Structural analysis of the SAM domain of the Arabidopsis mitochondrial tRNA import receptor.

Mitochondria are membrane-bound organelles of endosymbiotic origin with limited protein-coding capacity. The import of nuclear-encoded proteins and nucleic acids is required and essential for maintaining organelle mass, number, and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAalavia conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with six monomers per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of the Tric1 SAM domain may play a role in protein function whereby mutation of Gly241 introducing a larger side chain at this position disrupted the oligomer and resulted in the loss of RNA binding capability. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype. AlphaFold2 structure prediction of both the SAM domain and Tric1 support a cyclic pentameric or hexameric structure. In the case of a hexameric structure, a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane is observed. Our results highlight the importance of oligomerization of Tric1 for protein function.

Molecular basis of PRC1 targeting to Polycomb response elements by PhoRC.

Polycomb group (PcG) protein complexes repress transcription by modifying target gene chromatin. In Drosophila, this repression requires association of PcG protein complexes with cis-regulatory Polycomb response elements (PREs), but the interactions permitting formation of these assemblies are poorly understood. We show that the Sfmbt subunit of the DNA-binding Pho-repressive complex (PhoRC) and the Scm subunit of the canonical Polycomb-repressive complex 1 (PRC1) directly bind each other through their SAM domains. The 1.9 Å crystal structure of the Scm-SAM:Sfmbt-SAM complex reveals the recognition mechanism and shows that Sfmbt-SAM lacks the polymerization capacity of the SAM domains of Scm and its PRC1 partner subunit, Ph. Functional analyses in Drosophila demonstrate that Sfmbt-SAM and Scm-SAM are essential for repression and that PhoRC DNA binding is critical to initiate PRC1 association with PREs. Together, this suggests that PRE-tethered Sfmbt-SAM nucleates PRC1 recruitment and that Scm-SAM/Ph-SAM-mediated polymerization then results in the formation of PRC1-compacted chromatin.

Cancer-Related Mutations in the Sam Domains of EphA2 Receptor and Ship2 Lipid Phosphatase: A Computational Study.

The lipid phosphatase Ship2 interacts with the EphA2 receptor by forming a heterotypic Sam (sterile alpha motif)-Sam complex. Ship2 works as a negative regulator of receptor endocytosis and consequent degradation, and anti-oncogenic effects in cancer cells should be induced by hindering its association with EphA2. Herein, a computational approach is presented to investigate the relationship between Ship2-Sam/EphA2-Sam interaction and cancer onset and further progression. A search was first conducted through the COSMIC (Catalogue of Somatic Mutations in Cancer) database to identify cancer-related missense mutations positioned inside or close to the EphA2-Sam and Ship2-Sam reciprocal binding interfaces. Next, potential differences in the chemical-physical properties of mutant and wild-type Sam domains were evaluated by bioinformatics tools based on analyses of primary sequences. Three-dimensional (3D) structural models of mutated EphA2-Sam and Ship2-Sam domains were built as well and deeply analysed with diverse computational instruments, including molecular dynamics, to classify potentially stabilizing and destabilizing mutations. In the end, the influence of mutations on the EphA2-Sam/Ship2-Sam interaction was studied through docking techniques. This in silico approach contributes to understanding, at the molecular level, the mutation/cancer relationship by predicting if amino acid substitutions could modulate EphA2 receptor endocytosis.

Sex comb on midleg (Scm) is a functional link between PcG-repressive complexes in Drosophila.

The Polycomb group (PcG) proteins are key regulators of development in Drosophila and are strongly implicated in human health and disease. How PcG complexes form repressive chromatin domains remains unclear. Using cross-linked affinity purifications of BioTAP-Polycomb (Pc) or BioTAP-Enhancer of zeste [E(z)], we captured all PcG-repressive complex 1 (PRC1) or PRC2 core components and Sex comb on midleg (Scm) as the only protein strongly enriched with both complexes. Although previously not linked to PRC2, we confirmed direct binding of Scm and PRC2 using recombinant protein expression and colocalization of Scm with PRC1, PRC2, and H3K27me3 in embryos and cultured cells using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing). Furthermore, we found that RNAi knockdown of Scm and overexpression of the dominant-negative Scm-SAM (sterile α motif) domain both affected the binding pattern of E(z) on polytene chromosomes. Aberrant localization of the Scm-SAM domain in long contiguous regions on polytene chromosomes revealed its independent ability to spread on chromatin, consistent with its previously described ability to oligomerize in vitro. Pull-downs of BioTAP-Scm captured PRC1 and PRC2 and additional repressive complexes, including PhoRC, LINT, and CtBP. We propose that Scm is a key mediator connecting PRC1, PRC2, and transcriptional silencing. Combined with previous structural and genetic analyses, our results strongly suggest that Scm coordinates PcG complexes and polymerizes to produce broad domains of PcG silencing.

