Huntingtin-Interacting Protein 1 Promotes Vpr-Induced G2 Arrest and HIV-1 Infection in Macrophages

Human immunodeficiency virus type 1 (HIV-1) modulates the host cell cycle. The HIV-1 accessory protein Vpr arrests the cell cycle at the G2 phase in dividing cells, and the ability of Vpr to induce G2 arrest is well conserved among primate lentiviruses. Additionally, Vpr-mediated G2 arrest likely correlates with enhanced HIV-1 infection in monocyte-derived macrophages. Here, we screened small-interfering RNA to reveal candidates that suppress Vpr-induced G2 arrest and identified Huntingtin-interacting protein 1 (HIP1) required for efficient G2 arrest. Interestingly, HIP1 was not essential for Vpr-induced DNA double-strand breaks, which are required for activation of the DNA-damage checkpoint and G2 arrest. Furthermore, HIP1 knockdown suppressed HIV-1 infection in monocyte-derived macrophages. This study identifies HIP1 as a factor promoting Vpr-induced G2 arrest and HIV-1 infection in macrophages.

Rescue of aberrant huntingtin palmitoylation ameliorates mutant huntingtin-induced toxicity

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion in the HTT gene that codes for an elongated polyglutamine tract in the huntingtin (HTT) protein. HTT is subject to multiple post-translational modifications (PTMs) that regulate its cellular function. Mutating specific PTM sites within mutant HTT (mHTT) in HD mouse models can modulate disease phenotypes, highlighting the key role of HTT PTMs in the pathogenesis of HD. These findings have led to increased interest in developing small molecules to modulate HTT PTMs in order to decrease mHTT toxicity. However, the therapeutic efficacy of pharmacological modulation of HTT PTMs in preclinical HD models remains largely unknown. HTT is palmitoylated at cysteine 214 by the huntingtin-interacting protein 14 (HIP14 or ZDHHC17) and 14-like (HIP14L or ZDHHC13) acyltransferases. Here, we assessed if HTT palmitoylation should be regarded as a therapeutic target to treat HD by (1) investigating palmitoylation dysregulation in rodent and human HD model systems, (2) measuring the impact of mHTT-lowering therapy on brain palmitoylation, and (3) evaluating if HTT palmitoylation can be pharmacologically modulate. We show that palmitoylation of mHTT and some HIP14/HIP14L-substrates is decreased early in multiple HD mouse models, and that aging further reduces HTT palmitoylation. Lowering mHTT in the brain of YAC128 mice is not sufficient to rescue aberrant palmitoylation. However, we demonstrate that mHTT palmitoylation can be normalized in COS-7 cells, in YAC128 cortico-striatal primary neurons and HD patient-derived lymphoblasts using an acyl-protein thioesterase (APT) inhibitor. Moreover, we show that modulating palmitoylation reduces mHTT aggregation and mHTT-induced cytotoxicity in COS-7 cells and YAC128 neurons.HighlightsPalmitoylation of mHTT is reduced in multiple transgenic HD mouse modelsHTT palmitoylation decreases with increasing polyQ length in HD patient cellsmHTT-lowering in mouse brains does not rescue aberrant palmitoylationmHTT palmitoylation in HD patient-derived cells can be rescued via APT inhibitionPromoting palmitoylation reduces mHTT aggregation and cytotoxicity in vitro

The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

The methyltransferase SET domain–containing 2 (SETD2) was originally identified as Huntingtin (HTT) yeast partner B. However, a SETD2 function associated with the HTT scaffolding protein has not been elucidated, and no linkage between HTT and methylation has yet been uncovered. Here, we show that SETD2 is an actin methyltransferase that trimethylates lysine-68 (ActK68me3) in cells via its interaction with HTT and the actin-binding adapter HIP1R. ActK68me3 localizes primarily to the insoluble F-actin cytoskeleton in cells and regulates actin polymerization/depolymerization dynamics. Disruption of the SETD2-HTT-HIP1R axis inhibits actin methylation, causes defects in actin polymerization, and impairs cell migration. Together, these data identify SETD2 as a previously unknown HTT effector regulating methylation and polymerization of actin filaments and provide new avenues for understanding how defects in SETD2 and HTT drive disease via aberrant cytoskeletal methylation.

Huntingtin-interacting Protein 1 Promotes Vpr-induced G2 Arrest and HIV-1 Infection in Macrophages

Background: Human immunodeficiency virus type 1 (HIV-1) modulates the host cell cycle. The HIV-1 accessory protein Vpr arrests the cell cycle at G2 phase, which is important for efficient viral replication in dividing CD4+ T cells, because the transcriptional activity of the HIV-1 long terminal repeat is most active in G2 phase. Additionally, Vpr-mediated G2 arrest likely correlates with enhanced HIV-1 infection in monocyte-derived macrophages.Results: Here, we screened small-interfering RNA to reveal candidates that suppress Vpr-induced G2 arrest and identified Huntingtin-interacting protein 1 (HIP1) as both directly interacting with Vpr and required for efficient G2 arrest. Interestingly, HIP1 was not essential for Vpr-induced DNA double-strand breaks, which are required for activation of the DNA-damage checkpoint and G2 arrest. Furthermore, HIP1 knockdown suppressed HIV-1 infection in monocyte-derived macrophages.Conclusions: These results suggest that HIP1 operates in the downstream step(s) of DNA-damage induction to promote Vpr-induced G2 arrest and enhances HIV-1 infection in macrophages.

