Search

Candida auris is an emerging fungal pathogen responsible for health care-associated outbreaks that arise from persistent surface and skin colonization. We characterized the arsenal of adhesins used by C. auris and discovered an uncharacterized adhesin, Surface Colonization Factor (Scf1), and a conserved adhesin, Iff4109, that are essential for the colonization of inert surfaces and mammalian hosts. SCF1 is apparently specific to C. auris, and its expression mediates adhesion to inert and biological surfaces across isolates from all five clades. Unlike canonical fungal adhesins, which function through hydrophobic interactions, Scf1 relies on exposed cationic residues for surface association. SCF1 is required for C. auris biofilm formation, skin colonization, virulence in systemic infection, and colonization of inserted medical devices.

Abstract Background Staphylococci cause a wide range of infections, including implant-associated infections which are difficult to treat due to the presence of biofilms. Whilst some proteins involved in biofilm formation are known, the differences in biofilm production between staphylococcal species remains understudied. Currently biofilm formation by Staphylococcus aureus is better understood than other members of the genus as more research has focused on this species. Results We assembled a panel of 385 non-aureus Staphylococcus isolates of 19 species from a combination of clinical sources and reference strains. We used a high-throughput crystal violet assay to assess the biofilm forming ability of all strains and assign distinct biofilm formation categories. We compared the prevalence of Pfam domains between the categories and used machine learning to identify amino acid 20-mers linked to biofilm formation. This identified some domains within proteins already linked to biofilm formation and important domains not previously linked to biofilm formation in staphylococci. RT-qPCR confirmed the expression of selected genes predicted to encode important domains within biofilms in Staphylococcus epidermidis. The prevalence and distribution of biofilm associated domains showed a link to phylogeny, suggesting different Staphylococcus species have independently evolved different mechanisms of biofilm production. Conclusions This work has identified different routes to biofilm formation in diverse species of Staphylococcus and suggests independent evolution of biofilm has occurred multiple times across the genus. Understanding the mechanisms of biofilm formation in any given species is likely to require detailed study of relevant strains and the ability to generalise across the genus may be limited.

Abstract Background One of the major hurdles in clinical genetics is interpreting the clinical consequences associated with germline missense variants in humans. Recent significant advances have leveraged natural variation observed in large-scale human populations to uncover genes or genomic regions that show a depletion of natural variation, indicative of selection pressure. We refer to this as “genetic constraint”. Although existing genetic constraint metrics have been demonstrated to be successful in prioritising genes or genomic regions associated with diseases, their spatial resolution is limited in distinguishing pathogenic variants from benign variants within genes. Methods We aim to identify missense variants that are significantly depleted in the general human population. Given the size of currently available human populations with exome or genome sequencing data, it is not possible to directly detect depletion of individual missense variants, since the average expected number of observations of a variant at most positions is less than one. We instead focus on protein domains, grouping homologous variants with similar functional impacts to examine the depletion of natural variations within these comparable sets. To accomplish this, we develop the Homologous Missense Constraint (HMC) score. We utilise the Genome Aggregation Database (gnomAD) 125 K exome sequencing data and evaluate genetic constraint at quasi amino-acid resolution by combining signals across protein homologues. Results We identify one million possible missense variants under strong negative selection within protein domains. Though our approach annotates only protein domains, it nonetheless allows us to assess 22% of the exome confidently. It precisely distinguishes pathogenic variants from benign variants for both early-onset and adult-onset disorders. It outperforms existing constraint metrics and pathogenicity meta-predictors in prioritising de novo mutations from probands with developmental disorders (DD). It is also methodologically independent of these, adding power to predict variant pathogenicity when used in combination. We demonstrate utility for gene discovery by identifying seven genes newly significantly associated with DD that could act through an altered-function mechanism. Conclusions Grouping variants of comparable functional impacts is effective in evaluating their genetic constraint. HMC is a novel and accurate predictor of missense consequence for improved variant interpretation.

Meiotic progression requires coordinated assembly and disassembly of protein complexes involved in chromosome synapsis and meiotic recombination. Mouse TRIP13 and its ortholog Pch2 are instrumental in remodeling HORMA domain proteins. HORMAD proteins are associated with unsynapsed chromosome axes but depleted from the synaptonemal complex (SC) of synapsed homologs. Here we report that TRIP13 localizes to the synapsed SC in early pachytene spermatocytes and to telomeres throughout meiotic prophase I. Loss of TRIP13 leads to meiotic arrest and thus sterility in both sexes. Trip13-null meiocytes exhibit abnormal persistence of HORMAD1 and HOMRAD2 on synapsed SC and chromosome asynapsis that preferentially affects XY and centromeric ends. These major phenotypes are consistent with reported phenotypes of Trip13 hypomorph alleles. Trip13 heterozygous mice exhibit meiotic defects that are less severe than the Trip13-null mice, showing that TRIP13 is a dosage-sensitive regulator of meiosis. Localization of TRIP13 to the synapsed SC is independent of SC axial element proteins such as REC8 and SYCP2/ SYCP3. Terminal FLAG-tagged TRIP13 proteins are functional and recapitulate the localization of native TRIP13 to SC and telomeres. Therefore, the evolutionarily conserved localization of TRIP13/ Pch2 to the synapsed chromosomes provides an explanation for dissociation of HORMA domain proteins upon synapsis in diverse organisms. [ABSTRACT FROM AUTHOR]

The SET domain genes (SDGs) are significant contributors to various aspects of plant growth and development, mainly includes flowering, pollen development, root growth, regulation of the biological clock and branching patterns. To clarify the biological functions of the chrysanthemum SDG family, the SDG family members of four chrysanthemum cultivars and three related wild species were identified; their physical and chemical properties, protein domains and conserved motifs were predicted and analyzed. The results showed that 59, 67, 67, 102, 106, 114, and 123 SDGs were identified from Chrysanthemum nankingense, Chrysanthemum lavandulifolium, Chrysanthemum seticuspe, Chrysanthemum × morifolium cv. 'Hechengxinghuo', 'Zhongshanzigui', 'Quanxiangshuichang' and 'Jinbeidahong', respectively. The SDGs were divided into 5–7 subfamilies by cluster analysis; different conserved motifs were observed in particular families. The SDGs of C. lavandulifolium and C. seticuspe were distributed unevenly on 9 chromosomes. SDG promoters of different species include growth and development, photo-response, stress response and hormone responsive elements, among them, the cis-acting elements related to MeJA response had the largest proportion. The expression of chrysanthemum SDG genes was observed for most variable selected genes which has close association with important Arabidopsis thaliana genes related to flowering regulation. The qPCR results showed that the expression trend of SDG genes varied in different tissues at different growth stages with high expression in the flowering period. The ClSDG29 showed higher expression in the flower and bud tissues, which indicate that ClSDG29 might be associated with flowering regulation in chrysanthemum. In summary, the results of this study can provide a basis for subsequent research on chrysanthemum flowering time regulation. [ABSTRACT FROM AUTHOR]