Biophysical characterization of the ETV6 PNT domain polymerization interfaces.

ETV6 is an E26 transformation specific family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein-protein interaction. Herein, we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, along with amide hydrogen exchange measurements, we demonstrate that monomeric PNT domain variants adopt very stable helical bundle folds that do not change in conformation upon self-association into heterodimer models of the ETV6 polymer. Surface plasmon resonance-monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.

Hunting for Novel Routes in Anticancer Drug Discovery: Peptides against Sam-Sam Interactions.

Among the diverse protein binding modules, Sam (Sterile alpha motif) domains attract attention due to their versatility. They are present in different organisms and play many functions in physiological and pathological processes by binding multiple partners. The EphA2 receptor contains a Sam domain at the C-terminus (EphA2-Sam) that is able to engage protein regulators of receptor stability (including the lipid phosphatase Ship2 and the adaptor Odin). Ship2 and Odin are recruited by EphA2-Sam through heterotypic Sam-Sam interactions. Ship2 decreases EphA2 endocytosis and consequent degradation, producing chiefly pro-oncogenic outcomes in a cellular milieu. Odin, through its Sam domains, contributes to receptor stability by possibly interfering with ubiquitination. As EphA2 is upregulated in many types of tumors, peptide inhibitors of Sam-Sam interactions by hindering receptor stability could function as anticancer therapeutics. This review describes EphA2-Sam and its interactome from a structural and functional perspective. The diverse design strategies that have thus far been employed to obtain peptides targeting EphA2-mediated Sam-Sam interactions are summarized as well. The generated peptides represent good initial lead compounds, but surely many efforts need to be devoted in the close future to improve interaction affinities towards Sam domains and consequently validate their anticancer properties.

Viral protein X reduces the incorporation of mutagenic noncanonical rNTPs during lentivirus reverse transcription in macrophages.

Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain- and HD domain-containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase-mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for ∼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages.

DLC1 SAM domain-binding peptides inhibit cancer cell growth and migration by inactivating RhoA.

Deleted-in-liver cancer 1 (DLC1) exerts its tumor suppressive function mainly through the Rho-GTPase-activating protein (RhoGAP) domain. When activated, the domain promotes the hydrolysis of RhoA-GTP, leading to reduced cell migration. DLC1 is kept in an inactive state by an intramolecular interaction between its RhoGAP domain and the DLC1 sterile α motif (SAM) domain. We have shown previously that this autoinhibited state of DLC1 may be alleviated by tensin-3 (TNS3) or PTEN. We show here that the TNS3/PTEN-DLC1 interactions are mediated by the C2 domains of the former and the SAM domain of the latter. Intriguingly, the DLC1 SAM domain was capable of binding to specific peptide motifs within the C2 domains. Indeed, peptides containing the binding motifs were highly effective in blocking the C2-SAM domain-domain interaction. Importantly, when fused to the tat protein-transduction sequence and subsequently introduced into cells, the C2 peptides potently promoted the RhoGAP function in DLC1, leading to decreased RhoA activation and reduced tumor cell growth in soft agar and migration in response to growth factor stimulation. To facilitate the development of the C2 peptides as potential therapeutic agents, we created a cyclic version of the TNS3 C2 domain-derived peptide and showed that this peptide readily entered the MDA-MB-231 breast cancer cells and effectively inhibited their migration. Our work shows, for the first time, that the SAM domain is a peptide-binding module and establishes the framework on which to explore DLC1 SAM domain-binding peptides as potential therapeutic agents for cancer treatment.

The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing WT SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-α, IFN-ß, and TNF-α mRNA levels induced by Sendai virus infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in lipopolysaccharide-treated or Sendai virus-infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-ß mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1's nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.

