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Stimulator of interferon genes (STING) mediates innate immune response upon binding to cyclic GMP-AMP (cGAMP). It recruits tank-binding kinase 1 (TBK1) and transcription factor interferon regulatory factor 3 (IRF3) through its C-terminal tail and facilitates TBK1-dependent phosphorylation of IRF3 via forming STING polymers in mammalian cells. However, the mechanism behind STING-mediated activation of NF-κB transcription factor, Relish, in insect cells is unknown. Our study revealed that insect STING formed oligomers and the cryptic RIP homotypic interaction motif (cRHIM) was required for its oligomerization and its anti-viral functions. Cells expressing cRHIM-deficient mutants exhibited lower levels of anti-viral molecules, higher viral load after viral infection and weak activation of Relish. Moreover, we observed that under cGAMP stimulation, insect STING interacted with IMD, and deletion of the cRHIM motif on either protein prevented this interaction. Finally, we demonstrated that cGAMP enhanced the amyloid-like property of insect STING aggregates by ThT staining. In summary, our research showed that insect STING employed a homotypic motif to form intermolecular interactions that are essential for its antiviral signaling., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Short Linear Motifs (SLiMs) are the smallest structural and functional components of modular eukaryotic proteins. They are also the most abundant, especially when considering post-translational modifications. As well as being found throughout the cell as part of regulatory processes, SLiMs are extensively mimicked by intracellular pathogens. At the heart of the Eukaryotic Linear Motif (ELM) Resource is a representative (not comprehensive) database. The ELM entries are created by a growing community of skilled annotators and provide an introduction to linear motif functionality for biomedical researchers. The 2024 ELM update includes 346 novel motif instances in areas ranging from innate immunity to both protein and RNA degradation systems. In total, 39 classes of newly annotated motifs have been added, and another 17 existing entries have been updated in the database. The 2024 ELM release now includes 356 motif classes incorporating 4283 individual motif instances manually curated from 4274 scientific publications and including >700 links to experimentally determined 3D structures. In a recent development, the InterPro protein module resource now also includes ELM data. ELM is available at: http://elm.eu.org., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The polysialyltransferases ST8SIA2 and ST8SIA4 and their product, polysialic acid (polySia), are known to be related to cancers and mental disorders. ST8SIA2 and ST8SIA4 have conserved amino acid (AA) sequence motifs essential for the synthesis of the polySia structures on the neural cell adhesion molecule. To search for a new motif in the polysialyltransferases, we adopted the in silico Individual Meta Random Forest program that can predict disease-related AA substitutions. The Individual Meta Random Forest program predicted a new eight-amino-acids sequence motif consisting of highly pathogenic AA residues, thus designated as the pathogenic (P) motif. A series of alanine point mutation experiments in the pathogenic motif (P motif) showed that most P motif mutants lost the polysialylation activity without changing the proper enzyme expression levels or localization in the Golgi. In addition, we evaluated the enzyme stability of the P motif mutants using newly established calculations of mutation energy, demonstrating that the subtle change of the conformational energy regulates the activity. In the AlphaFold2 model, we found that the P motif was a buried β-strand underneath the known surface motifs unique to ST8SIA2 and ST8SIA4. Taken together, the P motif is a novel buried β-strand that regulates the full activity of polysialyltransferases from the inside of the molecule., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Multiple genetic factors control tillering, a key agronomy trait for wheat ( Triticum aestivum L.) yield. Previously, we reported a dwarf-monoculm mutant ( dmc ) derived from wheat cultivar Guomai 301, and found that the contents of gibberellic acid 3 (GA 3 ) in the tiller primordia of dmc were significantly higher. Transcriptome analysis indicated that some wheat gibberellin oxidase ( TaGAox ) genes TaGA20ox-A2 , TaGA20ox-B2 , TaGA3ox-A2 , TaGA20ox-A4 , TaGA2ox-A10 and TaGA2ox-B10 were differentially expressed in dmc . Therefore, this study systematically analyzed the roles of gibberellin oxidase genes during wheat tillering. A total of 63 TaGAox genes were identified by whole genome analysis. The TaGAoxs were clustered to four subfamilies, GA20oxs, GA2oxs, GA3oxs and GA7oxs, including seven subgroups based on their protein structures. The promoter regions of TaGAox genes contain a large number of cis -acting elements closely related to hormone, plant growth and development, light, and abiotic stress responses. Segmental duplication events played a major role in TaGAoxs expansion. Compared to Arabidopsis , the gene collinearity degrees of the GAoxs were significantly higher among wheat, rice and maize. TaGAox genes showed tissue-specific expression patterns. The expressions of TaGAox genes ( TaGA20ox-B2 , TaGA7ox-A1 , TaGA2ox10 and TaGA3ox-A2 ) were significantly affected by exogenous GA 3 applications, which also significantly promoted tillering of Guomai 301, but didn't promote dmc . TaGA7ox-A1 overexpression transgenic wheat lines were obtained by Agrobacterium mediated transformation. Genomic PCR and first-generation sequencing demonstrated that the gene was integrated into the wheat genome. Association analysis of TaGA7ox-A1 expression level and tiller number per plant demonstrated that the tillering capacities of some TaGA7ox-A1 transgenic lines were increased. These data demonstrated that some TaGAoxs as well as GA signaling were involved in regulating wheat tillering, but the GA signaling pathway was disturbed in dmc . This study provided valuable clues for functional characterization of GAox genes in wheat., Competing Interests: The authors declare that they have no competing interests., (© 2023 Wang et al.)

Glutathione peroxidase-like enzyme is an important enzymatic antioxidant in plants. It is involved in scavenging reactive oxygen species, which can effectively prevent oxidative damage and improve resistance. GPXL has been studied in many plants but has not been reported in potatoes, the world's fourth-largest food crop. This study identified eight StGPXL genes in potatoes for the first time through genome-wide bioinformatics analysis and further studied the expression patterns of these genes using qRT-PCR. The results showed that the expression of StGPXL1 was significantly upregulated under high-temperature stress, indicating its involvement in potato defense against high-temperature stress, while the expression levels of StGPXL4 and StGPXL5 were significantly downregulated. The expression of StGPXL1, StGPXL2, StGPXL3, and StGPXL6 was significantly upregulated under drought stress, indicating their involvement in potato defense against drought stress. After MeJA hormone treatment, the expression level of StGPXL6 was significantly upregulated, indicating its involvement in the chemical defense mechanism of potatoes. The expression of all StGPXL genes is inhibited under biotic stress, which indicates that GPXL is a multifunctional gene family, which may endow plants with resistance to various stresses. This study will help deepen the understanding of the function of the potato GPXL gene family, provide comprehensive information for the further analysis of the molecular function of the potato GPXL gene family as well as a theoretical basis for potato molecular breeding.