Background: Staphylococci cause a wide range of infections, including implant-associated infections which are difficult to treat due to the presence of biofilms. Whilst some proteins involved in biofilm formation are known, the differences in biofilm production between staphylococcal species remains understudied. Currently biofilm formation by Staphylococcus aureus is better understood than other members of the genus as more research has focused on this species. Results: We assembled a panel of 385 non-aureus Staphylococcus isolates of 19 species from a combination of clinical sources and reference strains. We used a high-throughput crystal violet assay to assess the biofilm forming ability of all strains and assign distinct biofilm formation categories. We compared the prevalence of Pfam domains between the categories and used machine learning to identify amino acid 20-mers linked to biofilm formation. This identified some domains within proteins already linked to biofilm formation and important domains not previously linked to biofilm formation in staphylococci. RT-qPCR confirmed the expression of selected genes predicted to encode important domains within biofilms in Staphylococcus epidermidis. The prevalence and distribution of biofilm associated domains showed a link to phylogeny, suggesting different Staphylococcus species have independently evolved different mechanisms of biofilm production. Conclusions: This work has identified different routes to biofilm formation in diverse species of Staphylococcus and suggests independent evolution of biofilm has occurred multiple times across the genus. Understanding the mechanisms of biofilm formation in any given species is likely to require detailed study of relevant strains and the ability to generalise across the genus may be limited. [ABSTRACT FROM AUTHOR]

This review discusses how research around the oxysterol-binding protein family has evolved. We briefly summarize how this protein family, designated OSBP-related (ORP) or OSBP-like (OSBPL) proteins, was discovered, how protein domains highly conserved among family members between taxa paved the way for understanding their mechanisms of action, and how insights into protein structural and functional features help to understand their versatility as lipid transporters. We also discuss questions and future avenues of research opened by these findings. The investigations on oxysterol-binding protein family serve as a real-life example of the notion that science often advances as a collective effort of multiple lines of enquiry, including serendipitous routes. While original articles invariably explain the motivation of the research undertaken in rational terms, the actual paths to findings may be less intentional. Fortunately, this does not reduce the impact of the discoveries made. Besides hopefully providing a useful account of ORP family proteins, we aim to convey this message. [ABSTRACT FROM AUTHOR]

Anthracnose (ANTH) caused by Colletotrichum lindemuthianum is a major disease of common bean (Phaseolus vulgaris L.). The genetic basis of ANTH resistance in the Middle American Diversity Panel (MDP) is unknown. The objectives of this study were to identify (1) Middle American accessions resistant to races 7, 19, 51, 63, 167, and 1085 of C. lindemuthianum and (ii) genomic regions and positional candidate genes associated with resistance to these races. The MDP composed of 240 Middle American accessions was evaluated for resistance to races 7, 19, 51, 63, 167, and 1085. The MDP was genotyped with 211,763 single nucleotide polymorphisms (SNPs), and mixed linear model analysis was conducted to identify genomic regions associated with resistance to the six races. Seven accessions were highly resistant to all six races, and these can be used as sources of resistance to improve specific market classes in the Middle American gene pool. The genomic region (385,894 bp) on chromosome Pv04 was significantly associated with resistance to race 167. Genomic regions on Pv02 (41,570,325 bp), Pv07 (24,122,343 bp), and Pv11 (51,707,917 bp) were significantly associated with resistance to race 19. Disease resistance (R) genes with the nucleotide binding‐APAF resistance protein and CED‐4 domain were identified as positional candidate genes on Pv04 and Pv11. There were no SNPs significantly associated with resistance to races 7, 51, 63, and 1085. Pyramiding the identified genomic regions on Pv04, Pv07, and Pv11 could provide durable ANTH resistance in Middle American varieties for races 19 and 167. [ABSTRACT FROM AUTHOR]

The Drosophila corneal lens is entirely composed of chitin and other apical extracellular matrix components, and it is not known how it acquires the biconvex shape that enables it to focus light onto the retina. We show here that the zona pellucida domain-containing protein Dusky-like is essential for normal corneal lens morphogenesis. Dusky-like transiently localizes to the expanded apical surfaces of the corneal lens-secreting cells and prevents them from undergoing apical constriction and apicobasal contraction. Dusky-like also controls the arrangement of two other zona pellucida domain proteins, Dumpy and Piopio, external to the developing corneal lens. Loss of either dusky-like or dumpy delays chitin accumulation and disrupts the outer surface of the corneal lens. We find that artificially inducing apical constriction by activating myosin contraction is sufficient to similarly alter chitin deposition and corneal lens morphology. These results demonstrate the importance of cell shape in controlling the morphogenesis of overlying apical extracellular matrix structures such as the corneal lens. [ABSTRACT FROM AUTHOR]

SCY1‐like protein 2 (SCYL2) is a member of the SCY1‐like pseudokinase family which regulates secretory protein trafficking. It plays a crucial role in the nervous system by suppressing excitotoxicity in the developing brain. Scyl2 knockout mice have excess prenatal mortality and survivors show severe neurological dysfunction. Bi‐allelic loss‐of‐function (LOF) variants in SCYL2 were recently associated with arthrogryposis multiplex congenita‐4 (AMC4) following the report of 6 individuals from two consanguineous unrelated families. The AMC4 phenotype described included severe arthrogryposis, corpus callosum agenesis, epilepsy and frequently, early death. We describe here two additional similarly affected individuals with AMC4, including one diagnosed in the prenatal period, with bi‐allelic LOF variants in SCYL2, and two individuals homozygous for missense variants in the protein kinase domain of SCYL2 and presenting with developmental delay only. Our study confirms the association of SCYL2 with AMC4 and suggests a milder phenotype can occur, extending the phenotypic spectrum of autosomal recessive SCYL2‐related disorders. [ABSTRACT FROM AUTHOR]

The most extensively studied β‐d‐galactosidases (EC3.2.1.23) belonging to four glycoside hydrolase (GH) families 1, 2, 35, and 42 are widely distributed among Bacteria, Archaea and Eukaryotes. Here, we report a novel GH35 family β‐galactosidase from the hyperthermophilic Thermoprotei archaeon Desulfurococcus amylolyticus (DaβGal). Unlike fungal monomeric six‐domain β‐galactosidases, the DaβGal enzyme is a dimer; it has an extra jelly roll domain D7 and three composite domains (D4, D5, and D6) that are formed by the distantly located polypeptide chain regions. The enzyme possesses a high specificity for β‐d‐galactopyranosides, and its distinguishing feature is the ability to cleave pNP‐β‐d‐fucopyranoside. DaβGal efficiently catalyzes the hydrolysis of lactose at high temperatures, remains stable and active at 65 °С, and retains activity at 95 °С with a half‐life time value equal to 73 min. These properties make archaeal DaβGal a more attractive candidate for biotechnology than the widely used fungal β‐galactosidases. [ABSTRACT FROM AUTHOR]