The C-terminal domain of feline and bovine SAMHD1 proteins has a crucial role in lentiviral restriction.

SAM and HD domain-containing protein 1 (SAMHD1) is a host factor that restricts reverse transcription of lentiviruses such as HIV in myeloid cells and resting T cells through its dNTP triphosphohydrolase (dNTPase) activity. Lentiviruses counteract this restriction by expressing the accessory protein Vpx or Vpr, which targets SAMHD1 for proteasomal degradation. SAMHD1 is conserved among mammals, and the feline and bovine SAMHD1 proteins (fSAM and bSAM) restrict lentiviruses by reducing cellular dNTP concentrations. However, the functional regions of fSAM and bSAM that are required for their biological functions are not well-characterized. Here, to establish alternative models to investigate SAMHD1 in vivo, we studied the restriction profile of fSAM and bSAM against different primate lentiviruses. We found that both fSAM and bSAM strongly restrict primate lentiviruses and that Vpx induces the proteasomal degradation of both fSAM and bSAM. Further investigation identified one and five amino acid sites in the C-terminal domain (CTD) of fSAM and bSAM, respectively, that are required for Vpx-mediated degradation. We also found that the CTD of bSAM is directly involved in mediating bSAM's antiviral activity by regulating dNTPase activity, whereas the CTD of fSAM is not. Our results suggest that the CTDs of fSAM and bSAM have important roles in their antiviral functions. These findings advance our understanding of the mechanism of fSAM- and bSAM-mediated viral restriction and might inform strategies for improving HIV animal models.

Loss of a highly conserved sterile alpha motif domain gene (WEEP) results in pendulous branch growth in peach trees.

Plant shoots typically grow upward in opposition to the pull of gravity. However, exceptions exist throughout the plant kingdom. Most conspicuous are trees with weeping or pendulous branches. While such trees have long been cultivated and appreciated for their ornamental value, the molecular basis behind the weeping habit is not known. Here, we characterized a weeping tree phenotype in Prunus persica (peach) and identified the underlying genetic mutation using a genomic sequencing approach. Weeping peach tree shoots exhibited a downward elliptical growth pattern and did not exhibit an upward bending in response to 90° reorientation. The causative allele was found to be an uncharacterized gene, Ppa013325, having a 1.8-Kb deletion spanning the 5' end. This gene, dubbed WEEP, was predominantly expressed in phloem tissues and encodes a highly conserved 129-amino acid protein containing a sterile alpha motif (SAM) domain. Silencing WEEP in the related tree species Prunus domestica (plum) resulted in more outward, downward, and wandering shoot orientations compared to standard trees, supporting a role for WEEP in directing lateral shoot growth in trees. This previously unknown regulator of branch orientation, which may also be a regulator of gravity perception or response, provides insights into our understanding of how tree branches grow in opposition to gravity and could serve as a critical target for manipulating tree architecture for improved tree shape in agricultural and horticulture applications.

Calcium influx mediates the chemoattractant-induced translocation of the arrestin-related protein AdcC in Dictyostelium.

Arrestins are key adaptor proteins that control the fate of cell-surface membrane proteins and modulate downstream signaling cascades. The Dictyostelium discoideum genome encodes six arrestin-related proteins, harboring additional modules besides the arrestin domain. Here, we studied AdcB and AdcC, two homologs that contain C2 and SAM domains. We showed that AdcC - in contrast to AdcB - responds to various stimuli (such as the chemoattractants cAMP and folate) known to induce an increase in cytosolic calcium by transiently translocating to the plasma membrane, and that calcium is a direct regulator of AdcC localization. This response requires the calcium-dependent membrane-targeting C2 domain and the double SAM domain involved in AdcC oligomerization, revealing a mode of membrane targeting and regulation unique among members of the arrestin clan. AdcB shares several biochemical properties with AdcC, including in vitro binding to anionic lipids in a calcium-dependent manner and auto-assembly as large homo-oligomers. AdcB can interact with AdcC; however, its intracellular localization is insensitive to calcium. Therefore, despite their high degree of homology and common characteristics, AdcB and AdcC are likely to fulfill distinct functions in amoebae.

Ankyloblepharon-ectodermal Defects-cleft Lip-palate Syndrome Due to a Novel Missense Mutation in the SAM Domain of the TP63 Gene.