Kinesin family motors are microtubule (MT)-stimulated ATPases known best as transporters of cellular cargoes through the cytoplasm, regulators of MT dynamics, organizers of the mitotic spindle, and for insuring equal division of DNA during mitosis. Several kinesins have also been shown to regulate transcription by interacting with transcriptional cofactors and regulators, nuclear receptors, or with specific promotor elements on DNA. We previously showed that an LxxLL nuclear receptor box motif in the kinesin-2 family motor KIF17 mediates binding to the orphan nuclear receptor estrogen related receptor alpha (ERR1) and is responsible for the suppression of ERR1-dependent transcription by KIF17. Analysis of all kinesin family proteins revealed that multiple kinesins contain this LxxLL motif, raising the question as to whether additional kinesin motors contribute to the regulation of ERR1. In this study, we interrogate the effects of multiple kinesins with LxxLL motifs on ERR1-mediated transcription. We demonstrate that the kinesin-3 family motor KIF1B contains two LxxLL motifs, one of which binds to ERR1. In addition, we show that expression of a KIF1B fragment containing this LxxLL motif inhibits ERR1-dependent transcription by regulating nuclear entry of ERR1. We also provide evidence that the effects of expressing the KIF1B-LxxLL fragment on ERR1 activity are mediated by a mechanism distinct from that of KIF17. Since LxxLL domains are found in many kinesins, our data suggest an expanded role for kinesins in nuclear receptor mediated transcriptional regulation.

Preeclampsia is a pregnancy-specific disorder involving placental abnormalities. Elevated placental Sialic acid immunoglobulin-like lectin (Siglec)-6 expression has been correlated with preeclampsia. Siglec-6 is a transmembrane receptor, expressed predominantly by the trophoblast cells in the human placenta. It interacts with sialyl glycans such as sialyl-TN glycans as well as binds leptin. Siglec-6 overexpression has been shown to influence proliferation, apoptosis, and invasion in the trophoblast (BeWo) cell model. However, there is no direct evidence that Siglec-6 plays a role in preeclampsia pathogenesis and its signaling potential is still largely unexplored. Siglec-6 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif in its cytoplasmic tail suggesting a signaling function. Site-directed mutagenesis and transfection were employed to create a series of Siglec-6 expressing HTR-8/SVneo trophoblastic cell lines with mutations in specific functional residues to explore the signaling potential of Siglec-6. Co-immunoprecipitation and inhibitory assays were utilized to investigate the association of Src-kinases and SH-2 domain-containing phosphatases with Siglec-6. In this study, we show that Siglec-6 is phosphorylated at ITIM and ITIM-like domains by Src family kinases. Phosphorylation of both ITIM and ITIM-like motifs is essential for the recruitment of phosphatases like Src homology region 2 containing protein tyrosine phosphatase 2 (SHP-2), which has downstream signaling capabilities. These findings suggest Siglec-6 as a signaling molecule in human trophoblasts. Further investigation is warranted to determine which signaling pathways are activated downstream to SHP-2 recruitment and how overexpression of Siglec-6 in preeclamptic placentas impacts pathogenesis.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein mediates viral entry and membrane fusion. Its cleavage at S1/S2 and S2' sites during the biosynthesis in virus producer cells and viral entry are critical for viral infection and transmission. In contrast, the biological significance of the junction region between both cleavage sites for S protein synthesis and function is less understood. By analyzing the conservation and structure of S protein, we found that intrachain contacts formed by the conserved tyrosine (Y) residue 756 (Y756) with three α-helices contribute to the spike's conformational stability. When Y756 is mutated to an amino acid residue that can provide hydrogen bonds, S protein could be expressed as a cleaved form, but not vice versa . Also, the L753 mutation linked to the Y756 hydrogen bond prevents the S protein from being cleaved. Y756 and L753 mutations alter S protein subcellular localization. Importantly, Y756 and L753 mutations are demonstrated to reduce the infectivity of the SARS-CoV-2 pseudoviruses by interfering with the incorporation of S protein into pseudovirus particles and causing the pseudoviruses to lose their sensitivity to neutralizing antibodies. Furthermore, both mutations affect the assembly and production of SARS-CoV-2 virus-like particles in cell culture. Together, our findings reveal for the first time a critical role for the conserved L753-LQ-Y756 motif between S1/S2 and S2' cleavage sites in S protein synthesis and processing as well as virus assembly and infection. IMPORTANCE The continuous emergence of SARS-CoV-2 variants such as the delta or lambda lineage caused the continuation of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. Logically, the spike (S) protein mutation has attracted much concern. However, the key amino acids in S protein for its structure and function are still not very clear. In this study, we discovered for the first time that the conserved residues Y756 and L753 at the junction between the S1/S2 and S2' sites are very important, like the S2' cleavage site R815, for the synthesis and processing of S protein such as protease cleavage, and that the mutations severely interfered with the incorporation of S protein into pseudotyped virus particles and SARS-CoV-2 virus-like particles. Consequently, we delineate the novel potential target for the design of broad-spectrum antiviral drugs in the future, especially in the emergence of SARS-CoV-2 variants.

WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries. Despite their significance in physiological and disease-like conditions, their reversible interactions with other proteins remain modestly examined. Here, we show the development and validation of a protein nanopore for the detection and quantification of WD40 repeat protein 5 (WDR5), a chromatin-associated hub involved in epigenetic regulation of histone methylation. Our nanopore sensor is equipped with a 14-residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4 Win ), a WDR5 ligand. Our approach reveals a broad dynamic range of MLL4 Win -WDR5 interactions and three distant subpopulations of binding events, representing three modes of protein recognition. The three binding events are confirmed as specific interactions using a weakly binding WDR5 derivative and various environmental contexts. These outcomes demonstrate the substantial sensitivity of our nanopore sensor, which can be utilized in protein analytics., (© 2022. The Author(s).)

β-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking., (© 2022. The Author(s).)

Tousled-like kinases (TLKs) are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants. A major function of TLKs is to phosphorylate the histone chaperone proteins ASF1a and ASF1b to facilitate DNA replication-coupled nucleosome assembly, but how TLKs selectively target these critical substrates is unknown. Here, we show that TLK2 selectivity towards ASF1 substrates is achieved in two ways. First, the TLK2 catalytic domain recognizes consensus phosphorylation site motifs in the ASF1 C-terminal tail. Second, a short sequence at the TLK2 N-terminus docks onto the ASF1a globular N-terminal domain in a manner that mimics its histone H3 client. Disrupting either catalytic or non-catalytic interactions through mutagenesis hampers ASF1 phosphorylation by TLK2 and cell growth. Our results suggest that the stringent selectivity of TLKs for ASF1 is enforced by an unusual interaction mode involving mutual recognition of a short sequence motifs by both kinase and substrate., (© 2022. The Author(s).)