Arsenic (As) has been classified as a carcinogen for humans. There is abundant evidence indicating that arsenic increases the risk of bladder cancer among human populations. However, the underlying mechanisms have yet to be fully understood and elucidated. NADPH oxidases (NOXs) are the main enzymes for ROS production in the body. NADPH Oxidase 2 (NOX2), which is the most distinctive and ubiquitously expressed subunit of NOXs, can promote the formation and development of tumors. The utilization of NOX2 as a therapeutic target has been proposed to modulate diseases resulting from the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3). Matrine has been reported to exhibit various pharmacological effects, including anti-inflammatory, antifibrotic, antitumor, and analgesic properties. However, it has not been reported whether matrine can inhibit malignant transformation induced by arsenic in uroepithelial cells through NOX2. We have conducted a series of experiments using both a sub-chronic NaAsO2 exposure rat model and a long-term NaAsO2 exposure cell model. Our findings indicate that arsenic significantly increases cell proliferation, migration, and angiogenesis in vivo and in vitro. Arsenic exposure resulted in an upregulation of reactive oxygen species (ROS), NOX2, and NLRP3 inflammasome expression. Remarkably, both in vivo and in vitro, the administration of matrine demonstrated a significant improvement in the detrimental impact of arsenic on bladder epithelial cells. This was evidenced by the downregulation of proliferation, migration, and angiogenesis, as well as the expression of the NOX2 and NLRP3 inflammasomes. Collectively, these findings indicate that matrine possesses the ability to reduce NOX2 levels and inhibit the transformation of bladder epithelial cells. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by symptoms such as bradykinesia, resting tremor, and rigidity, primarily driven by the degradation of dopaminergic (DA) neurons in the substantia nigra. A significant contributor to familial autosomal dominant PD cases is mutations in the LRRK2 gene, making it a primary therapeutic target. This study explores the role of microRNAs (miRNAs) in regulating the proteomic stress responses associated with neurodegeneration in PD using C. elegans models. Our focus is on miR-71, a miRNA known to affect stress resistance and act as a pro-longevity factor in C. elegans. We investigated miR-71's function in C. elegans models of PD, where mutant LRRK2 expression correlates with dopaminergic neuronal death. Our findings reveal that miR-71 overexpression rescues motility defects and slows dopaminergic neurodegeneration in these models, suggesting its critical role in mitigating the proteotoxic effects of mutant LRRK2. Conversely, miR-71 knockout exacerbates neuronal death caused by mutant LRRK2. Additionally, our data indicate that miR-71's neuroprotective effect involves downregulating the toll receptor domain protein tir-1, implicating miR-71 repression of tir-1 as vital in the response to LRRK2-induced proteotoxicity. These insights into miR-71's role in C. elegans models of PD not only enhance our understanding of molecular mechanisms in neurodegeneration but also pave the way for potential research into human neurodegenerative diseases, leveraging the conservation of miRNAs and their targets across species. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid–liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated. [ABSTRACT FROM AUTHOR]

Current tools for functionally profiling T cell receptors with respect to cytotoxic potency and cross-reactivity are hampered by difficulties in establishing model systems to test these proteins in the contexts of different HLA alleles and against broad arrays of potential antigens. We have implemented a granzyme-activatable sensor of T cell cytotoxicity in a universal prototyping platform which enables facile recombinant expression of any combination of TCR-, peptide-, and class I MHC-coding sequences and direct assessment of resultant responses. This system consists of an engineered cell platform based on the immortalized natural killer cell line, YT-Indy, and the MHC-null antigen-presenting cell line, K562. These cells were engineered to furnish the YT-Indy/K562 pair with appropriate protein domains required for recombinant TCR expression and function in a non-T cell chassis, integrate a fluorescence-based target-centric early detection reporter of cytotoxic function, and deploy a set of protective genetic interventions designed to preserve antigen-presenting cells for subsequent capture and downstream characterization. Our data show successful reconstitution of the surface TCR complex in the YT-Indy cell line at biologically relevant levels. We also demonstrate successful induction and highly sensitive detection of antigen-specific response in multiple distinct model TCRs. Additionally, we monitored destruction of targets in co-culture and found that our survival-optimized system allowed for complete preservation after 24 h exposure to cytotoxic effectors. With this bioplatform, we anticipate investigators will be empowered to rapidly express and characterize T cell receptor responses, generate knowledge regarding the patterns of T cell receptor recognition, and optimize therapeutic T cell receptors. [ABSTRACT FROM AUTHOR]

Rationale: Idiopathic pulmonary fibrosis (IPF) causes irreversible fibrosis of the lung parenchyma. Although antifibrotic therapy can slow IPF progression, treatment response is variable. There exists a critical need to develop a precision medicine approach to IPF. Objectives: To identify and validate biologically driven molecular endotypes of IPF. Methods: Latent class analysis (LCA) was independently performed in prospectively recruited discovery (n = 875) and validation (n = 347) cohorts. Twenty-five plasma biomarkers associated with fibrogenesis served as class-defining variables. The association between molecular endotype and 4-year transplant-free survival was tested using multivariable Cox regression adjusted for baseline confounders. Endotype-dependent differential treatment response to future antifibrotic exposure was then assessed in a pooled cohort of patients naive to antifibrotic therapy at the time of biomarker measurement (n = 555). Measurements and Main Results: LCA independently identified two latent classes in both cohorts (P < 0.0001). WFDC2 (WAP four-disulfide core domain protein 2) was the most important determinant of class membership across cohorts. Membership in class 2 was characterized by higher biomarker concentrations and a higher risk of death or transplant (discovery, hazard ratio [HR], 2.02; 95% confidence interval [CI], 1.64–2.48; P < 0.001; validation, HR, 1.95; 95% CI, 1.34–2.82; P < 0.001). In pooled analysis, significant heterogeneity in treatment effect was observed between endotypes (P = 0.030 for interaction), with a favorable antifibrotic response in class 2 (HR, 0.64; 95% CI, 0.45–0.93; P = 0.018) but not in class 1 (HR, 1.19; 95% CI, 0.77–1.84; P = 0.422). Conclusions: In this multicohort study, we identified two novel molecular endotypes of IPF with divergent clinical outcomes and responses to antifibrotic therapy. Pending further validation, these endotypes could enable a precision medicine approach for future IPF clinical trials. [ABSTRACT FROM AUTHOR]

The Synaptotagmin-like mitochondrial-lipid binding protein (SMP) domain is found in a group of ER-resident lipid transfer proteins that are recruited to membrane contact sites (MCSs) by adaptors. Deciphering the molecular basis underlying the recruitment of SMP proteins to specific MCS sheds light not only on their cellular localization but also on their biological functions at these sites. Here we summarize recent advances in SMP domain-containing lipid transfer proteins, focusing on a recent study showing the localization, regulation and cellular function of a specific SMP protein named testis expressed protein 2 (Tex2). TMEM55, a potential PIP phosphatase on late endosome/lysosomal (LE/lys) membranes, was identified as an adaptor that enables the recruitment of Tex2 to ER- LE/lys MCS. In addition, we have summarized several important questions about the regulation and physiological functions of Tex2 that remained unanswered. [ABSTRACT FROM AUTHOR]

The mechanisms of plant drought resistance are unclear but may involve membrane trafficking and metabolic reprogramming, including proline accumulation. Forward genetic screening using a proline dehydrogenase 1 (ProDH1) promoter:reporter identified a drought hypersensitive mutant with a single-amino acid substitution (P335L) in the nonphototrophic hypocotyl 3 (NPH3) domain of NPH3/root phototropism 2-like 5 (NRL5)/naked pins in Yucca 8 (NPY8). Further experiments found that NRL5 and other NPH3 domain proteins are guanosine triphosphatases (GTPases). NRL5, but not NRL5P335L, interacted with the RABE1c and RABH1b GTPases and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Vesicle-Associated Membrane Protein (VAMP)721/722. These proteins controlled NRL5 localization and connection to trafficking while also being genetically downstream of, and potentially regulated by, NRL5. These data demonstrate that NRL5-mediated restraint of proline catabolism is required for drought resistance and also reveal unexpected functions of the NPH3 domain such that the role of NPH3 domain proteins in signaling, trafficking, and cellular polarity can be critically reevaluated. [ABSTRACT FROM AUTHOR]

