Sterile Alpha Motif Domain Research Articles

NAD+ cleavage activity by animal and plant TIR domains in cell death pathways.

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

Conserved Herpesvirus Protein Kinases Target SAMHD1 to Facilitate Virus Replication

SUMMARYTo ensure a successful infection, herpesviruses have developed elegant strategies to counterbalance the host anti-viral responses. Sterile alpha motif and HD domain 1 (SAMHD1) was recently identified as an intrinsic restriction factor for a variety of viruses. Aside from HIV-2 and the related simian immunodeficiency virus (SIV) Vpx proteins, the direct viral countermeasures against SAMHD1 restriction remain unknown. Using Epstein-Barr virus (EBV) as a primary model, we discover that SAMHD1-mediated anti-viral restriction is antagonized by EBV BGLF4, a member of the conserved viral protein kinases encoded by all herpesviruses. Mechanistically, we find that BGLF4 phosphorylates SAMHD1 and thereby inhibits its deoxynucleotide triphosphate triphosphohydrolase (dNTPase) activity. We further demonstrate that the targeting of SAMHD1 for phosphorylation is a common feature shared by beta- and gamma-herpesviruses. Together, our findings uncover an immune evasion mechanism whereby herpesviruses exploit the phosphorylation of SAMHD1 to thwart host defenses.

DprA Is Essential for Natural Competence in Riemerella anatipestifer and Has a Conserved Evolutionary Mechanism.

Riemerella anatipestifer ATCC11845 (RA ATCC11845) is naturally competent. However, the genes involved in natural transformation in this species remain largely unknown. Bioinformatic analysis predicts that DprA of RA (DprARa) has three domains: a sterile alpha motif (SAM), a Rossmann fold (RF) domain and a Z-DNA-binding domain (Zα). Inactivation of dprA abrogated natural transformation in RA ATCC11845, and this effect was restored by the expression of dprA in trans. The dprA with SAM and RF domains of Streptococcus pneumoniae and the dprA with RF and Zα domains of Helicobacter pylori was able to restore natural transformation in the RA ATCC11845 dprA mutant. An Arg123 mutation in the RF domain of R. anatipestifer was not able to restore natural transformation of the RA ATCC11845 dprA mutant. Furthermore, DprAR123E abolished its ability to bind DNA, suggesting that the RF domain is essential for the function of DprA. Finally, the dprA of Fusobacterium naviforme which has not been reported to be natural competent currently was partially able to restore natural transformation in RA ATCC11845 dprA mutant. These results collectively suggest that DprA has a conserved evolutionary mechanism.

Molecular dynamics investigation of a redox switch in the anti-HIV protein SAMHD1.

HIV-1 is restricted in macrophages and certain quiescent myeloid cells due to a "Scorched Earth" dNTP starvation strategy attributed to the sterile alpha motif and HD domain protein-SAMHD1. Active SAMHD1 tetramers are assembled by GTP-Mg+2-dNTP cross bridges and cleave the triphosphate groups of dNTPs at a K m of ~10 μM, which is consistent with dNTP concentrations in cycling cells, but far higher than the equivalent concentration in quiescent cells. Given the substantial disparity between the dNTP concentrations required to activate SAMHD1 tetramers (~10 μM) and the dNTP concentrations in noncycling cells (~10 nM), the possibility of alternate enzymatically active forms of SAMHD1, including monomers remains open. In particular, the possibility of redox regulation of such monomers is also an open question. There have been experimental studies on the regulation of SAMHD1 by Glutathione driven redox reactions recently. Therefore, in this work, we have performed all-atom molecular dynamics simulations to study the dynamics of monomeric SAMHD1 constructs in the context of the three redox-susceptible Cysteine residues and compared them to monomers assembled within a tetramer. Our results indicate that assembly into a tetramer causes ordering of the catalytic core and increased solvent accessibility of the Catalytic Site. We have also found that glutathionylation of surface exposed C522 causes long range allosteric disruptions extending into the protein core. Finally, we see evidence suggesting a transient interaction between C522 and C341. Such a disulfide linkage has been hypothesized by experimental models, but has never been observed in crystal structures before.