SARS-CoV-2 harbors a unique S1/S2 furin cleavage site within its spike protein, which can be cleaved by furin and other proprotein convertases. Proteolytic activation of SARS-CoV-2 spike protein at the S1/S2 boundary facilitates interaction with host ACE2 receptor for cell entry. To address this, high titer antibody was generated against the SARS-CoV-2-specific furin motif. Using a series of innovative ELISA-based assays, this furin site blocking antibody displayed high sensitivity and specificity for the S1/S2 furin cleavage site, including with a P681R mutation, and demonstrated effective blockage of both enzyme-mediated cleavage and spike-ACE2 interaction. The results suggest that immunological blocking of the furin cleavage site may afford a suitable approach to stem proteolytic activation of SARS-CoV-2 spike protein and curtail viral infectivity., (Copyright © 2022 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and in vitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The signal peptide (SP) of integrated membrane proteins is removed cotranslationally or posttranslationally in the endoplasmic reticulum, while GP64, a membrane fusion protein of Bombyx mori nucleopolyhedrovirus (BmNPV), retains its SP in the mature protein and virion. In this study, we revealed that uncleaved SP is a key determinant with additional functions in infection. First, uncleaved SP endows BmNPV with strong virulence; second, SP retention-induced BmNPV infection depends on cholesterol recognition amino acid consensus domain 1 (CRAC1) and CRAC2. In contrast, the recombinant virus with SP-cleaved GP64 has reduced infectivity, and only CRAC2 is required for BmNPV infection. Furthermore, we showed that cholesterol in the plasma membrane is an important fusion receptor that interacts with CRAC2 of GP64. Our study suggested that BmNPV GP64 is a key cholesterol-binding protein and uncleaved SP determines GP64's unique dependence on the CRAC domains. IMPORTANCE BmNPV is a severe pathogen that mainly infects silkworms. GP64 is the key membrane fusion protein that mediates BmNPV infection, and some studies have indicated that cholesterol and lipids are involved in BmNPV infection. A remarkable difference from other membrane fusion proteins is that BmNPV GP64 retains its SP in the mature protein, but the cause is still unclear. In this study, we investigated the reason why BmNPV retains this SP, and its effects on protein targeting, virulence, and CRAC dependence were revealed by comparison of recombinant viruses harboring SP-cleaved or uncleaved GP64. Our study provides a basis for understanding the dependence of BmNPV infection on cholesterol/lipids and host specificity.

Mitochondrial ultrastructure is highly adaptable and undergoes dynamic changes upon physiological and energetic cues. MICOS (mitochondrial contact site and cristae organizing system), a large oligomeric protein complex, maintains mitochondrial ultrastructure as it is required for formation of crista junctions (CJs) and contact sites. MIC13 acts as a critical bridge between two MICOS subcomplexes. Deletion of MIC13 causes loss of CJs resulting in cristae accumulating as concentric rings and specific destabilization of the MIC10-subcomplex. Mutations in MIC13 are associated with infantile lethal mitochondrial hepato-encephalopathy, yet functional regions within MIC13 were not known. To identify and characterize such regions, we systemically generated 20 amino-acids deletion variants across the length of MIC13. While deletion of many of these regions of MIC13 is dispensable for its stability, the N-terminal region and a stretch between amino acid residues 84 and 103 are necessary for the stability and functionality of MIC13. We could further locate conserved motifs within these regions and found that a GxxxG motif in the N-terminal transmembrane segment and an internal WN motif are essential for stability of MIC13, formation of the MIC10-subcomplex, interaction with MIC10- and MIC60-subcomplexes and maintenance of cristae morphology. The GxxxG motif is required for membrane insertion of MIC13. Overall, we systematically found important conserved residues of MIC13 that are required to perform the bridging between the two MICOS subcomplexes. The study improves our understanding of the basic molecular function of MIC13 and has implications for its role in the pathogenesis of a severe mitochondrial disease., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chromosomal rearrangements can generate genetic fusions composed of two distinct gene sequences, many of which have been implicated in tumorigenesis and progression. Our study proposes a model whereby oncogenic gene fusions frequently alter the protein stability of the resulting fusion products, via exchanging protein degradation signal (degron) between gene sequences. Computational analyses of The Cancer Genome Atlas (TCGA) identify 2,406 cases of degron exchange events and reveal an enrichment of oncogene stabilization due to loss of degrons from fusion. Furthermore, we identify and experimentally validate that some recurrent fusions, such as BCR-ABL, CCDC6-RET and PML-RARA fusions, perturb protein stability by exchanging internal degrons. Likewise, we also validate that EGFR or RAF1 fusions can be stabilized by losing a computationally-predicted C-terminal degron. Thus, complementary to enhanced oncogene transcription via promoter swapping, our model of degron loss illustrates another general mechanism for recurrent fusion proteins in driving tumorigenesis., (© 2021. The Author(s).)

RNA-binding proteins (RBPs) have a significant role in various regulatory tasks. However, the mechanism by which RBPs identify the subsequence target RNAs is still not clear. In recent years, several machine and deep learning-based computational models have been proposed for understanding the binding preferences of RBPs. These methods required integrating multiple features with raw RNA sequences such as secondary structure and their performances can be further improved. In this paper, we propose an efficient and simple convolution neural network, RBPCNN, that relies on the combination of the raw RNA sequence and evolutionary information. We show that conservation scores (evolutionary information) for the RNA sequences can significantly improve the overall performance of the proposed predictor. In addition, the automatic extraction of the binding sequence motifs can enhance our understanding of the binding specificities of RBPs. The experimental results show that RBPCNN outperforms significantly the current state-of-the-art methods. More specifically, the average area under the receiver operator curve was improved by 2.67 percent and the mean average precision was improved by 8.03 percent. The datasets and results can be downloaded from https://home.jbnu.ac.kr/NSCL/RBPCNN.htm.

The large (L) polymerase proteins of most nonsegmented, negative-stranded (NNS) RNA viruses have conserved methyltransferase motifs, (G)-G-G-D and K-D-K-E, which are important for the stabilization and translation of mRNA. However, the function of the (G)-G-G-D and K-D-K-E motifs in the NNS RNA virus Newcastle disease virus (NDV) remains unclear. We observed G-G-D and K-D-K-E motifs in all NDV genotypes. By using the infection cloning system of NDV rSG10 strain, recombinant NDVs with a single amino acid mutated to alanine in one motif (G-G-D or K-D-K-E) were rescued. The intracerebral pathogenicity index and mean death time assay results revealed that the G-G-D motif and K-D-K-E motif attenuate the virulence of NDV to various degrees. The replication, transcription, and translation levels of the K-D-K-E motif-mutant strains were significantly higher than those of wild-type virus owing to their altered regulation of the affinity between nucleocapsid protein and eukaryotic translation initiation factor 4E. When the infection dose was changed from a multiplicity of infection (MOI) of 10 to an MOI of 0.01, the cell-to-cell spread abilities of G-G-D- and K-D-K-E-mutant strains were reduced, according to plaque assay and dynamic indirect immunofluorescence assay results. Finally, we found that NDV strains with G-G-D or K-D-K-E motif mutations had less pathogenicity in 3-week-old specific-pathogen-free chickens than wild-type NDV. Therefore, these methyltransferase motifs can affect virulence by regulating the translation and cell-to-cell spread abilities of NDV. This work provides a feasible approach for generating vaccine candidates for viruses with methyltransferase motifs. IMPORTANCE Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts. Our study found that a deficiency in its methyltransferase motifs (G-G-D and K-D-K-E motifs) can attenuate NDV and revealed the molecular mechanism by which these motifs affect pathogenicity, which provides a new direction for the development of NDV vaccines. In addition to the (G)-G-G-D and K-D-K-E motifs of many nonsegmented, negative-stranded RNA viruses, similar motifs have been found in dengue virus, Zika virus, Japanese encephalitis virus (JEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This suggests that such motifs may be present in more viruses. Our finding also provides a molecular basis for the discovery and functional study of (G)-G-G-D and K-D-K-E motifs of other viruses.

Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases involved in many biological processes, such as inflammation and antiviral immunity. In the present study, a novel TRIM protein homolog named CgTRIM1 was identified from Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgTRIM1 was of 1914 bp encoding a putative polypeptide of 637 amino acid residues. There were three classical domains in the predicted CgTRIM1 protein, including one RING domain, two b-box domains and one coiled-coil domain in N-terminal. For the lack of C-terminal domains, the CgTRIM1 was classified as the member of C-V TRIM subfamily. The mRNA transcripts of CgTRIM1 were detected in all the tested tissues and haemocytes, with the highest expression level in gill. The mRNA and protein levels of CgTRIM1 in gill were significantly up-regulated at 6 h after poly (I:C) stimulation. Moreover, the nuclear translocation of CgTRIM1 was observed in haemocytes of oysters after poly (I:C) stimulation. After IFN-like protein (CgIFNLP) was knocked down by RNA interference (RNAi), the expression of CgTRIM1 in gill was markedly inhibited in both mRNA (0.14-fold, p < 0.001) and protein levels after poly (I:C) stimulation. Furthermore, after knocking down of CgTRIM1, the mRNA expression levels of IFN-stimulated genes, including myxovirus resistance of oyster (CgMx) and Interferon-induced protein 44 (CgIFI44) were significantly down-regulated post poly (I:C) stimulation, while no significant change of the CgIFNLP expression was observed. These results indicated that CgTRIM1 participated in the antiviral response of C. gigas by regulating the mRNA expressions of IFN-stimulated genes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Protein arginine methyltransferase 6 (PRMT6) catalyses the asymmetric dimethylation of arginines on numerous substrate proteins within the human cell. In particular, PRMT6 methylates histone H3 arginine 2 (H3R2) which affects both gene repression and activation. However, the substrate specificity of PRMT6 has not been comprehensively analysed. Here, we systematically characterise the substrate recognition motif of PRMT6, finding that it has broad specificity and recognises the RG motif. Working with a H3 tail peptide as a template, on which we made 204 amino acid substitutions, we use targeted mass spectrometry to measure their effect on PRMT6 in vitro activity. We first show that PRMT6 methylates R2 and R8 in the H3 peptide, although H3R8 is methylated with lower efficiency and is not an in vivo PRMT6 substrate. We then quantify the effect of 194 of these amino acid substitutions on methylation at both H3R2 and H3R8. In both cases, we find that PRMT6 tolerates essentially any amino acid substitution in the H3 peptide, but that positively charged and bulky residues are preferred near the target arginine. We show that PRMT6 also has preference for glycine, but only in the position immediately following the target arginine. This indicates that PRMT6 recognises the RG motif rather than the RGG motif. We further confirm this preference for the RG motif on another PRMT6 substrate, histone H4R3. This broad specificity and recognition of RG rather than RGG are distinctive among the PRMT family and has implications for the development of drugs to selectively target PRMT6. DATABASES: Panorama Public (https://panoramaweb.org/PRMT6motif.url); ProteomeXchange (PXD016711)., (© 2021 Federation of European Biochemical Societies.)

The geminivirus replication protein, Rep, has long been recognized as a high-value target for control of geminivirus infections as this protein is highly conserved and essential for viral replication and proliferation. In addition, inhibition of viral replication has been pursued through various antiviral strategies with varying degrees of success, including inhibitory peptides that target Rep. While much effort has centered around sequence characterization of the Rep protein and inhibitory peptides, detailed structural analysis has been missing. This study computationally investigated the presence of common structural features within these inhibitory peptides and if these features could inform if a particular peptide will bind Rep and/or interfere with viral replication. Molecular dynamics simulations of the inhibitory peptide library showed that simply possessing stable structural features does not inform interference of viral replication regardless of the binding of Rep. Additionally, nearly all known Rep inhibitory peptides sample a conserved β-sheet structural motif, possibly informing structure-function relationships in binding Rep. In particular, two peptides (A22 and A64) characterized by this structural motif were computationally docked against a wide variety of geminivirus Rep proteins to determine a mechanism of action. Computational docking revealed these peptides utilize a common Rep protein sequence motif for binding, HHN-x 1/2 -Q. The results identified residues in both Rep and the inhibitory peptides that play a significant role in the interaction, establishing the foundation for a rational structure-based design approach for the construction of both broadly reactive and geminivirus species-specific inhibitors.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 is a huge public health crisis for the globe. The receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein plays a vital role in viral infection and serves as a major target for developing neutralizing antibodies. In this study, the antibody response to the RBD of SARS-CoV-2 S protein was analyzed by a panel of sera from animals immunized with RBD-based antigens and four linear B-cell epitope peptides (R345, R405, R450 and R465) were revealed. The immunogenicity of three immunodominant peptides (R345, R405, R465) was further accessed by peptide immunization in mice, and all of them could induced potent antibody response to SARS-CoV-2 S protein, indicating that the three determinants in the RBD were immunogenic. We further generated and characterized monoclonal antibodies (15G9, 12C10 and 10D2) binding to these epitope peptides, and finely mapped the three immunodominant epitopes using the corresponding antibodies. Neutralization assays showed that all three monoclonal antibodies had neutralization activity. Results from IFA and western blotting showed that 12C10 was a cross-reactive antibody against both of SARS-CoV-2 and SARS-CoV. Results from conservative and structural analysis showed that 350 VYAWN 354 was a highly conserved epitope and exposed on the surface of SARS-CoV-2 S trimer, whereas 473 YQAGSTP 479 located in the receptor binding motif (RBM) was variable among different SARS-CoV-2 strains. 407 VRQIAP 412 was a highly conserved, but cryptic epitope shared between SARS-CoV-2 and SARS-CoV. These findings provide important information for understanding the humoral antibody response to the RBD of SARS-CoV-2 S protein and may facilitate further efforts to design SARS-CoV-2 vaccines and the target of COVID-19 diagnostic., Competing Interests: HL, YL, YT and YW were employed by Henan Zhongze Bioengineering Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jiang, Zhang, Liu, Ding, Liu, Tian, Wang and Wang.)