The cell has multiple mechanisms for sensing and responding to dynamic changes in the mechanical environment. In the process, intracellular signaling is activated to modulate gene expression. Recent studies have shown that multifunctional signaling molecules that link intracellular force and gene expression are important for understanding cellular functions in the mechanical environment. This review discusses recent studies on one of the mechanotransducers, Fourand-a-half LIM domains 2 (FHL2), which localizes to focal adhesions (FAs), actin cytoskeleton, and nucleus. FHL2 localizes to FAs and the actin cytoskeleton in the cell on stiff substrate. In this situation, intracellular tension of F-actin by Myosin II is critical for FHL2 localization to FAs and actin stress fibers. In the case, a conserved phenylalanine in each LIM domain is responsible for its localization to F-actin. On the other hand, lower tension of F-actin in the cell on a soft substrate causes FHL2 to be released into the cytoplasm, resulting in its localization in the nucleus. At the molecular level, phosphorylation of specific tyrosine in FHL2 by FAK, non-receptor tyrosine kinase, is critical to nuclear localization. Finally, by binding to transcription factors, FHL2 modulates gene expression for cell proliferation as a transcriptional co-factor. Thus, FHL2 is involved inmechano-sensing and -transduction in the cell in a mechanical environment. [ABSTRACT FROM AUTHOR]

Introduction: Research on Glycyrrhiza uralensis, a nonhalophyte that thrives in saline-alkaline soil and a traditional Chinese medicinal component, is focused on improving its ability to tolerate salt stress to increase its productivity and preserve its "Dao-di" characteristics. Furthermore, the inoculation of bioagents such as Bacillus subtilis to increase plant responses to abiotic stressors is currently a mainstream strategy. Mitogen-activated protein kinase (MAPK), a highly conserved protein kinase, plays a significant role in plant responses to various abiotic stress pathways. Methods: This investigation involved the identification of 21 members of the GuMAPK family from the genome of G. uralensis, with an analysis of their protein conserved domains, gene structures, evolutionary relationships, and phosphorylation sites using bioinformatics tools. Results: Systematic evolutionary analysis of the 21 GuMAPKs classified them into four distinct subgroups, revealing significant differences in gene structure and exon numbers. Collinearity analysis highlighted the crucial role of segmental duplication in expanding the GuMAPK gene family, which is particularly evident in G. uralensis and shows a close phylogenetic relationship with Arabidopsis thaliana, tomato, and cucumber. Additionally, the identification of phosphorylation sites suggests a strong correlation between GuMAPK and various physiological processes, including hormonal responses, stress resistance, and growth and development. Protein interaction analysis further supported the role of GuMAPK proteins in regulating essential downstream genes. Through examination of transcriptome expression patterns, GuMAPK16-2 emerged as a prospective pivotal regulatory factor in the context of salt stress and B. subtilis inoculation, a finding supported by its subcellular localization within the nucleus. Discussion: These discoveries offer compelling evidence for the involvement of GuMAPK in the salt stress response and for the exploration of the mechanisms underlying B. subtilis' enhancement of salt tolerance in G. uralensis. [ABSTRACT FROM AUTHOR]

Powerful associations that link drugs of abuse with cues in the drug-paired environment often serve as prepotent relapse triggers. Drug-associated contexts and cues activate ensembles of nucleus accumbens (NAc) neurons, including D1-class medium spiny neurons (MSNs) that typically promote, and D2-class MSNs that typically oppose, drug seeking. We found that in mice, cocaine conditioning upregulated transiently the activity-regulated transcription factor, Neuronal PAS Domain Protein 4 (NPAS4), in a small subset of NAc neurons. The NPAS4+ NAc ensemble was required for cocaine conditioned place preference. We also observed that NPAS4 functions within NAc D2-, but not D1-, MSNs to support cocaine-context associations and cue-induced cocaine, but not sucrose, seeking. Together, our data show that the NPAS4+ ensemble of NAc neurons is essential for cocaine-context associations in mice, and that NPAS4 itself functions in NAc D2-MSNs to support cocaine-context associations by suppressing drug-induced counteradaptations that oppose relapse-related behaviour. Neuronal PAS domain protein 4 (NPAS4) is an immediate early gene, but its role in striatal circuits in drug seeking behaviour is not understood. Here the authors show that exposure to cocaine in mice results in induction of NPAS4 in a subset of nucleus accumbens (NAc) neurons, and in D2-neurons of the NAc it contributes to cocaine-associations and cocaine seeking behaviour. [ABSTRACT FROM AUTHOR]

Previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and vii-ulence in many gram-negative bacteria. However, the role of T6SS in vii ulence in Pectobacterium atr<>septicum remains controversial. We examined a closely related strain, PccS 1, and discovered that its T6SS comprises a single-copy cluster of 17 core genes with ahigher identity to homologs from/1 atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS 1, we found that three of the five VgrGs could be classified into group 1 Vit ·Gs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and Previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and vii-ulence in many gram-negative bacteria. However, the role of T6SS in vii ulence in Pectobacterium atr<>septicum remains controversial. We examined a closely related strain, PccS 1, and discovered that its T6SS comprises a single-copy cluster of 17 core genes with ahigher identity to homologs from/1 atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS 1, we found that three of the five VgrGs could be classified into group 1 Vit ·Gs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either the T6SS or type IV secretion system (T+SS). This interaction directly governed vii·ulence and competition. Meanwhile, supernatant proteomic analyses with strains defective in the T6SS and/or T4SS confirmed that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose deposition in the course of infection. Notably. this redundant secretion mechanism between the T6SS and T4SS is believed to be the first of its kind in bacteria. [ABSTRACT FROM AUTHOR]

Transcription Termination Factor 1 (TTF1) is a multifunctional mammalian protein with vital roles in various cellular processes, including Pol I-mediated transcription initiation and termination, pre-rRNA processing, chromatin remodelling, DNA damage repair, and polar replication fork arrest. It comprises two distinct functional regions; the N-terminal regulatory region (1-445 aa), and the C-terminal catalytic region (445-859 aa). The Myb domain located at the C-terminal region is a conserved DNA binding domain spanning from 550 to 732 aa (183 residues). Despite its critical role in various cellular processes, the physical structure of TTF1 remains unsolved. Attempts to purify the functional TTF1 protein have been unsuccessful till date. Therefore, we focused on characterizing the Myb domain of this essential protein. We started with predicting a 3-D model of the Myb domain using homology modelling, and ab-initio method. We then determined its stability through MD simulation in an explicit solvent. The model predicted is highly stable, which stabilizes at 200ns. To experimentally validate the computational model, we cloned and expressed the codon optimized Myb domain into a bacterial expression vector and purified the protein to homogeneity. Further, characterization of the protein shows that, Myb domain is predominantly helical (65%) and is alone sufficient to bind the Sal Box DNA. This is the first-ever study to report a complete in silico model of the Myb domain, which is physically characterized. The above study will pave the way towards solving the atomic structure of this essential mammalian protein. [ABSTRACT FROM AUTHOR]

