primate immunodeficiency viruses (HIV and SIV). This potent inhibition is counteracted by the Vpx accessory virulence factor of HIV-2/SIVsm viruses, which targets SAMHD1 for proteasome-dependent degradation, by reprogramming cellular CRL4(DCAF1) E3 ubiquitin ligase. However, the precise mechanism of Vpx-dependent recruitment of human SAMHD1 onto the ligase, and the molecular interfaces on the respective molecules have not been defined. Here, we show that human SAMHD1 is recruited to the CRL4(DCAF1-Vpx) E3 ubiquitin ligase complex by interacting with the DCAF1 substrate receptor subunit in a Vpx-dependent manner. No stable association is detectable with DCAF1 alone. The SAMHD1 determinant for the interaction is a short peptide located distal to the SAMHD1 catalytic domain and requires the presence of Vpx for stable engagement. This peptide is sufficient to confer Vpx-dependent recruitment to CRL4(DCAF1) and ubiquitination when fused to heterologous proteins. The precise amino acid sequence of the peptide diverges among SAMHD1 proteins from different vertebrate species, explaining selective down-regulation of human SAMHD1 levels by Vpx. Critical amino acid residues of SAMHD1 and Vpx involved in the DCAF1-Vpx-SAMDH1 interaction were identified by mutagenesis. Our findings show that the N terminus of Vpx, bound to DCAF1, recruits SAMHD1 via its C terminus to CRL4, in a species-specific manner for proteasomal degradation.

Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.

Macrophages and dendritic cells have key roles in viral infections, providing virus reservoirs that frequently resist antiviral therapies and linking innate virus detection to antiviral adaptive immune responses. Human immunodeficiency virus 1 (HIV-1) fails to transduce dendritic cells and has a reduced ability to transduce macrophages, due to an as yet uncharacterized mechanism that inhibits infection by interfering with efficient synthesis of viral complementary DNA. In contrast, HIV-2 and related simian immunodeficiency viruses (SIVsm/mac) transduce myeloid cells efficiently owing to their virion-associated Vpx accessory proteins, which counteract the restrictive mechanism. Here we show that the inhibition of HIV-1 infection in macrophages involves the cellular SAM domain HD domain-containing protein 1 (SAMHD1). Vpx relieves the inhibition of lentivirus infection in macrophages by loading SAMHD1 onto the CRL4(DCAF1) E3 ubiquitin ligase, leading to highly efficient proteasome-dependent degradation of the protein. Mutations in SAMHD1 cause Aicardi-Goutières syndrome, a disease that produces a phenotype that mimics the effects of a congenital viral infection. Failure to dispose of endogenous nucleic acid debris in Aicardi-Goutières syndrome results in inappropriate triggering of innate immune responses via cytosolic nucleic acids sensors. Thus, our findings show that macrophages are defended from HIV-1 infection by a mechanism that prevents an unwanted interferon response triggered by self nucleic acids, and uncover an intricate relationship between innate immune mechanisms that control response to self and to retroviral pathogens.

A novel role of CCN3 in regulating endothelial inflammation.

The vascular endothelium plays a fundamental role in the health and disease of the cardiovascular system. The molecular mechanisms regulating endothelial homeostasis, however, remain incompletely understood. CCN3, a member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators, has been shown to play an important role in numerous cell types. The function of CCN3 in endothelial cells has yet to be elucidated. Immunohistochemical analysis of CCN3 expression in mouse tissues revealed robust immunoreactivity in the endothelium of large arteries, small resistance vessels, and veins. We found that CCN3 expression in human umbilical vein endothelial cells (HUVECs) is transcriptionally induced by laminar shear stress (LSS) and HMG CoA-reductase inhibitors (statins). Promoter analyses identified the transcription factor Kruppel-like factor 2 (KLF2) as a direct regulator of CCN3 expression. In contrast to LSS, proinflammatory cytokines reduced CCN3 expression. Adenoviral overexpression of CCN3 in HUVEC markedly inhibited the cytokine-mediated induction of vascular adhesion molecule-1 (VCAM-1). Consistent with this observation, CCN3 significantly reduced monocyte adhesion. Conversely, CCN3 knockdown in HUVECs resulted in enhancement of cytokine-induced VCAM-1 expression. Concordant effects were observed on monocyte adhesion. Gain and loss-of-function mechanistic studies demonstrated that CCN3 negatively regulates nuclear factor kappaB (NF-κB) activity by reducing its translocation into the nucleus and subsequent binding to the VCAM-1 promoter, suggesting that CCN3's anti-inflammatory effects occur secondary to inhibition of NF-κB nuclear accumulation. This study identifies CCN3 as a novel regulator of endothelial proinflammatory activation.