Background: Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations., Methods: Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined., Results: Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents., Conclusions: The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues., Funding: Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV)., Competing Interests: EG, KT, KP, MS, SV, SV, GK, JP, TS, NB, TB is named as an inventor with The Medical University of Vienna and the University of Natural Resources and Life Sciences of a patent describing the rhDAO heparin-binding motif mutants presented herein (patent pending WO2020169577A1), KS none, MR None, BJ is named as an inventor with The Medical University of Vienna and the University of Natural Resources and Life Sciences of a patent describing the rhDAO heparin-binding motif mutants presented herein (patent pending WO2020169577A1)., (© 2021, Gludovacz et al.)

PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3'-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

GATA transcription factors (TFs) are widespread eukaryotic regulators whose DNA-binding domain is a class IV zinc finger motif (CX 2 CX 17-20 CX 2 C) followed by a basic region. We identified 262 GATA genes (389 GATA TFs) from seven Populus genomes using the pipeline of GATA-TFDB. Alternative splicing forms of Populus GATA genes exhibit dynamics of GATA gene structures including partial or full loss of GATA domain and additional domains. Subfamily III of Populus GATA genes display lack CCT and/or TIFY domains. 21 Populus GATA gene clusters (PCs) were defined in the phylogenetic tree of GATA domains, suggesting the possibility of subfunctionalization and neofunctionalization. Expression analysis of Populus GATA genes identified the five PCs displaying tissue-specific expression, providing the clues of their biological functions. Amino acid patterns of Populus GATA motifs display well conserved manner of Populus GATA genes. The five Populus GATA genes were predicted as membrane-bound GATA TFs. Biased chromosomal distributions of GATA genes of three Populus species. Our comparative analysis approaches of the Populus GATA genes will be a cornerstone to understand various plant TF characteristics including evolutionary insights., (© 2021. The Author(s).)

AP-2 complex subunit mu-A (AP2M1A) is a component of the adaptor complexes that link clathrin to receptors in coated vesicles. It has recently been shown to be involved in the resistance to oxidative damage, challenging the conventional role of AP2M1A. Here we demonstrated that AP2M1A was a heparin-binding protein abundantly stored in eggs and embryos of zebrafish, and its gene expression was markedly up-regulated by LPS and LTA treatment. We also showed that recombinant AP2M1A (rAP2M1A) was not only able to interact with Gram-negative and Gram-positive bacteria as well as their signature molecules LPS and LTA, but also able to inhibit the growth of the bacteria. Additionally, we found that AP2M1A 354-382 that contained 2 closely positioned heparin-binding motifs could also bind to LPS and LTA, and inhibit the bacterial growth. Both rAP2M1A and AP2M1A 354-382 were shown to execute antibacterial activity by a combined action of destabilization/destruction of bacterial cell wall through interaction with LPS and LTA, disturbance of the usually polarized membrane through depolarization, and apoptosis/necrosis through intracellular ROS production. Finally, we showed that AP2M1A could protect zebrafish developing embryos/larvae against attack by the potential pathogen Aeromonas hydrophila. All these demonstrate for the first time that AP2M1A is a maternal antimicrobial protein previously uncharacterized. It also establishes a correlation between antibacterial activity and heparin-binding motifs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In the immune system, C-type lectins, as pattern recognition receptors, have an important function. Carbohydrate-recognition domains (CRDs) endow C-type lectins with the function of recognizing and scavenging non-self factors. In the present study, a new C-type lectin (designated as PtCTL-9 according to the order of discovery) from swimming crab (Portunus trituberculatus) was characterized. QPN (Gln-Pro-Asn) and FHS (Phe-His-Ser) were identified as the key motifs that determine carbohydrate binding. Motif QPN was mutated to QPD (Gln-Pro-Asp) (M1) and EPN (Glu-Pro-Asn) (M2) to study its immune function and for comparative analysis. The results showed that PtCTL-9 displayed broad non-self immunity. PtCTL-9 could also function as an opsonin to promote phagocytosis and the in vitro encapsulation of hemocytes. These results indicated that PtCTL-9 has an extensive nonself-recognition ability, regulates pathogen clearance, and its QPN motif is important in PtCTL-9's immune function., (Copyright © 2021. Published by Elsevier Ltd.)

PB1 functions as the catalytic subunit of influenza virus RNA polymerase complex and plays an essential role in viral RNA transcription and replication. To determine plasticity in the PB1 enzymatic site and map catalytically important residues, 658 mutants were constructed, each with one to seven mutations in the enzymatic site of PB1. The polymerase activities of these mutants were quantified using a minigenome assay, and polymerase activity-associated residues were identified using sparse learning. Results showed that polymerase activities are affected by the residues not only within the conserved motifs, but also across the inter-motif regions of PB1, and the latter are primarily located at the base of the palm domain, a region that is conserved in avian PB1 but with high sequence diversity in swine PB1. Our results suggest that mutations outside the PB1 conserved motifs may affect RNA replication and could be associated with influenza virus host adaptation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

DEP domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in regulating metabolism, growth, autophagy and apoptosis by functions as an endogenous inhibitor of mTOR signaling pathway. Activated by phosphatidic acid, a second messenger in mTOR signaling, DEPTOR dissociates from mTORC1 complex with unknown mechanism. Here, we present a 1.5 Å resolution crystal structure, which shows that the N-terminal two tandem DEP domains of hDEPTOR fold into a dumbbell-shaped structure, protruding the characteristic β-hairpin arms of DEP domains on each side. An 18 amino acids DDEX motif at the end of DEP2 interacts with DEP1 and stabilizes the structure. Biochemical studies showed that the tandem DEP domains directly interact with phosphatidic acid using two distinct positively charged patches. These results provide insights into mTOR activation upon phosphatidic acid stimulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Transmissible cancers are malignant cells that can spread between individuals of a population, akin to both a parasite and a mobile graft. The survival of the Tasmanian devil, the largest remaining marsupial carnivore, is threatened by the remarkable emergence of two independent lineages of transmissible cancer, devil facial tumour (DFT) 1 and devil facial tumour 2 (DFT2). To aid the development of a vaccine and to interrogate how histocompatibility barriers can be overcome, we analysed the peptides bound to major histocompatibility complex class I (MHC-I) molecules from Tasmanian devil cells and representative cell lines of each transmissible cancer. Here, we show that DFT1 + IFN-γ and DFT2 cell lines express a restricted repertoire of MHC-I allotypes compared with fibroblast cells, potentially reducing the breadth of peptide presentation. Comparison of the peptidomes from DFT1 + IFNγ, DFT2 and host fibroblast cells demonstrates a dominant motif, despite differences in MHC-I allotypes between the cell lines, with preference for a hydrophobic leucine residue at position 3 and position Ω of peptides. DFT1 and DFT2 both present peptides derived from neural proteins, which reflects a shared cellular origin that could be exploited for vaccine design. These results suggest that polymorphisms in MHC-I molecules between tumours and host can be 'hidden' by a common peptide motif, providing the potential for permissive passage of infectious cells and demonstrating complexity in mammalian histocompatibility barriers., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