Plant height, spike, leaf, stem and grain morphologies are key components of plant architecture and related to wheat yield. A wheat (Triticum aestivum L.) mutant, wpa1, displaying temperature-dependent pleiotropic developmental anomalies, was isolated. The WPA1 gene, encoding a von Willebrand factor type A (vWA) domain protein, was located on chromosome arm 7DS and isolated by map-based cloning. The functionality of WPA1 was validated by multiple independent EMS-induced mutants and gene editing. Phylogenetic analysis revealed that WPA1 is monocotyledon-specific in higher plants. The identification of WPA1 provides opportunity to study the temperature regulated wheat development and grain yield. [ABSTRACT FROM AUTHOR]

Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation, and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain of SulM, which catalyzes an unusual β-lactam–forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for β-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the β-lactone–forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized β-lactam–forming biosynthetic gene clusters by genome mining. [ABSTRACT FROM AUTHOR]

Isoforms of microtubule-associated protein 2 (MAP2) differ from their homolog Tau in the sequence and interactions of the N-terminal region. Binding of the N-terminal region of MAP2c (N-MAP2c) to the dimerization/docking domains of the regulatory subunit RIIa of cAMP-dependent protein kinase (RIIDD2) and to the Src-homology domain 2 (SH2) of growth factor receptor-bound protein 2 (Grb2) have been described long time ago. However, the structural features of the complexes remained unknown due to the disordered nature of MAP2. Here, we provide structural description of the complexes. We have solved solution structure of N-MAP2c in complex with RIIDD2, confirming formation of an amphiphilic α-helix of MAP2c upon binding, defining orientation of the ahelix in the complex and showing that its binding register differs from previous predictions. Using chemical shift mapping, we characterized the binding interface of SH2-Grb2 and rat MAP2c phosphorylated by the tyrosine kinase Fyn in their complex and proposed a model explaining differences between SH2-Grb2 complexes with rat MAP2c and phosphopeptides with a Grb2-specific sequence. The results provide the structural basis of a potential role of MAP2 in regulating cAMP-dependent phosphorylation cascade via interactions with RIIDD2 and Ras signaling pathway via interactions with SH2-Grb2. [ABSTRACT FROM AUTHOR]

Src homology 3 (SH3) domains play a critical role in mediating protein–protein interactions (PPIs) involved in cell proliferation, migration, and the cytoskeleton. Despite their abundance in the human proteome, the functions and molecular interactions of many SH3 domains remain unknown, and this is in part due to the lack of SH3-domain-specific reagents available for their study. Affimer proteins have been developed as affinity reagents targeting a diverse range of targets, including those involved in PPIs. In this study, Affimer proteins were isolated against both the N- and C-terminal SH3 domains (NSH3 and CSH3) of growth-factor-receptor-bound protein 2 (Grb2), an adapter protein that provides a critical link between cell surface receptors and Ras signalling pathways. Targeting the CSH3 alone for the inhibition of PPIs appeared sufficient for curtailing Ras signalling in mammalian cell lines stimulated with human epidermal growth factor (EGF), which conflicts with the notion that the predominant interactions with Ras activating Son of sevenless (SOS) occur via the NSH3 domain. This result supports a model in which allosteric mechanisms involved in Grb2-SOS1 interaction modulate Ras activation. [ABSTRACT FROM AUTHOR]

Simple Summary: CRM domain proteins play key roles in plant growth and chloroplast development. To explore the role of CRM domain protein in the chloroplast development of rice, we cloned a rice CRM domain protein gene, YL4, and then conducted a tissue-specific expression analysis, subcellular localization, and transcription analysis. The experimental results showed that YL4 was highly expressed in leaves and localized to the chloroplast, and its mutation affected chloroplast-related gene transcription levels and some agronomic traits. These results indicate that the YL4 gene is a pivotal regulatory factor in chloroplast development and rice growth. Chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are a family of plant-specific proteins associated with RNA binding. In this study, we have conducted a detailed characterization of a novel rice CRM gene (LOC_Os04g39060) mutant, yl4, which showed yellow-green leaves at all the stages, had fewer tillers, and had a decreased plant height. Map-based cloning and CRISPR/Cas9 editing techniques all showed that YL4 encoded a CRM domain protein in rice. In addition, subcellular localization revealed that YL4 was in chloroplasts. YL4 transcripts were highly expressed in all leaves and undetectable in roots and stems, and the mutation of YL4 affected the transcription of chloroplast-development-related genes. This study indicated that YL4 is essential for chloroplast development and affects some agronomic traits. [ABSTRACT FROM AUTHOR]

Lectins are sugar interacting proteins which bind specific glycans reversibly and have ubiquitous presence in all forms of life. They have diverse biological functions such as cell signaling, molecular recognition, etc. C-type lectins (CTL) are a group of proteins from the lectin family which have been studied extensively in animals and are reported to be involved in immune functions, carcinogenesis, cell signaling, etc. The carbohydrate recognition domain (CRD) in CTL has a highly variable protein sequence and proteins carrying this domain are also referred to as C-type lectin domain containing proteins (CTLD). Because of this low sequence homology, identification of CTLD from hypothetical proteins in the sequenced genomes using homology based programs has limitations. Machine learning (ML) tools use characteristic features to identify homologous sequences and it has been used to develop a tool for identification of CTLD. Initially 500 sequences of well annotated CTLD and 500 sequences of non CTLD were used in developing the machine learning model. The classifier program Linear SVC from sci kit library of python was used and characteristic features in CTLD sequences like dipeptide and tripeptide composition were used as training attributes in various classifiers. A precision, recall and multiple correlation coefficient (MCC) value of 0.92, 0.91 and 0.82 respectively were obtained when tested on external test set. On fine tuning of the parameters like kernel, C value, gamma, degree and increasing number of non CTLD sequences there was improvement in precision, recall and MCC and the corresponding values were 0.99, 0.99 and 0.96. New CTLD have also been identified in the hypothetical segment of human genome using the trained model. The tool is available on our local server for interested users. [ABSTRACT FROM AUTHOR]

The interaction between SARS-CoV PDZ-binding motifs (PBMs) and cellular PDZs is responsible for virus virulence. The PBM sequence present in the 3a and envelope (E) proteins of SARS-CoV can potentially bind to over 400 cellular proteins containing PDZ domains. The role of SARS-CoV 3a and E proteins was studied. SARS-CoVs, in which 3a-PBM and E-PMB have been deleted (3a-PBM-/E-PBM-), reduced their titer around one logarithmic unit but still were viable. In addition, the absence of the E-PBM and the replacement of 3a-PBM with that of E did not allow the rescue of SARS-CoV. E protein PBM was necessary for virulence, activating p38-MAPK through the interaction with Syntenin-1 PDZ domain. However, the presence or absence of the homologous motif in the 3a protein, which does not bind to Syntenin-1, did not affect virus pathogenicity. Mutagenesis analysis and in silico modeling were performed to study the extension of the PBM of the SARS-CoV E protein. Alanine and glycine scanning was performed revealing a pair of amino acids necessary for optimum virus replication. The binding of E protein with the PDZ2 domain of the Syntenin-1 homodimer induced conformational changes in both PDZ domains 1 and 2 of the dimer. [ABSTRACT FROM AUTHOR]