Huntingtin-interacting protein family members have a conserved pro-viral function fromCaenorhabditis elegansto humans

Huntingtin-interacting protein family members are evolutionarily conserved from yeast to humans, and they are known to be key factors in clathrin-mediated endocytosis. Here we identified theCaenorhabditis elegansprotein huntingtin-interacting protein-related 1 (HIPR-1) as a host factor essential for Orsay virus infection ofC. elegans. Ablation of HIPR-1 resulted in a greater than 10,000-fold reduction in viral RNA, which could be rescued by ectopic expression of HIPR-1. Viral RNA replication from an endogenous transgene replicon system was not affected by lack of HIPR-1, suggesting that HIPR-1 plays a role during an early, prereplication virus life-cycle stage. Ectopic expression of HIPR-1 mutants demonstrated that neither the clathrin light chain-binding domain nor the clathrin heavy chain-binding motif were needed for virus infection, whereas the inositol phospholipid-binding and F-actin–binding domains were essential. In human cell culture, deletion of the human HIP orthologs HIP1 and HIP1R led to decreased infection by Coxsackie B3 virus. Finally, ectopic expression of a chimeric HIPR-1 harboring the human HIP1 ANTH (AP180 N-terminal homology) domain rescued Orsay infection inC. elegans, demonstrating conservation of its function through evolution. Collectively, these findings further our knowledge of cellular factors impacting viral infection inC. elegansand humans.

Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.

HYPK scaffolds the Nedd8 and LC3 proteins to initiate the formation of autophagosome around the poly-neddylated huntingtin exon1 aggregates

ABSTRACTSelective autophagy of protein aggregates is necessary for maintaining the cellular proteostasis. Several regulatory proteins play critical roles in this process. Here, we report that the huntingtin interacting protein K (HYPK) modulates the autophagic degradation of poly-neddylated huntingtin exon1 aggregates. HYPK functions as a scaffolding protein that binds to the Nedd8 and LC3 proteins. The C-terminal ubiquitin-associated (UBA) domain of HYPK binds to the Nedd8, whereas an N-terminal tyrosine-type (Y-type) LC3 interacting region (LIR) of HYPK binds to the LC3. Several conserved amino acids in the UBA domain of HYPK are necessary to mediate the efficient binding of HYPK to Nedd8. The autophagy inducing properties of HYPK are manifested by the increased lipidation of LC3 protein, increased expression of beclin-1 and ATG-5 proteins, and generation of puncta-like granules of LC3 in the HYPK overexpressing cells. Association of the ‘H-granules’ of HYPK with the poly-neddylated huntingtin exon1 aggregates results in the formation of autophagosome around the huntingtin exon1 aggregates, thereby clearing the aggregates by aggrephagy. Poly-neddylation of huntingtin exon1 is required for its autophagic degradation by HYPK. Thus, overexpression of Nedd8 also increases the basal level of cellular autophagy, other than maintaining the autophagy flux. The poly-neddylation dependent autophagic clearance of huntingtin exon1 by HYPK leads to better cell physiology and survival. Taken together, our study describes a novel mechanism of HYPK mediated autophagy of poly-neddylated huntingtin exon1 aggregates.

Deficiency of the Endocytic Protein Hip1 Leads to DecreasedGdpd3Expression, Low Phosphocholine, and Kypholordosis

ABSTRACTDeficiency of huntingtin-interacting protein 1 (Hip1) results in degenerative phenotypes. Here we generated aHip1deficiency allele where a floxed transcriptional stop cassette and a humanHIP1cDNA were knocked into intron 1 of the mouseHip1locus.CMV-Cre-mediated germ line excision of the stop cassette resulted in expression of HIP1 and rescue of theHip1knockout phenotype.Mx1-Cre-mediated excision led to HIP1 expression in spleen, kidney and liver, and also rescued the phenotype. In contrast,hGFAP-Cre-mediated, brain-specific HIP1 expression did not rescue the phenotype. Metabolomics and microarrays of severalHip1knockout tissues identified low phosphocholine (PC) levels and low glycerophosphodiester phosphodiesterase domain containing 3 (Gdpd3) gene expression. Since Gdpd3 has lysophospholipase D activity that results in the formation of choline, a precursor of PC,Gdpd3downregulation could lead to the low PC levels. To test whetherGdpd3contributes to theHip1deficiency phenotype, we generatedGdpd3knockout mice. Double knockout ofGdpd3andHip1worsened the Hip1 phenotype. This suggests that Gdpd3 compensates for Hip1 loss. More-detailed knowledge of howHip1deficiency leads to low PC will improve our understanding of HIP1 in choline metabolism in normal and disease states.