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome is a rare genetic disease with an autosomal dominant transmission, characterized by several congenital anomalies. Clinical features include ectodermal defects affecting the skin, hair, teeth, nails and sweat glands, associated with typical eyelid fusion in addition to a cleft lip and/or palate. The diagnosis is based on clinical criteria and molecular genetic testing of TP63 gene, the gene related to AEC syndrome. In this context, most reported mutations induce an amino acid change in the sterile alpha motif (SAM) domain, and are predicted to disrupt protein-protein interactions. We here describe the case of a 2-year-old Moroccan girl diagnosed with AEC syndrome on the basis of clinical features. The molecular studies and bioinformatics tools revealed a novel heterozygous missense mutation c.1798G>C (p.Gly600Arg) in exon 14 of the TP63 gene, that was not found in her parents. The molecular analysis and the early diagnosis of this syndrome are important to offer appropriate genetic counseling and management to patients and their families.

Interplay of ancestral non-primate lentiviruses with the virus-restricting SAMHD1 proteins of their hosts.

Lentiviruses infect both dividing CD4+ T cells and nondividing myeloid cells, and the infected myeloid cells serve as long-living viral reservoirs. Host sterile alpha motif- and histidine-aspartate domain-containing protein 1 (SAMHD1) kinetically restricts reverse transcription of primate lentiviruses, including human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus (SIV), in nondividing myeloid cells. SAMHD1 enforces this restriction through its dNTP triphosphohydrolase (dNTPase) activity that depletes cellular dNTPs. Some primate lentiviruses, such as HIV-2, SIVsm, and SIVagm, counteract SAMHD1 restriction by using their viral accessory proteins (Vpx or Vpr) that induce the proteosomal degradation of SAMHD1 and increase dNTP levels. SAMHD1 is conserved among non-primate mammals such as cats, cows, and horses that also carry their own lentiviruses (feline and bovine immunodeficiency viruses and equine infectious anemia viruses, respectively). However, whether these viruses also target SAMHD1 is unknown. Here, we tested whether these ancestral non-primate lentiviruses also can counteract their host SAMHD1 proteins by promoting their proteosomal degradation. Using biochemical and various cell-based assays, we observed that SAMHD1 proteins from the non-primate host species display dGTP-dependent dNTPase activity, but that the non-primate lentiviruses fail to proteosomally degrade the SAMHD1 proteins of their hosts. Our findings suggest that accessory protein-mediated proteosomal degradation of SAMHD1 did not exist among the ancestral non-primate lentiviruses and was uniquely gained by some primate lentiviruses after their transmission to primate species.

Complex formation of sphingomyelin synthase 1 with glucosylceramide synthase increases sphingomyelin and decreases glucosylceramide levels.

Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer). Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) are key enzymes that catalyze the conversion of Cer to SM and GlcCer, respectively. GlcCer synthesis has been postulated to occur mainly in cis-Golgi, and SM synthesis is thought to occur in medial/trans-Golgi; however, SMS1 and GCS are known to partially co-localize in cisternae, especially in medial/trans-Golgi. Here, we report that SMS1 and GCS can form a heteromeric complex, in which the N terminus of SMS1 and the C terminus of GCS are in close proximity. Deletion of the N-terminal sterile α-motif of SMS1 reduced the stability of the SMS1-GCS complex, resulting in a significant reduction in SM synthesis in vivo In contrast, chemical-induced heterodimerization augmented SMS1 activity, depending on an increase in the amount and stability of the complex. Fusion of the SMS1 N terminus to the GCS C terminus via linkers of different lengths increased SM synthesis and decreased GlcCer synthesis in vivo These results suggest that formation of the SMS1-GCS heteromeric complex increases SM synthesis and decreases GlcCer synthesis. Importantly, this regulation of relative Cer levels by the SMS1-GCS complex was confirmed by CRISPR/Cas9-mediated knockout of SMS1 or GCS combined with pharmacological inhibition of Cer transport protein in HEK293T cells. Our findings suggest that complex formation between SMS1 and GCS is part of a critical mechanism controlling the metabolic fate of Cer in the Golgi.

The SAM domain inhibits EphA2 interactions in the plasma membrane.