This study presents in a systematic manner the key modeling results corroborated by experimental biophysical data and obtained by the authors during long-term research on bitopic (single-pass) membrane proteins (BMPs), which are the crucial elements of cell signaling. The manuscript does not claim to be a comprehensive review of the subject of interest, whereby the authors did not aim to describe accurately the current state of the art, given the numerous reliable publications. Rather, this is an essay illustrating the authors' understanding of the basic principles of organization of protein transmembrane domains (TMDs) and of their contribution to the cell functioning. Among the key topics highlighted in the present study are the fine-tuned processes of TMD oligomerization and the direct contribution from the dynamic membrane environment to this process, as well as the key role of TMD in the functioning of cell receptors and mutual relations between all the components of proteinmembrane complexes during the signal transduction in normal and pathological conditions. [ABSTRACT FROM AUTHOR]

Numerous studies have been devoted to individual cases of horizontally acquired genes in fungi. It has been shown that such genes expand the hosts' metabolic capabilities and contribute to their adaptations as parasites or symbionts. Some studies have provided an extensive characterization of the horizontal gene transfer (HGT) in Dikarya. However, in the early diverging fungi (EDF), a similar characterization is still missing. In order to fill this gap, we have designed a computational pipeline to obtain a statistical sample of reliable HGT events with a low false discovery rate. We have analyzed 44 EDF proteomes and identified 829 xenologs in fungi ranging from Chytridiomycota to Mucoromycota. We have identified several patterns and statistical properties of EDF HGT. We show that HGT is driven by bursts of gene exchange and duplication, resulting in highly divergent numbers and molecular properties of xenologs between fungal lineages. Ancestrally aquatic fungi are generally more likely to acquire foreign genetic material than terrestrial ones. Endosymbiotic bacteria can be a source of useful xenologs, as exemplified by NOD-like receptors transferred to Mortierellomycota. Closely related fungi have similar rates of intronization of xenologs. Posttransfer gene fusions and losses of protein domains are common and may influence the encoded proteins' functions. We argue that there is no universal approach for HGT identification and inter- and intra-kingdom transfers require tailored identification methods. Our results help to better understand how and to what extent HGT has shaped the metabolic, adaptive, and immune capabilities of fungi. [ABSTRACT FROM AUTHOR]

Statistical analyses of homologous protein sequences can identify amino acid residue positions that co-evolve to generate family members with different properties. Based on the hypothesis that the coevolution of residue positions is necessary for maintaining protein structure, coevolutionary traits revealed by statistical models provide insight into residue–residue interactions that are important for understanding protein mechanisms at the molecular level. With the rapid expansion of genome sequencing databases that facilitate statistical analyses, this sequence-based approach has been used to study a broad range of protein families. An emerging application of this approach is to design hybrid transcriptional regulators as modular genetic sensors for novel wiring between input signals and genetic elements to control outputs. Among many allosterically regulated regulator families, the members contain structurally conserved and functionally independent protein domains, including a DNA-binding module (DBM) for interacting with a specific genetic element and a ligand-binding module (LBM) for sensing an input signal. By hybridizing a DBM and an LBM from two different family members, a hybrid regulator can be created with a new combination of signal-detection and DNA-recognition properties not present in natural systems. In this review, we present recent advances in the development of hybrid regulators and their applications in cellular engineering, especially focusing on the use of statistical analyses for characterizing DBM–LBM interactions and hybrid regulator design. Based on these studies, we then discuss the current limitations and potential directions for enhancing the impact of this sequence-based design approach. [ABSTRACT FROM AUTHOR]

The Azotobacter vinelandii FeSII protein forms an oxygen‐resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin‐type ferredoxin that forms a dimer in solution. Previously, the crystal structure was solved [Schlesier et al. (2016), J. Am. Chem. Soc.138, 239–247] with five copies in the asymmetric unit. One copy is a normal adrenodoxin domain that forms a dimer with its crystallographic symmetry mate. The other four copies are in an 'open' conformation with a loop flipped out exposing the 2Fe–2S cluster. The open and closed conformations were interpreted as oxidized and reduced, respectively, and the large conformational change in the open configuration allowed binding to nitrogenase. Here, the structure of FeSII was independently solved in the same crystal form. The positioning of the atoms in the unit cell is similar to the earlier report. However, the interpretation of the structure is different. The 'open' conformation is interpreted as the product of a crystallization‐induced domain swap. The 2Fe–2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. It is also cautioned that crystal structures should be interpreted in terms of the contents of the entire crystal rather than of one asymmetric unit. [ABSTRACT FROM AUTHOR]

Atypical porcine pestivirus (APPV) can cause congenital tremor type A-II in neonatal piglets, posing a significant threat to swine herd health globally. Our previous study demonstrated that the Mut domains, comprising 112 amino acids at the N-terminus, are the primary functional regions of the E2 protein of APPV. This study identified 14 host cellular proteins that exhibit potential interactions with the Mut domains of the E2 protein using yeast two-hybrid screening. Using bioinformatics analysis, we discovered that the Mut domains of the E2 protein might exert regulatory effects on apoptosis by modulating energy metabolism within the mitochondria. We also conducted co-immunoprecipitation, glutathione S-transferase pull-down, and immunofluorescence assays to confirm the interaction between the Mut domains of the E2 protein and cathepsin H and signal sequence receptor subunit 4 (SSR4). Ultimately, SSR4 enhanced APPV replication in vitro. In summary, our study successfully elucidated the interactions between the Mut domains of the E2 protein and host cell protein, predicted the potential pathways implicated in these interactions, and demonstrated SSR4 involvement in APPV infection. These significant findings contribute valuable knowledge toward a deeper understanding of APPV pathogenesis and the role of the Mut domains of the E2 protein in this intricate process. [ABSTRACT FROM AUTHOR]

The cyclic nucleotides 3′,5′-cAMP/cGMP are now established as essential components of cellular processes in plants. The discovery of 2′,3′-cAMP/cGMP synthetases in Toll/interleukin-1 receptor (TIR) proteins supports an increasing recognition of both isomers as signaling molecules. Amino acid motifs diagnostic for adenylate cyclases (ACs) and guanylate cyclases (GCs) have enabled the discovery of moonlighting catalytic activities in both well-characterized and new proteins. 3′,5′-cAMP/cGMP operate as intramolecular tuners in microenvironments, while the 2′,3′ isoforms operate at the systems level in defense responses. Certain phosphodiesterases (PDEs) that hydrolyze 3′,5′-cAMP/cGMP function in the same protein as the enzymes that generate it. The twin AC-PDE domain architecture enables the tuning of cyclic nucleotide activities in a transient, dynamic, and compartmentalized manner. Research on cyclic nucleotide moonlighting enzymes is expected to drive biotechnological innovations and crop improvements. Cyclic nucleotides 3′,5′-cAMP and 3′,5′-cGMP are now established signaling components of the plant cell while their 2′,3′ positional isomers are increasingly recognized as such. 3′,5′-cAMP/cGMP is generated by adenylate cyclases (ACs) or guanylate cyclases (GCs) from ATP or GTP, respectively, whereas 2′,3′-cAMP/cGMP is produced through the hydrolysis of double-stranded DNA or RNA by synthetases. Recent evidence suggests that the cyclic nucleotide generating and inactivating enzymes moonlight in proteins with diverse domain architecture operating as molecular tuners to enable dynamic and compartmentalized regulation of cellular signals. Further characterization of such moonlighting enzymes and extending the studies to noncanonical cyclic nucleotides promises new insights into the complex regulatory networks that underlie plant development and responses, thus offering exciting opportunities for crop improvement. [ABSTRACT FROM AUTHOR]

Background: The modification of N6‐methyladenosine (m6A) plays a pivotal role in tumor by altering both innate and adaptive immune systems through various pathways, including the regulation of messenger RNA. The YTH domain protein family, acting as "readers" of m6A modifications, affects RNA splicing, stability, and immunogenicity, thereby playing essential roles in immune regulation and antitumor immunity. Despite their significance, the impact of the YTH domain protein family on tumor initiation and progression, as well as their involvement in tumor immune regulation and therapy, remains underexplored and lacks comprehensive review. Conclusion: This review introduces the molecular characteristics of the YTH domain protein family and their physiological and pathological roles in biological behavior, emphasizing their mechanisms in regulating immune responses and antitumor immunity. Additionally, the review discusses the roles of the YTH domain protein family in immune‐related diseases and tumor resistance, highlighting that abnormal expression or dysfunction of YTH proteins is closely linked to tumor resistance. Key points: This review provides an in‐depth understanding of the YTH domain protein family in immune regulation and antitumor immunity, suggesting new strategies and directions for immunotherapy of related diseases.These insights not only deepen our comprehension of m6A modifications and YTH protein functions but also pave the way for future research and clinical applications. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is a common cause of respiratory infection that often leads to hospitalization of infected younger children and older adults. RSV is classified into two strains, A and B, each with several subgroups or genotypes. One issue with the definition of these subgroups is the lack of a unified method of identification or genotyping. We propose that genotyping strategies based on the genes coding for replication-associated proteins could provide critical information on the replication capacity of the distinct subgroups, while clearly distinguishing genotypes. Here, we analyzed the virus replication-associated genes N, P, M2, and L from de novo assembled RSV A sequences obtained from 31 newly sequenced samples from hospitalized patients in Philadelphia and 78 additional publicly available sequences from different geographic locations within the United States. In-depth analysis and annotation of variants in the replication-associated proteins identified the polymerase protein L as a robust target for genotyping RSV subgroups. Importantly, our analysis revealed non-synonymous variations in L that were consistently accompanied by conserved changes in its cofactor P or the M2-2 protein, suggesting associations and interactions between specific domains of these proteins. Similar associations were seen among sequences of the related human metapneumovirus. These results highlight L as an alternative to other RSV genotyping targets and demonstrate the value of in-depth analyses and annotations of RSV sequences as it can serve as a foundation for subsequent in vitro and clinical studies on the efficiency of the polymerase and fitness of different virus isolates. IMPORTANCE Given the historical heterogeneity of respiratory syncytial virus (RSV) and the disease it causes, there is a need to understand the properties of the circulating RSV strains each season. This information would benefit from an informative and consensus method of genotyping the virus. Here, we carried out a variant analysis that shows a pattern of specific variations among the replication-associated genes of RSV A across different seasons. Interestingly, these variation patterns, which were also seen in human metapneumovirus sequences, point to previously defined interactions of domains within these genes, suggesting co-variation in the replication-associated genes. Our results also suggest a genotyping strategy that can prove to be particularly important in understanding the genotype-phenotype correlation in the era of RSV vaccination, where selective pressure on the virus to evolve is anticipated. More importantly, the categorization of pneumoviruses based on these patterns may be of prognostic value. [ABSTRACT FROM AUTHOR]

GABAA receptors (γ-aminobutyric acid-gated receptors type A; GABAARs), the major structural and functional postsynaptic components of inhibitory synapses in the mammalian brain, belong to a family of GABA-gated Cl-/HCO3 - ion channels. They are assembled as heteropentamers from a family of subunits including: α (1-6), β(1-3), γ(1-3), δ, ε, π, θ and ρ(1-3). GABAARs together with the postsynaptic adhesion protein Neuroligin 2 (NL2) and many other pre- and postsynaptic proteins guide the initiation and functional maturation of inhibitory GABAergic synapses. This study examined how GABAARs and NL2 interact with each other to initiate the formation of synapses. Two functionally distinct GABAAR subtypes, the synaptic type α2β2γ2-GABAARs versus extrasynaptic type α4β3δ-GABAARs were expressed in HEK293 cells alone or together with NL2 and co-cultured with striatal GABAergic medium spiny neurons to enable innervation of HEK293 cells by GABAergic axons. When expressed alone, only the synaptic α2β2γ2-GABAARs induced innervation of HEK293 cells. However, when GABAARs were co-expressed with NL2, the effect on synapse formation exceeded the individual effects of these proteins indicating a synergistic interaction, with α2β2γ2-GABAAR/NL2 showing a significantly greater synaptogenic activity than α4β3δ-GABAAR/NL2 or NL2 alone. To investigate the molecular basis of this interaction, different combinations of GABAAR subunits and NL2 were co-expressed, and the degree of innervation and synaptic activity assessed, revealing a key role of the γ2 subunit. In biochemical assays, the interaction between NL2 and α2β2γ2-GABAAR was established and mapped to the large intracellular domain of the γ2 subunit. [ABSTRACT FROM AUTHOR]

Maternal vaccinations administered prior to conception or during pregnancy enhance the immune protection of newborn infants against many pathogens. A feasibility experiment was conducted to determine if monkeys can be used to model the placental transfer of maternal antibody against SARS-CoV-2. Six adult rhesus monkeys were immunized with adjuvanted recombinant-protein antigens comprised of receptor-binding domain human IgG1-Fc fusion proteins (RBD-Fc) containing protein sequences from the ancestral-Wuhan or Gamma variants. The female monkeys mounted robust and sustained anti-SARS-CoV-2 antibody responses. Blood samples collected from their infants after delivery verified prenatal transfer of high levels of spike-specific IgG, which were positively correlated with maternal IgG titers at term. In addition, an in vitro test of ACE2 neutralization indicated that the infants' IgG demonstrated antigen specificity, reflecting prior maternal immunization with either Wuhan or Gamma-variant antigens. All sera showed stronger ACE2-RBD binding inhibition when variants in the assay more closely resembled the vaccine RBD sequence than with more distantly related variants (i.e., Delta and Omicron). Monkeys are a valuable animal model for evaluating new vaccines that can promote maternal and infant health. Further, the findings highlight the enduring nature and safety of the immune protection elicited by an adjuvanted recombinant RBD-Fc vaccine. [ABSTRACT FROM AUTHOR]