Geographically, East Asia had the highest liver cancer burden in 2017. Besides this, liver cancer-related deaths were high in Japan, accounting for 3.90% of total deaths. The development of liver cancer is influenced by several factors, and genetic alteration is one of the critical factors among them. Therefore, the detailed mechanism driving the oncogenic transformation of liver cells needs to be elucidated. Recently, many researchers have focused on investigating the liver cancer genome and identified somatic mutations (MTs) of several transcription factors. In this line, next-generation sequencing of the cancer genome identified that oxidative stress-related transcription factor NRF2 (NFE2L2) is mutated in different cancers, including hepatocellular carcinoma (HCC). Here, we demonstrated that NRF2 DLG motif mutations (NRF2 D29A and L30F), found in Japanese liver cancer patients, upregulate the transcriptional activity of NRF2 in HCC cell lines. Moreover, the transcriptional activity of NRF2 mutations is not suppressed by KEAP1, presumably because NRF2 MTs disturb proper NRF2-KEAP1 binding and block KEAP1-mediated degradation of NRF2. Additionally, we showed that both MTs upregulate the transcriptional activity of NRF2 on the MMP9 promoter in Hepa1-6 and Huh7 cells, suggesting that MT derived gain-of-function of NRF2 may be important for liver tumor progression. We also found that ectopic overexpression of oncogenic BRAF WT and V600E increases the transcriptional activity of NRF2 WT on both the 3xARE reporter and MMP9 promoter. Interestingly, NRF2 D29A and L30F MTs with oncogenic BRAF V600E MT synergistically upregulate the transcription activity of NRF2 on the 3xARE reporter and MMP9 promoter in Hepa1-6 and Huh7 cells. In summary, our findings suggest that MTs in NRF2 have pathogenic effects, and that NRF2 MTs together with oncogenic BRAF V600E MT synergistically cause more aberrant transcriptional activity. The high activity of NRF2 MTs in HCC with BRAF MT warrants further exploration of the potential diagnostic, prognostic, and therapeutic utility of this pathway in HCC.

The transition of amniotes to a fully terrestrial lifestyle involved the adaptation of major molecular innovations to the epidermis, often in the form of epidermal appendages such as hair, scales and feathers. Feathers are diverse epidermal structures of birds, and their evolution has played a key role in the expansion of avian species to a wide range of lifestyles and habitats. As with other epidermal appendages, feather development is a complex process which involves many different genetic and protein elements. In mammals, many of the genetic elements involved in epidermal development are located at a specific genetic locus known as the epidermal differentiation complex (EDC). Studies have identified a homologous EDC locus in birds, which contains several genes expressed throughout epidermal and feather development. A family of avian EDC genes rich in aromatic amino acids that also contain MTF amino acid motifs (EDAAs/EDMTFs), that includes the previously reported histidine-rich or fast-protein (HRP/fp), an important marker in feather development, has expanded significantly in birds. Here, we characterize the EDAA gene family in birds and investigate the evolutionary history and possible functions of EDAA genes using phylogenetic and sequence analyses. We provide evidence that the EDAA gene family originated in an early archosaur ancestor, and has since expanded in birds, crocodiles and turtles, respectively. Furthermore, this study shows that the respective amino acid compositions of avian EDAAs are characteristic of structural functions associated with EDC genes and feather development. Finally, these results support the hypothesis that the genes of the EDC have evolved through tandem duplication and diversification, which has contributed to the evolution of the intricate avian epidermis and epidermal appendages.

DNA N6-methylation (6mA) in Adenine nucleotide is a post replication modification responsible for many biological functions. Automated and accurate computational methods can help to identify 6mA sites in long genomes saving significant time and money. Our study develops a convolutional neural network (CNN) based tool i6mA-CNN capable of identifying 6mA sites in the rice genome. Our model coordinates among multiple types of features such as PseAAC (Pseudo Amino Acid Composition) inspired customized feature vector, multiple one hot representations and dinucleotide physicochemical properties. It achieves auROC (area under Receiver Operating Characteristic curve) score of 0.98 with an overall accuracy of 93.97% using fivefold cross validation on benchmark dataset. Finally, we evaluate our model on three other plant genome 6mA site identification test datasets. Results suggest that our proposed tool is able to generalize its ability of 6mA site identification on plant genomes irrespective of plant species. An algorithm for potential motif extraction and a feature importance analysis procedure are two by products of this research. Web tool for this research can be found at: https://cutt.ly/dgp3QTR .

High mobility group A2 (HMGA2) is a chromatin-associated protein involved in the regulation of stem cell function, embryogenesis and cancer development. Although the protein does not contain a consensus SUMOylation site, it is shown to be SUMOylated. In this study, we demonstrate that the first lysine residue in the reported K 66 KAE SUMOylation motif in HMGA2 can be methylated in vitro and in vivo by the Set7/9 methyltransferase. By editing the lysine, the increased hydrophobicity of the resulting 6-N-methyl-lysine transforms the sequence into a consensus SUMO motif. This post-translational editing dramatically increases the subsequent SUMOylation of this site. Furthermore, similar putative methylation-dependent SUMO motifs are found in a number of other chromatin factors, and we confirm methylation-dependent SUMOylation of a site in one such protein, the Polyhomeotic complex 1 homolog (PHC1). Together, these results suggest that crosstalk between methylation and SUMOylation is a general mode for regulation of chromatin function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Significance: Unique to the branched-chain aminotransferase (BCAT) proteins is their redox-active CXXC motif. Subjected to post-translational modification by reactive oxygen species and reactive nitrogen species, these proteins have the potential to adopt numerous cellular roles, which may be fundamental to their role in oncogenesis and neurodegenerative diseases. An understanding of the interplay of the redox regulation of BCAT with important cell signaling mechanisms will identify new targets for future therapeutics. Recent Advances: The BCAT proteins have been assigned novel thiol oxidoreductase activity that can accelerate the refolding of proteins, in particular when S-glutathionylated, supporting a chaperone role for BCAT in protein folding. Other metabolic proteins were also shown to have peroxide-mediated redox associations with BCAT, indicating that the cellular function of BCAT is more diverse. Critical Issues: While the role of branched-chain amino acid metabolism and its metabolites has dominated aspects of cancer research, less is known about the role of BCAT. The importance of the CXXC motif in regulating the BCAT activity under hypoxic conditions, a characteristic of tumors, has not been addressed. Understanding how these proteins operate under various cellular redox conditions will become important, in particular with respect to their moonlighting roles. Future Directions: Advances in the quantification of thiols, their measurement, and the manipulation of metabolons that rely on redox-based interactions should accelerate the investigation of the cellular role of moonlighting proteins such as BCAT. Given the importance of cross talk between signaling pathways, research should focus more on these "housekeeping" proteins paying attention to their wider application. Antioxid. Redox Signal. 34, 1048-1067.

Activity-regulated cytoskeleton-associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self-assembly into virus-like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self-association and capsid formation is largely unknown. Here, we identified a 28-amino-acid stretch in the mammalian Arc N-terminal (NT) domain that is necessary and sufficient for self-association. Within this region, we identified a 7-residue oligomerization motif, critical for the formation of virus-like capsids. Purified wild-type Arc formed capsids as shown by transmission and cryo-electron microscopy, whereas mutant Arc with disruption of the oligomerization motif formed homogenous dimers. An atomic-resolution crystal structure of the oligomerization region peptide demonstrated an antiparallel coiled-coil interface, strongly supporting NT-NT domain interactions in Arc oligomerization. The NT coil-coil interaction was also validated in live neurons using fluorescence lifetime FRET imaging, and mutation of the oligomerization motif disrupted Arc-facilitated endocytosis. Furthermore, using single-molecule photobleaching, we show that Arc mRNA greatly enhances higher-order oligomerization in a manner dependent on the oligomerization motif. In conclusion, a helical coil in the Arc NT domain supports self-association above the dimer stage, mRNA-induced oligomerization, and formation of virus-like capsids. DATABASE: The coordinates and structure factors for crystallographic analysis of the oligomerization region were deposited at the Protein Data Bank with the entry code 6YTU., (© 2020 Federation of European Biochemical Societies.)

LcrF is the master regulator that positively regulates the Ysc type III secretion system (T3SS) in Yersinia and shares a high similarity with the DNA-binding domain of the T3SS master regulator ExsA in Pseudomonas aeruginosa. Based on these features, bioinformatics analysis has predicted a putative LcrF-binding site in its target promoters. Here, we experimentally characterized its binding motif. An adenine-rich LcrF-binding region in the lcrG promoter sequence, a typical regulatory target of LcrF, was first confirmed. To obtain detailed information, this binding region was cloned into a synthetized promoter and mutations in this region were further constructed. We demonstrated that the 5'-AAAAA-n5-GnCT-3' sequence is required for LcrF regulation and this motif is strictly located 4-bp upstream of a noncanonical promoter, in which the -35 and -10 elements are separated by a 21-bp spacer. Consistently, the putative binding motif was found in promoters of nine T3SS related operons or genes positively regulated by LcrF. Transcriptome analysis further confirmed that LcrF specifically activates T3SS genes in Yersinia. Collectively, our data suggest that LcrF has evolved to be a specific T3SS activator with a stringent sequence requirement for transcriptional regulation., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Gluten-specific CD4+ T cells are drivers of celiac disease (CeD). Previous studies of gluten-specific T-cell receptor (TCR) repertoires have found public TCRs shared across multiple individuals, biased usage of particular V-genes and conserved CDR3 motifs. The CDR3 motifs within the gluten-specific TCR repertoire, however, have not been systematically investigated. In the current study, we analyzed the largest TCR database of gluten-specific CD4+ T cells studied so far consisting of TCRs of 3122 clonotypes from 63 CeD patients. We established a TCR database from CD4+ T cells isolated with a mix of HLA-DQ2.5:gluten tetramers representing four immunodominant gluten epitopes. In an unbiased fashion we searched by hierarchical clustering for common CDR3 motifs among 2764 clonotypes. We identified multiple CDR3α, CDR3β, and paired CDR3α:CDR3β motif candidates. Among these, a previously known conserved CDR3β R-motif used by TRAV26-1/TRBV7-2 TCRs specific for the DQ2.5-glia-α2 epitope was the most prominent motif. Furthermore, we identified the epitope specificity of altogether 16 new CDR3α:CDR3β motifs by comparing with TCR sequences of 231 T-cell clones with known specificity and TCR sequences of cells sorted with single HLA-DQ2.5:gluten tetramers. We identified 325 public TCRα and TCRβ sequences of which 145, 102 and 78 belonged to TCRα, TCRβ and paired TCRαβ sequences, respectively. While the number of public sequences was depended on the number of clonotypes in each patient, we found that the proportion of public clonotypes from the gluten-specific TCR repertoire of given CeD patients appeared to be stable (median 37%). Taken together, we here demonstrate that the TCR repertoire of CD4+ T cells specific to immunodominant gluten epitopes in CeD is diverse, yet there is clearly biased V-gene usage, presence of public TCRs and existence of conserved motifs of which R-motif is the most prominent., Competing Interests: SD-K, LR, RN, AC, KL, S-WQ, GS and LS are holders of a patent application entitled "Method of diagnosing celiac disease" (US20210010077A1) on the use of the gluten-specific T cell receptor sequences described in the current work for diagnosis of celiac disease., (Copyright © 2021 Dahal-Koirala, Risnes, Neumann, Christophersen, Lundin, Sandve, Qiao and Sollid.)

De novo protein design has enabled the creation of new protein structures. However, the design of functional proteins has proved challenging, in part due to the difficulty of transplanting structurally complex functional sites to available protein structures. Here, we used a bottom-up approach to build de novo proteins tailored to accommodate structurally complex functional motifs. We applied the bottom-up strategy to successfully design five folds for four distinct binding motifs, including a bifunctionalized protein with two motifs. Crystal structures confirmed the atomic-level accuracy of the computational designs. These de novo proteins were functional as components of biosensors to monitor antibody responses and as orthogonal ligands to modulate synthetic signaling receptors in engineered mammalian cells. Our work demonstrates the potential of bottom-up approaches to accommodate complex structural motifs, which will be essential to endow de novo proteins with elaborate biochemical functions, such as molecular recognition or catalysis.

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

Ovine pulmonary adenomatosis (OPA) is caused by jaagsiekte sheep retrovirus (JSRV) and is a chronic, progressive, and infectious neoplastic lung disease in sheep, which causes significant economic losses to the sheep industry. Neither a vaccine nor serological diagnostic methods to detect OPA are available. We performed a JSRV infection survey in sheep using blood samples (n = 1,372) collected in the three northeastern provinces of China (i.e., Inner Mongolia, Heilongjiang, and Jilin) to determine JSRV infection status in sheep herds using a real-time PCR assay targeting the gag gene of JSRV. The ovine endogenous retrovirus sequence was successfully amplified in all sheep samples tested (296 from the Inner Mongolia Autonomous Region, 255 from Jilin province, and 821 from Heilongjiang province). Subsequently, we attempted to distinguish exogenous JSRV (exJSRV) and endogenous JSRV (enJSRV) infections in these JSRV-positive samples using a combination assay that identifies a ScaI restriction site in an amplified 229-bp fragment of the gag gene of JSRV and a "LHMKYXXM" motif in the cytoplasmic tail region of the JSRV envelope protein. The ScaI restriction site is present in all known oncogenic JSRVs but absent in ovine endogenous retroviruses, while the "LHMKYXXM" motif is in all known exJSRVs but not in enJSRVs. Interestingly, one JSRV strain (HH13) from Heilongjiang province contained the "LHMKYXXM" motif but not the ScaI enzyme site. Phylogenetic analysis showed that strain HH13 was closely related to strain enJSRV-21 reported in the USA, indicating that HH13 could be an exogenous virus. Our results provide valuable information for further research on the genetic evolution and pathogenesis of JSRV.