SUN-268 Severe Infantile Insulin Resistance: A Novel Feature of MIRAGE Syndrome

Background MIRAGE syndrome is a rare multisystem disorder characterized by myelodysplasia, infection, growth restriction, adrenal hypoplasia, genital phenotypes, and enteropathy. The syndrome is associated with mutations in Sterile Alpha Motif Domain Containing 9 (SAMD9) gene, encoding an endosome fusion facilitator protein with additional function in growth factor signaling. To date, there are no reported cases of MIRAGE syndrome associated with severe insulin resistance in infancy. Clinical Case The patient was a premature female infant born at 30 6/7 weeks gestation with a history of IUGR and brain abnormalities. Her hospital course was complicated by respiratory failure, impaired immune function, chronic diarrhea due to pancreatic exocrine deficiency, anemia, and primary adrenal insufficiency. Whole exome sequencing revealed a novel, de novo missense mutation in SAMD9 (R1293Q), confirming the clinical diagnosis of MIRAGE syndrome. At 5 months of age, she developed severe hyperglycemia with insulin resistance (insulin 303 µIU/mL, normal < 13 µIU/mL, and C-peptide 18.2 ng/mL, normal < 2.2 ng/mL). Markers of insulin receptor defects were normal, including adiponectin, IGFBP-1, and SHBG, and no mutations were identified in INSR by whole exome sequencing. The patient had no episodes of diabetic ketosis or metabolic acidosis, and had no lipodystrophy or acanthosis nigricans on exam. Initially, an insulin infusion of 0.1-0.2 U/kg/hr was sufficient to maintain euglycemia. By age 6 months, persistent hyperglycemia led to a rapid increase of the insulin infusion to 18 U/kg/hr over the course of 3 days. On day 2 of insulin up titration, glucophage was started at 15 mg/kg/day. After 72 hours of escalating insulin doses and 48 hours of glucophage, both her hyperglycemia and insulin resistance rapidly improved, with insulin requirements decreasing to < 0.5 U/kg/hr over the next 2 days to maintain glucose in the 100-200 mg/dL range. In the following months, her insulin requirement varied greatly. While fasting, no insulin was needed to maintain euglycemia. However, while on parental nutrition or enteral feeds, insulin infusion requirement varied at 0.03 U/kg/hr - 0.5 U/kg/hr. At 8 months, she transitioned to subcutaneous insulin while enteral feeds were optimized and condensed. Subcutaneous insulin doses were gradually increased to 20-24 U/kg/day in an effort to maintain blood glucose <200 mg/dl. Unfortunately, at 9 months of age, the patient expired from sepsis. Conclusion This is the first case of severe insulin resistance presenting as a manifestation of MIRAGE syndrome due to a missense mutation in SAMD9. This patient had improvement in glycemic control after treatment with glucophage and insulin. SAMD9 mutations should be considered in patients with multiple endocrinopathies including adrenal hypoplasia, severe insulin resistance, and pancreatic exocrine deficiency.

The C-terminal SAM domain of p73 binds to the N terminus of MDM2

BackgroundThe p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing key roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); this domain, SAMp73, formed by five helices (α1 to α5), is thought to mediate in protein-protein interactions. The E3-ligase MDM2 binds to p73 at its N terminus transactivation domain (TA), but it does not promote its degradation via ubiquitination; however, the details of such MDM2/p73 interaction are not fully known. MethodsWe studied the binding of SAMp73 with N-terminal MDM2, by several biophysical techniques, namely, fluorescence, far-UV circular dichroism (CD), NMR and bio-layer interferometry (BLI). ResultsOur results obtained by fluorescence, T2-relaxation measurements and BLI show that there was binding between both proteins with a dissociation constant of ~10 μM. Furthermore, the binding region of SAMp73 involved mainly residues in the major α-helix, α5, and the nearby α4, as shown by HSQC-NMR. The binding was so specific that an isolated peptide comprising α4 and α5 helices of SAMp73, α4α5, did also bind to the N terminus of MDM2, although with weaker affinity than the entire domain. ConclusionsA new interaction between MDM2 and SAMp73 has been found, which could have potential therapeutic applications in cancers involving inactivated p53. General significanceA novel interaction between the C-terminal SAM of p73 and N-terminal MDM2 is described. The interaction could be used to modulate the functions where the wild-type, intact p73 is involved.

Multiple Autoimmune Disorders in Aicardi-Goutières Syndrome

BackgroundAicardi-Goutières syndrome is an early-onset encephalopathy with presumed immune pathogenesis caused by inherited defects in nucleic acid metabolism. It is a model disease to study systemic autoimmunity, and there are many clinical, genetic, and basic science considerations that underline a possible overlap between Aicardi-Goutières syndrome and systemic lupus erythematosus. ResultsWe describe a 15-year-old girl with Aicardi-Goutières syndrome due to compound heterozygous pathogenic variants in SAMHD1 (sterile alpha motif domain and HD domain-containing protein 1). Over time, she developed multiple autoimmune diseases (vitiligo, alopecia areata, immune thrombocytopenia, positive antithyroglobulin antibodies) without positive antinuclear antibody or features of systemic lupus erythematosus. Her thrombocytopenia was refractory to treatment with corticosteroids and intravenous immunoglobulin but responded to a standard course of rituximab. ConclusionThis is the first report of a multiple autoimmune syndrome in a patient with molecularly proven Aicardi-Goutières syndrome. This study illustrates an emerging pattern of the natural history of Aicardi-Goutières syndrome characterized by early encephalopathic presentation followed by symptoms of systemic autoimmunity.

Structure of the SLy1 SAM homodimer reveals a new interface for SAM domain self-association

Sterile alpha motif (SAM) domains are protein interaction modules that are involved in a diverse range of biological functions such as transcriptional and translational regulation, cellular signalling, and regulation of developmental processes. SH3 domain-containing protein expressed in lymphocytes 1 (SLy1) is involved in immune regulation and contains a SAM domain of unknown function. In this report, the structure of the SLy1 SAM domain was solved and revealed that this SAM domain forms a symmetric homodimer through a novel interface. The interface consists primarily of the two long C-terminal helices, α5 and α5′, of the domains packing against each other. The dimerization is characterized by a dissociation constant in the lower micromolar range. A SLy1 SAM domain construct with an extended N-terminus containing five additional amino acids of the SLy1 sequence further increases the stability of the homodimer, making the SLy1 SAM dimer two orders of magnitude more stable than previously studied SAM homodimers, suggesting that the SLy1 SAM dimerization is of functional significance. The SLy1 SAM homodimer contains an exposed mid-loop surface on each monomer, which may provide a scaffold for mediating interactions with other SAM domain-containing proteins via a typical mid-loop–end-helix interface.

Genome-wide association study of degenerative mitral valve disease in Maltese dogs.

Genome-wide association study (GWAS) is a powerful tool for identifying the genetic causes of various diseases. This study was conducted to identify genomic variation in Maltese dog genomes associated with degenerative mitral valve disease (DMVD) development and to evaluate the association of each biological condition with DMVD in Maltese dogs. DNA was extracted from blood samples obtained from 48 Maltese dogs (32 with DMVD and 16 controls). Genome-wide single nucleotide polymorphism (SNP) genotyping was performed. The top 30 SNPs from each association of various conditions and genetic variations were mapped to their gene locations. A total of 173,662 loci were successfully genotyped, with an overall genotype completion rate of 99.41%. Quality control analysis excluded 46,610 of these SNPs. Manhattan plots were produced using allelic tests with various candidate clinical conditions. A significant peak of association was observed between mitral valve prolapse (MVP) and SNPs on chromosome 17. The present study revealed significant SNPs in several genes associated with cardiac function, including PDZ2, Armadillo repeat protein detected in velo-cardio-facial syndrome, catenin (cadherin-associated protein) alpha 3, low-density lipoprotein receptor class A domain containing protein 4, and sterile alpha motif domain containing protein 3. To our knowledge, this is the first study of a genetic predisposition to DMVD in Maltese dogs. Although only a limited number of cases were analyzed, these data could be the basis for further research on the genetic predisposition to MVP and DMVD in Maltese dogs.

Silencing PsKPP4, a MAP kinase kinase kinase gene, reduces pathogenicity of the stripe rust fungus.

Many obligately parasitic pathogens absorb nutrients from host plants via specialized infection structures, called haustoria and infection hyphae, to further colonization and growth in the host plant. In the wheat (Triticum aestivum) stripe rust fungus, Puccinia striiformis f. sp. tritici (Pst), the mitogen-activated protein kinase kinase (MAPKK) PsFUZ7 is involved in the regulation of haustorium formation and invasive growth. Here, we functionally characterized PsKPP4 of Pst, which is homologous to the yeast MAPKKK STE11. Similar to the silencing of PsFUZ7, the knockdown of PsKPP4 was detected in the vegetative hyphae and haustoria, resulting in the reduced pathogenicity of Pst. Pst urediniospores treated with the STE11 MAPKKK activation inhibitor produced deformed germ tubes. In addition, overexpression of PsKPP4 in fission yeast resulted in the production of fusiform cells and increased tolerance of yeast cells to oxidative stress. The transformation of PsKPP4 into the mst11 mutant of Magnaporthe oryzae partially restored mst11 function. The PsKPP4 protein contains a sterile alpha motif (SAM), Ras association (RA) and kinase domains, similar to its homologues in other fungi. Yeast two-hybrid assays revealed that the SAM domain is essential for the interaction between PsKPP4 and PsUBC2, a homologue of Ustilago maydis UBC2, known to interact with KPP4, which is associated with the regulation of the Fus3 cascade. Host-induced gene silencing of PsUBC2 reduced the pathogenicity of Pst slightly, indicating that PsUBC2 also plays a minor role in the regulation of the infection pathway of Pst. These observations indicate that PsKPP4, interacting with PsUBC2, may play an important role in the regulation of infection-related morphogenesis in Pst.

Regulation of tankyrase activity by a catalytic domain dimer interface

Tankyrases (TNKS and TNKS2) are enzymes that catalyze poly-ADP-ribosylation (PARsylation) of their target proteins. Tankyrase-mediated PARsylation plays critical regulatory roles in cell signaling, particularly in the Wnt/β-catenin pathway. The sterile alpha motif (SAM) domain in tankyrases mediates their oligomerization, which is essential for tankyrase function. The oligomerization regulates the catalytic activity of tankyrases, but the underlying mechanism is unclear. Our analyses of crystal structures of the tankyrase catalytic domain suggest that formation of a head-to-head dimer regulates the catalytic activity. Our activity assays show that residues in the catalytic domain dimer interface are important for the PARsylation activity of tankyrases both in solution and cells. The dimer is weak and may only form in the context of the SAM domain-mediated oligomers of tankyrases, consistent with the dependence of the tankyrase activity on the SAM domain.

Sam domain-based stapled peptides: Structural analysis and interaction studies with the Sam domains from the EphA2 receptor and the lipid phosphatase Ship2

Sam (Sterile alpha motif) domains represent small helical protein-protein interaction modules which play versatile functions in different cellular processes. The Sam domain from the EphA2 receptor binds the Sam domain of the lipid phosphatase Ship2 and this interaction modulates receptor endocytosis and degradation primarily generating pro-oncogenic effects in cell. To identify molecule antagonists of the EphA2-Sam/Ship2-Sam complex with anti-cancer activity, we focused on hydrocarbon helical stapled peptides. EphA2-Sam and one of its interactors (i.e., the first Sam domain of the adaptor protein Odin) were used as model systems for peptide design. Increase in helicity in the stapled peptides, with respect to the corresponding linear/native-like regions, was proved by structural studies conducted through CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance). Interestingly, interaction assays by means of NMR, SPR (Surface Plasmon Resonance) and MST (MicroScale Thermophoresis) techniques led to the discovery of a novel ligand of Ship2-Sam.