All members of the Eph receptor family of tyrosine kinases contain a SAM domain near the C terminus, which has been proposed to play a role in receptor homotypic interactions and/or interactions with binding partners. The SAM domain of EphA2 is known to be important for receptor function, but its contribution to EphA2 lateral interactions in the plasma membrane has not been determined. Here we use a FRET-based approach to directly measure the effect of the SAM domain on the stability of EphA2 dimers on the cell surface in the absence of ligand binding. We also investigate the functional consequences of EphA2 SAM domain deletion. Surprisingly, we find that the EphA2 SAM domain inhibits receptor dimerization and decreases EphA2 tyrosine phosphorylation. This role is dramatically different from the role of the SAM domain of the related EphA3 receptor, which we previously found to stabilize EphA3 dimers and increase EphA3 tyrosine phosphorylation in cells in the absence of ligand. Thus, the EphA2 SAM domain likely contributes to a unique mode of EphA2 interaction that leads to distinct signaling outputs.

Interferons Induce Expression of SAMHD1 in Monocytes through Down-regulation of miR-181a and miR-30a.

SAMHD1 is a phosphohydrolase maintaining cellular dNTP homeostasis but also acts as a critical regulator in innate immune responses due to its antiviral activity and association with autoimmune disease, leading to aberrant activation of interferon. SAMHD1 expression is differentially regulated by interferon in certain primary cells, but the underlying mechanism is not understood. Here, we report a detailed characterization of the promotor region, the 5'- and 3'-untranslated region (UTR) of SAMHD1, and the mechanism responsible for the cell type-dependent up-regulation of SAMHD1 protein by interferon. We demonstrate that induction of SAMHD1 by type I and II interferons depends on 3'-UTR post-transcriptional regulation, whereas the promoter drives basal expression levels. We reveal novel functional target sites for the microRNAs miR-181a, miR-30a, and miR-155 in the SAMHD1 3'-UTR. Furthermore, we demonstrate that down-regulation of endogenous miR-181a and miR-30a levels inversely correlates with SAMHD1 protein up-regulation upon type I and II interferon stimulation in primary human monocytes. These miRNAs are not modulated by interferon in macrophages or dendritic cells, and consequently protein levels of SAMHD1 remain unchanged. These results suggest that SAMHD1 is a non-classical interferon-stimulated gene regulated through cell type-dependent down-regulation of miR-181a and miR-30a in innate sentinel cells.

A Highly Active Isoform of Lentivirus Restriction Factor SAMHD1 in Mouse.

The triphosphohydrolase SAMHD1 (sterile α motif and histidine-aspartate domain-containing protein 1) restricts HIV-1 replication in nondividing myeloid cells by depleting the dNTP pool, preventing reverse transcription. SAMHD1 is also reported to have ribonuclease activity that degrades the virus genomic RNA. Human SAMHD1 is regulated by phosphorylation of its carboxyl terminus at Thr-592, which abrogates its antiviral function yet has only a small effect on its phosphohydrolase activity. In the mouse, SAMHD1 is expressed as two isoforms (ISF1 and ISF2) that differ at the carboxyl terminus due to alternative splicing of the last coding exon. In this study we characterized the biochemical and antiviral properties of the two mouse isoforms of SAMHD1. Both are antiviral in nondividing cells. Mass spectrometry analysis showed that SAMHD1 is phosphorylated at several amino acid residues, one of which (Thr-634) is homologous to Thr-592. Phosphomimetic mutation at Thr-634 of ISF1 ablates its antiviral activity yet has little effect on phosphohydrolase activity in vitro dGTP caused ISF1 to tetramerize, activating its catalytic activity. In contrast, ISF2, which lacks the phosphorylation site, was significantly more active, tetramerized, and was active without added dGTP. Neither isoform nor human SAMHD1 had detectable RNase activity in vitro or affected HIV-1 genomic RNA stability in newly infected cells. These data support a model in which SAMHD1 catalytic activity is regulated through tetramer stabilization by the carboxyl-terminal tail, phosphorylation destabilizing the complexes and inactivating the enzyme. ISF2 may serve to reduce the dNTP pool to very low levels as a means of restricting virus replication.

Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.

BACKGROUND: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. METHODS: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat. RESULTS: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1ß, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6. CONCLUSIONS: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching.

Single-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Co-immunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of HeLa cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER.