ELM-the Eukaryotic Linear Motif resource-2024 update.

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.

Identification of a buried ß-strand as a novel disease-related motif in the human polysialyltransferases.

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 ß-strand underneath the known surface motifs unique to ST8SIA2 and ST8SIA4. Taken together, the P motif is a novel buried ß-strand that regulates the full activity of polysialyltransferases from the inside of the molecule.

Wheat gibberellin oxidase genes and their functions in regulating tillering.

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 (GA3) 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 GA3 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.

Genome-Wide Analysis and Expression Profiling of the Glutathione Peroxidase-like Enzyme Gene Family in Solanum tuberosum.

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.

Kinesins Modify ERR1-Dependent Transcription Using a Conserved Nuclear Receptor Box Motif.

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.

Siglec-6 Signaling Uses Src Kinase Tyrosine Phosphorylation and SHP-2 Recruitment.

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.

QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and 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.

Disentangling the recognition complexity of a protein hub using a nanopore.

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 (MLL4Win), a WDR5 ligand. Our approach reveals a broad dynamic range of MLL4Win-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.

Tousled-like kinase 2 targets ASF1 histone chaperones through client mimicry.

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.

An extended motif in the SARS-CoV-2 spike modulates binding and release of host coatomer in retrograde trafficking.

ß-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.

A novel antibody against the furin cleavage site of SARS-CoV-2 spike protein: Effects on proteolytic cleavage and ACE2 binding.

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.

The "LLQY" Motif on SARS-CoV-2 Spike Protein Affects S Incorporation into Virus Particles.

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.

Common sequence motifs of nascent chains engage the ribosome surface and trigger factor.

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.

Two Cholesterol Recognition Amino Acid Consensus Motifs of GP64 with Uncleaved Signal Peptide Are Required for Bombyx mori Nucleopolyhedrovirus Infection.

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.

Genetic fusions favor tumorigenesis through degron loss in oncogenes.

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.

Genomewide analysis of sperm whale E2 ubiquitin conjugating enzyme genes.

Marine mammals are exposed to the oxidative stress induced by hypoxia/reoxygenation cycles yet resist cellular damage. The availability of high-quality genomes promises to provide insights on how this is achieved. In this study, we considered the ubiquitinconjugating enzymes (E2) gene family, UBE2 genes, which encodes enzymes with critical roles in cellular physiology, including the oxidative stress response. The sperm whale was the first marine mammal with a chromosome-level genome, allowing the study of gene family repertories, phylogenetic relationships, chromosome gene organization, and other evolutionary patterns on a genomewide basis. Here, 39 UBE2 genes (similar to human, including 32 intact genes, one partial gene, six pseudogenes) were identified in sperm whale genome. These genes were found on 17 chromosomes and were assigned into 23 subfamilies, 16 subgroups, and four classes based on structural characteristics and functions, phylogeny and conserved domains, respectively. Although the gene structure and motif distribution of sperm whale UBE2 genes are conserved in each subfamily, motif variation and intron gain/loss may contribute to functional divergence. Segmental duplications were detected in six gene pairs, which could drive UBE2 gene innovation in the sperm whale. Contrasting seven cetaceans and five terrestrial taxa, we found that cetaceans have experienced shifts in selective constraint on UBE2 genes, which may contribute to oxidative stress tolerance during the adaptation to aquatic life. Our results provide the first comprehensive survey of cetacean UBE2 genes.

Epitope Profiling Reveals the Critical Antigenic Determinants in SARS-CoV-2 RBD-Based Antigen.

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 350VYAWN354 was a highly conserved epitope and exposed on the surface of SARS-CoV-2 S trimer, whereas 473YQAGSTP479 located in the receptor binding motif (RBM) was variable among different SARS-CoV-2 strains. 407VRQIAP412 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.

Mutations in the Methyltransferase Motifs of L Protein Attenuate Newcastle Disease Virus by Regulating Viral Translation and Cell-to-Cell Spread.

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.

Heparin-binding motif mutations of human diamine oxidase allow the development of a first-in-class histamine-degrading biopharmaceutical.

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).

Conserved Structural Motif Identified in Peptides That Bind to Geminivirus Replication Protein Rep.

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 ß-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-x1/2-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.