Abstract 5522: Cancer-associated point mutant in the START domain of DLC1 encodes a protein attenuated for tumor suppressor activity and for binding both Caveolin-1 and PLCD1 but not for Rho-GAP activity

Abstract The tumor suppressor gene DLC1 encodes a multi-domain protein, including a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, RhoB, and RhoC, and has been hypothesized to be the basis of its tumor suppressor functions. Other well recognized DLC1 domains are an N-terminal sterile alpha motif (SAM) domain, and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. DLC1 is down-regulated in several malignancies by gene deletion, epigenetic promoter methylation, or post-translational mechanisms. In addition, we have recently determined, by analyzing the TCGA database that point mutations in the DLC1 gene occur frequently, and have provided evidence that most of the mutant proteins display attenuated tumor suppressor function. Moreover, the mutations are distributed along the entire coding region, implying that regions of DLC1 in addition to its RhoGAP domain contribute to its full tumor suppressor activity. Several DLC1 binding partners, in addition to Rho-GTP, have been identified. They include Caveolin-1, the principal structural component and marker of caveolae, which interacts with the START domain of DLC1. The distribution of caveolae is closely related to intracellular events, such as MAPK activation or the control of intracellular Ca2+ levels. Phospholipase C delta 1 (PLCD1) transforms PtdIns(4,5)P2 (PIP2) into inositol triphosphate (IP3) and diacyglycerol (DAG), which participate in intracellular Ca2+ mobilization and protein kinase C activation, respectively. p122RhoGAP, the rat homolog of DLC1, was initially cloned as a novel PLCD1-interacting protein, but the interacting region of PLCD1 with p122RhoGAP was not identified. In addition, others have concluded the function of PLCD1 is not affected by human DLC1. In the current study, we have characterized a colon cancer-associated DLC1 mutant (R947C) whose mutation lies in the START domain. The mutant has an attenuated tumor suppressor phenotype, as determined by deficiency in reduction of growth in soft agar and cell migration. Compared with the wild type, the binding of the mutant to PLCD1 and to Caveolin-1 is reduced, but its RhoGAP activity is intact. In addition, phosphorylation of Caveolin-1 by Src at Tyr 14 reduces its interaction with wild type DLC1, and Caveolin-1 and PLCD1 interact in vivo more efficiently with each other in the absence of DLC1. Moreover, wild type DLC1, Caveolin-1 and PLCD1 possess cooperative tumor suppressor activities in cells expressing all three genes. Overall, this study provides new insight into the role of the START domain of DLC1, and reinforces the Rho-GAP-independent regulation of tumor growth by DLC1. Citation Format: Beatriz Sanchez-Solana, Xiaolan Qian, Dunrui Wang, Marian Durkin, Brajendra Tripathi, Alex Papageorge, Douglas R. Lowy. Cancer-associated point mutant in the START domain of DLC1 encodes a protein attenuated for tumor suppressor activity and for binding both Caveolin-1 and PLCD1 but not for Rho-GAP activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5522.

Circular RNA CpG island hypermethylation-associated silencing in human cancer.

Noncoding RNAs (ncRNAs), such as microRNAs and long noncoding RNAs (lncRNAs), participate in cellular transformation. Work done in the last decade has also demonstrated that ncRNAs with growth-inhibitory functions can undergo promoter CpG island hypermethylation-associated silencing in tumorigenesis. Herein, we wondered whether circular RNAs (circRNAs), a type of RNA transcripts lacking 5′-3′ ends and forming closed loops that are gaining relevance in cancer biology, are also a target of epigenetic inactivation in tumors. To tackle this issue, we have used cancer cells genetically deficient for the DNA methyltransferase enzymes in conjuction with circRNA expression microarrays. We have found that the loss of DNA methylation provokes a release of circRNA silencing. In particular, we have identified that promoter CpG island hypermethylation of the genes TUSC3 (tumor suppressor candidate 3), POMT1 (protein O-mannosyltransferase 1), ATRNL1 (attractin-like 1) and SAMD4A (sterile alpha motif domain containing 4A) is linked to the transcriptional downregulation of both linear mRNA and the hosted circRNA. Although some circRNAs regulate the linear transcript, we did not observe changes in TUSC3 mRNA levels upon TUSC3 circ104557 overexpression. Interestingly, we found circRNA-mediated regulation of target miRNAs and an in vivo growth inhibitory effect upon TUSC3 circ104557 transduction. Data mining for 5′-end CpG island methylation of TUSC3, ATRNL1, POMT1 and SAMD4A in cancer cell lines and primary tumors showed that the epigenetic defect was commonly observed among different tumor types in association with the diminished expression of the corresponding transcript. Our findings support a role for circRNA DNA methylation-associated loss in human cancer.