Klf15 deficiency is a molecular link between heart failure and aortic aneurysm formation.

Current therapies for diseases of heart muscle (cardiomyopathy) and aorta (aortopathy) include inhibitors of the renin-angiotensin system, beta-adrenergic antagonists, and the statin class of cholesterol-lowering agents. These therapies have limited efficacy, as adverse cardiovascular events continue to occur with some frequency in patients taking these drugs. Although cardiomyopathy and aortopathy can coexist in a number of conditions (for example, Marfan's syndrome, acromegaly, pregnancy, and aging), pathogenetic molecular links between the two diseases remain poorly understood. We reasoned that identification of common molecular perturbations in these two tissues could point to therapies for both conditions. Here, we show that deficiency of the transcriptional regulator Kruppel-like factor 15 (Klf15) in mice leads to both heart failure and aortic aneurysm formation through a shared molecular mechanism. Klf15 concentrations are markedly reduced in failing human hearts and in human aortic aneurysm tissues. Mice deficient in Klf15 develop heart failure and aortic aneurysms in a p53-dependent and p300 acetyltransferase-dependent fashion. KLF15 activation inhibits p300-mediated acetylation of p53. Conversely, Klf15 deficiency leads to hyperacetylation of p53 in the heart and aorta, a finding that is recapitulated in human tissues. Finally, Klf15-deficient mice are rescued by p53 deletion or p300 inhibition. These findings highlight a molecular perturbation common to the pathobiology of heart failure and aortic aneurysm formation and suggest that manipulation of KLF15 function may be a productive approach to treat these morbid diseases.

High-affinity insulin binding: insulin interacts with two receptor ligand binding sites.

The interaction of insulin with its receptor is complex. Kinetic and equilibrium binding studies suggest coexistence of high- and low-affinity binding sites or negative cooperativity. These phenomena and high-affinity interactions are dependent on the dimeric structure of the receptor. Structure-function studies of insulin analogs suggest insulin has two receptor binding sites, implying a bivalent interaction with the receptor. Alanine scanning studies of the secreted recombinant receptor implicate the L1 domain and a C-terminal peptide of the receptor alpha subunit as components of one ligand binding site. Functional studies suggest that the first and second type III fibronectin repeats of the receptor contain a second ligand binding site. We have used structure-directed alanine scanning mutagenesis to identify determinants in these domains involved in ligand interactions. cDNAs encoding alanine mutants of the holo-receptor were transiently expressed in 293 cells, and the binding properties of the expressed receptor were determined. Alanine mutations of Lys(484), Leu(552), Asp(591), Ile(602), Lys(616), Asp(620), and Pro(621) compromised affinities for insulin 2-5-fold. With the exception of Asp(620), none of these mutations compromised the affinity of the recombinant secreted receptor for insulin, indicating that the perturbation of the interaction is at the site of mutation and not an indirect effect on the interaction with the binding site of the secreted receptor. These residues thus form part of a novel ligand binding site of the insulin receptor. Complementation experiments demonstrate that insulin interacts in trans with both receptor binding sites to generate high-affinity interactions.