Low complexity regions (LCRs) are very frequent in protein sequences, generally having a lower propensity to form structured domains and tending to be much less evolutionarily conserved than globular domains. Their higher abundance in eukaryotes and in species with more cellular types agrees with a growing number of reports on their function in protein interactions regulated by post-translational modifications. LCRs facilitate the increase of regulatory and network complexity required with the emergence of organisms with more complex tissue distribution and development. Although the low conservation and structural flexibility of LCRs complicate their study, evolutionary studies of proteins across species have been used to evaluate their significance and function. To investigate how to apply this evolutionary approach to the study of LCR function in protein-protein interactions, we performed a detailed analysis for Huntingtin (HTT), a large protein that is a hub for interaction with hundreds of proteins, has a variety of LCRs, and for which partial structural information (in complex with HAP40) is available. We hypothesize that proteins RASA1, SYN2, and KAT2B may compete with HAP40 for their attachment to the core of HTT using similar LCRs. Our results illustrate how evolution might favor the interplay of LCRs with domains, and the possibility of detecting multiple modes of LCR-mediated protein-protein interactions with a large hub such as HTT when enough protein interaction data is available.

SAM68 is an mRNA-binding protein involved in mRNA processing in the nucleus that forms membraneless compartments called SAM68 Nuclear Bodies (SNBs). We found that intersectin 1 (ITSN1), a multidomain scaffold protein harboring five soluble SH3 domains, interacts with SAM68 proline-rich motifs (PRMs) surrounded by self-adhesive low complexity domains. While SAM68 is poorly soluble in vitro, the interaction of ITSN1 SH3 domains and mRNA with SAM68 enhances its solubility. In HeLa cells, the interaction between the first ITSN1 SH3 domain (SH3A) and P0, the N-terminal PRM of SAM68, induces the dissociation of SNBs. In addition, we reveal the ability of another SH3 domain (SH3D) of ITSN1 to bind to mRNAs. ITSN1 and mRNA may thus act in concert to promote SAM68 solubilization, consistent with the absence of mRNA in SNBs in cells. Together, these results support the notion of a specific chaperoning of PRM-rich SAM68 within nuclear ribonucleoprotein complexes by ITSN1 that may regulate the processing of a fraction of nuclear mRNAs, notably SAM68-controlled splicing events related to higher neuronal functions or cancer progression. This observation may also serve as a putative model of the interaction between other PRM-rich RBPs and signaling proteins harboring SH3 domains.

Immune checkpoint immunotherapy is a pillar of human oncology treatment with potential for non-human species. The first checkpoint immunotherapy approved for human cancers targeted the CTLA4 protein. CTLA4 can inhibit T cell activation by capturing and internalizing CD80 and CD86 from antigen presenting cells, a process called trans-endocytosis. Similarly, CD28 can capture CD80 and CD86 via trogocytosis and retain the captured ligands on the surface of the CD28-expressing cells. The wild Tasmanian devil (Sarcophilus harrisii) population has declined by 77% due to transmissible cancers that evade immune defenses despite genetic mismatches between the host and tumors. We used a live cell-based assay to demonstrate that devil CTLA4 and CD28 can capture CD80 and CD86. Mutation of evolutionarily conserved motifs in CTLA4 altered functional interactions with CD80 and CD86 in accordance with patterns observed in other species. These results suggest that checkpoint immunotherapies can be translated to evolutionarily divergent species., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Myxovirus resistance 1 (Mx1) gene plays an important role in uterine receptivity and conceptus development by creating a strong defense mechanism in the uterine environment. However, the specific role of Mx1 gene is not yet documented in the goat. Therefore, in the present study, full-length coding sequence (CDS) of the Mx1 gene was amplified, sequenced and characterized through various Bioinformatic tools. Temporal expression profile of Mx1 mRNA and protein was also examined by quantitative real-time PCR (qPCR) and western blot, respectively, in the endometrium of cyclic stage (non-pregnant), pregnancy stage I (16-24 days of gestation) and pregnancy stage II (25-40 days of gestation) of caprine (cp). A fragment of the cpMx1 gene, 2144 bp in length, was amplified from complementary DNA (cDNA) with a 1965 bp open reading frame. Coding and deduced amino acid sequences of the cpMx1 were aligned with other species and it exhibited 98.8-81.5 % identities with different species. On phylogenetic analysis, sheep and goat were found belonging to the same clade but differing from large ruminants. The cpMx1 protein possess the conserved signature motif (LPRGTGIVTR) of dynamin superfamily and the tripartite guanosine-5'-triphosphate (GTP) binding motif (GDQSSGKS, DLPG, TKPD) at the N-terminal end, and the leucine zipper motifs at the C-terminal end. Both cpMx1 mRNA and protein were found to be expressed maximally (P < 0.05) in the pregnancy stage I as compared to cyclic stage. It was concluded that the cpMx1 gene shares major structural and probably functional similarities with other species., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.

Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) to kill host cells. Here, we show that the WXG100 proteins EsxE and EsxF are essential for TNT secretion. EsxE and EsxF form a water-soluble heterodimer (EsxEF) that assembles into oligomers and long filaments, binds to membranes, and forms stable membrane-spanning channels. Electron microscopy of EsxEF reveals mainly pentameric structures with a central pore. Mutations of both WXG motifs and of a GXW motif do not affect dimerization, but abolish pore formation, membrane deformation and TNT secretion. The WXG/GXW mutants are locked in conformations with altered thermostability and solvent exposure, indicating that the WXG/GXW motifs are molecular switches controlling membrane interaction and pore formation. EsxF is accessible on the bacterial cell surface, suggesting that EsxEF form an outer membrane channel for toxin export. Thus, our study reveals a protein secretion mechanism in bacteria that relies on pore formation by small WXG proteins.

In the mouse, 129 functional class I odorant receptor (OR) genes reside in a ~ 3 megabase huge gene cluster on chromosome 7. The J element, a long-range cis-regulatory element governs the singular expression of class I OR genes by exerting its effect over the whole cluster. To elucidate the molecular mechanisms underlying class I-specific enhancer activity of the J element, we analyzed the J element sequence to determine the functional region and essential motif. The 430-bp core J element, that is highly conserved in mammalian species from the platypus to humans, contains a class I-specific conserved motif of AAACTTTTC, multiple homeodomain sites, and a neighboring O/E-like site, as in class II OR-enhancers. A series of transgenic reporter assays demonstrated that the class I-specific motif is not essential, but the 330-bp core J-H/O containing the homeodomain and O/E-like sites is necessary and sufficient for class I-specific enhancer activity. Further motif analysis revealed that one of homeodomain sequence is the Greek Islands composite motif of the adjacent homeodomain and O/E-like sequences, and mutations in the composite motif abolished or severely reduced class I-enhancer activity. Our results demonstrate that class I and class II enhancers share a functional motif for their enhancer activity.