An Alternative Homodimerization Interface of MnmG Reveals a Conformational Dynamics that Is Essential for Its tRNA Modification Function

The Escherichia coli homodimeric proteins MnmE and MnmG form a functional complex, MnmEG, that modifies tRNAs using GTP, methylene-tetrahydrofolate, FAD, and glycine or ammonium. MnmE is a tetrahydrofolate- and GTP-binding protein, whereas MnmG is a FAD-binding protein with each protomer composed of the FAD-binding domain, two insertion domains, and the helical C-terminal domain. The detailed mechanism of the MnmEG-mediated reaction remains unclear partially due to incomplete structural information on the free- and substrate-bound forms of the complex. In this study, we show that MnmG can adopt in solution a dimer arrangement (form I) different from that currently considered as the only biologically active (form II). Normal mode analysis indicates that form I can oscillate in a range of open and closed conformations. Using isothermal titration calorimetry and native red electrophoresis, we show that a form-I open conformation, which can be stabilized in vitro by the formation of an interprotomer disulfide bond between the catalytic C277 residues, appears to be involved in the assembly of the MnmEG catalytic center. We also show that residues R196, D253, R436, R554 and E585 are important for the stabilization of form I and the tRNA modification function. We propose that the form I dynamics regulates the alternative access of MnmE and tRNA to the MnmG FAD active site. Finally, we show that the C-terminal region of MnmG contains a sterile alpha motif domain responsible for tRNA–protein and protein–protein interactions.

Fish SAMHD1 performs as an activator for IFN expression

As a host limiting factor, Sterile Alpha Motif and Histidine-Aspartate Domain 1 protein (SAMHD1) is associated with IRF3-mediated antiviral and apoptotic responses in mammals. However, the antiviral mechanism of SAMHD1 remains indistinct in fish. In this study, we found the expression of Ctenopharyngodon idella SAMHD1 (MF326081) was up-regulated after transfection with poly I:C (dsRNA analog), B-DNA or Z-DNA into C. idella kidney cells (CIKs), but these expression profiles had no obvious change when the cells were incubated with these nucleic acids. These data may indicate that CiSAMHD1 participates in the intracellular PRR-mediated signaling pathway rather than extracellular PRR-mediated signaling pathway. Subcellular localization assay suggested that a part of over-expressed CiSAMHD1 were translocated from nuclear to cytoplasm when C. idella ovary cells (COs) were transfected with poly I:C, B-DNA or Z-DNA. Nucleic acid pulldown assays were performed to investigate the reason for nuclear-cytoplasm translocation of CiSAMHD1. The results showed that CiSAMHD1 had a high affinity with B-DNA, Z-DNA and ISD-PS (dsRNA analog). In addition, co-IP assays revealed the interaction of CiSAMHD1 with CiSTING (KF494194). Taken together, all these results suggest that grass carp SAMHD1 performs as an activator for innate immune response through STING-mediated signaling pathway.

SAMHD1 Phosphorylation Coordinates the Anti-HIV-1 Response by Diverse Interferons and Tyrosine Kinase Inhibition.

ABSTRACTMacrophages are susceptible to human immunodeficiency virus type 1 (HIV-1) infection despite abundant expression of antiviral proteins. Perhaps the most important antiviral protein is the restriction factor sterile alpha motif domain and histidine/aspartic acid domain-containing protein 1 (SAMHD1). We investigated the role of SAMHD1 and its phospho-dependent regulation in the context of HIV-1 infection in primary human monocyte-derived macrophages and the ability of various interferons (IFNs) and pharmacologic agents to modulate SAMHD1. Here we show that stimulation by type I, type II, and to a lesser degree, type III interferons share activation of SAMHD1 via dephosphorylation at threonine-592 as a consequence of signaling. Cyclin-dependent kinase 1 (CDK1), a known effector kinase for SAMHD1, was downregulated at the protein level by all IFN types tested. Pharmacologic inhibition or small interfering RNA (siRNA)-mediated knockdown of CDK1 phenocopied the effects of IFN on SAMHD1. A panel of FDA-approved tyrosine kinase inhibitors potently induced activation of SAMHD1 and subsequent HIV-1 inhibition. The viral restriction imposed via IFNs or dasatinib could be overcome through depletion of SAMHD1, indicating that their effects are exerted primarily through this pathway. Our results demonstrate that SAMHD1 activation, but not transcriptional upregulation or protein induction, is the predominant mechanism of HIV-1 restriction induced by type I, type II, and type III IFN signaling in macrophages. Furthermore, SAMHD1 activation presents a pharmacologically actionable target through which HIV-1 infection can be subverted.

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.

Novel host restriction factors implicated in HIV-1 replication

Human immunodeficiency virus-1 (HIV-1) is known to interact with multiple host cellular proteins during its replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to inhibit HIV-1 replication at various stages of its life cycle. These host cellular proteins, which are known as restriction factors, constitute an integral part of the host's first line of defence against the viral pathogen. Since the discovery of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) as an HIV-1 restriction factor, several human proteins have been identified that exhibit anti-HIV-1 restriction. While each restriction factor employs a distinct mechanism of inhibition, the HIV-1 virus has equally evolved complex counter strategies to neutralize their inhibitory effect. APOBEC3G, tetherin, sterile alpha motif and histidine-aspartate domain 1 (SAMHD1), and trim-5α are some of the best known HIV-1 restriction factors that have been studied in great detail. Recently, six novel restriction factors were discovered that exhibit significant antiviral activity: endoplasmic reticulum α1,2-mannosidase I (ERManI), translocator protein (TSPO), guanylate-binding protein 5 (GBP5), serine incorporator (SERINC3/5) and zinc-finger antiviral protein (ZAP). The focus of this review is to discuss the antiviral mechanism of action of these six restriction factors and provide insights into the probable counter-evasion strategies employed by the HIV-1 virus. The recent discovery of new restriction factors substantiates the complex host-pathogen interactions occurring during HIV-1 pathogenesis and makes it imperative that further investigations are conducted to elucidate the molecular basis of HIV-1 replication.

Conserved Herpesvirus Protein Kinases Target SAMHD1 to Facilitate Virus Replication

To ensure a successful infection, herpesviruses have developed elegant strategies to counterbalance the host anti-viral responses. Sterile alpha motif and HD domain 1 (SAMHD1) was recently identified as an intrinsic restriction factor for a variety of viruses. Aside from human immunodeficiency virus 2 (HIV-2) and the related simian immunodeficiency virus (SIV) Vpx proteins, the direct viral countermeasures against SAMHD1 restriction remain unknown. Using Epstein-Barr virus (EBV) as a primary model, we discovered that SAMHD1-mediated anti-viral restriction is antagonized by EBV BGLF4, a member of the conserved viral protein kinases encoded by all herpesviruses. Mechanistically, we found that BGLF4 phosphorylates SAMHD1 and thereby inhibits its dNTPase activity. We further demonstrated that the targeting of SAMHD1 for phosphorylation is a common feature shared by beta- and gamma- herpesviruses. Together, our findings uncover a unique immune evasion mechanism whereby herpesviruses exploit the phosphorylation of SAMHD1 to thwart host defenses.