The Cystathionine‐β‐Synthase (CBS) domain‐containing proteins (CDCPs) constitute a functionally diverse protein superfamily, sharing an evolutionary conserved CBS domain either in pair or quad. Rice genome (Oryza sativa subsp. indica) encodes 42 CDCPs; their functions remain largely unexplored. This study examines OsCBSCBS4, a quadruple CBS domain containing protein towards its role in regulating the abiotic stress tolerance in rice. Gene expression analyses revealed upregulation of OsCBSCBS4 in response to diverse abiotic stresses. Further, the cytoplasm‐localised OsCBSCBS4 showed interaction with two different kinases, a cytoplasmic localised cGMP‐dependant protein kinase (OsPKG) and the nucleo‐cytoplasmic catalytic subunit of sucrose‐nonfermentation 1‐related protein kinase 1 (OsSnRK1A). The interaction with the latter assisted in trafficking of OsCBSCBS4 to the nucleus as well. Overexpression of OsCBSCBS4 in rice resulted in enhanced tolerance to drought and salinity stress, via maintaining better physiological parameters and antioxidant activity. Additionally, OsCBSCBS4‐overexpressing rice plants exhibited reduced yield penalty under stress conditions. The in silico docking and in vitro binding analyses of OsCBSCBS4 with ATP suggest its involvement in cellular energy balance. Overall, this study provides novel insight into the unexplored functions of OsCBSCBS4 and demonstrates it as a new promising target for augmenting crop resilience. [ABSTRACT FROM AUTHOR]

T cell development in the thymus is dependent on the thymic microenvironment, in which thymic epithelial cells (TECs) are the major component. However, TECs undergo both a qualitative and quantitative loss during aging, which is believed to be the major factor responsible for age-dependent thymic atrophy. FOXN1 plays a critical role in TEC development and adult TECs maintenance. We have previously reported that intrathymic injection of a recombinant (r) protein containing murine FOXN1 and a protein transduction domain increases the number of TECs in mice, leading to enhanced thymopoiesis. However, intrathymic injection may not be an ideal choice for clinical applications. In this study, we produced a rFOXN1 fusion protein containing the N-terminal of CCR9, human FOXN1 and a protein transduction domain. When injected intravenously into 14-month-old mice, the rFOXN1 fusion protein enters the thymus and TECs, and enhances thymopoiesis, resulting in increased T cell generation in the thymus and increased number of T cells in peripheral lymphoid organ. Our results suggest that the rFOXN1 fusion protein has the potential to be used in preventing and treating T cell immunodeficiency in older adults. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cells have been used to successfully treat various blood cancers, but adverse effects have limited their potential. Here, we developed chimeric adaptor proteins (CAPs) and CAR tyrosine kinases (CAR-TKs) in which the intracellular ζ T cell receptor (TCRζ) chain was replaced with intracellular protein domains to stimulate signaling downstream of the TCRζ chain. CAPs contain adaptor domains and the kinase domain of ZAP70, whereas CAR-TKs contain only ZAP70 domains. We hypothesized that CAPs and CAR-TKs would be more potent than CARs because they would bypass both the steps that define the signaling threshold of TCRζ and the inhibitory regulation of upstream molecules. CAPs were too potent and exhibited high tonic signaling in vitro. In contrast, CAR-TKs exhibited high antitumor efficacy and significantly enhanced long-term tumor clearance in leukemia-bearing NSG mice as compared with the conventional CD19-28ζ-CAR-T cells. CAR-TKs were activated in a manner independent of the kinase Lck and displayed slower phosphorylation kinetics and prolonged signaling compared with the 28ζ-CAR. Lck inhibition attenuated CAR-TK cell exhaustion and improved long-term function. The distinct signaling properties of CAR-TKs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an antitumor chimeric receptor. Editor's summary: T cells that express chimeric antigen receptors (CARs) are therapeutically effective against only some types of cancers. Noting that downstream signaling molecules are recruited inefficiently to current CARs, Balagopalan et al. instead incorporated domains from these signaling proteins into the CAR itself. Inclusion of the kinase domain of ZAP70 to generate CAR tyrosine kinases resulted in cells with enhanced antitumor efficacy in mice compared with that of current CAR T cells. Thus, the signaling properties of CAR tyrosine kinases could be explored to enhance antitumor T cell efficacy. —John F. Foley [ABSTRACT FROM AUTHOR]

Cellulosic materials are attracting increasing research interest because of their abundance, biocompatibility, and biodegradability, making them suitable in multiple industrial and medical applications. Functionalization of cellulose is usually required to improve or expand its properties to meet the requirements of different applications. Cellulose-binding domains (CBDs) found in various proteins have been shown to be powerful tools in the functionalization of cellulose materials. In this review, we firstly introduce the structural characteristics of commonly used CBDs belonging to carbohydrate-binding module families 1, 2 and 3. Then, we summarize four main kinds of methodologies for employing CBDs to modify cellulosic materials (i.e., CBD only, genetic fusion, non-covalent linkage and covalent linkage). Via different approaches, CBDs have been used to improve the material properties of cellulose, immobilize enzymes for biocatalysis, and design various detection tools. To achieve industrial applications, researches for lowering the production cost of CBDs, improving their performance (e.g., stability), and expanding their application scenarios are still in need. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton regulates the integrity and repair of epithelial barriers by mediating the assembly of tight junctions (TJs), and adherens junctions (AJs), and driving epithelial wound healing. Actin filaments undergo a constant turnover guided by numerous actin-binding proteins, however, the roles of actin filament dynamics in regulating intestinal epithelial barrier integrity and repair remain poorly understood. Coactosin-like protein 1 (COTL1) is a member of the ADF/ cofilin homology domain protein superfamily that binds and stabilizes actin filaments. COTL1 is essential for neuronal and cancer cell migration, however, its functions in epithelia remain unknown. The goal of this study is to investigate the roles of COTL1 in regulating the structure, permeability, and repair of the epithelial barrier in human intestinal epithelial cells (IEC). COTL1 was found to be enriched at apical junctions in polarized IEC monolayers in vitro. The knockdown of COTL1 in IEC significantly increased paracellular permeability, impaired the steady state TJ and AJ integrity, and attenuated junctional reassembly in a calcium-switch model. Consistently, downregulation of COTL1 expression in Drosophila melanogaster increased gut permeability. Loss of COTL1 attenuated collective IEC migration and decreased cell-matrix attachment. The observed junctional abnormalities in COTL1-depleted IEC were accompanied by the impaired assembly of the cortical actomyosin cytoskeleton. Overexpression of either wild-type COTL1 or its actin-binding deficient mutant tightened the paracellular barrier and activated junction-associated myosin II. Furthermore, the actin-uncoupled COTL1 mutant inhibited epithelial migration and matrix attachment. These findings highlight COTL1 as a novel regulator of the intestinal epithelial barrier integrity and repair. [ABSTRACT FROM AUTHOR]

Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domains have been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (brassinosteroid insensitive 1-associated kinase 1/somatic embryogenesis receptor kinase 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting aC-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1. [ABSTRACT FROM AUTHOR]

Immune defence mechanisms exist across the tree of life in such diversity that prokaryotic antiviral responses have historically been considered unrelated to eukaryotic immunity. Mechanisms of defence in divergent eukaryotes were similarly believed to be largely clade specific. However, recent data indicate that a subset of modules (domains and proteins) from prokaryote defence systems are conserved in eukaryotes and populate many stages of innate immune pathways. In this Essay, we propose the notion of ancestral immunity, which corresponds to the set of immune modules conserved between prokaryotes and eukaryotes. After offering a typology of ancestral immunity, we speculate on the selective pressures that could have led to the differential conservation of specific immune modules across domains of life. The exploration of ancestral immunity is in its infancy and appears full of promises to illuminate immune evolution, and also to identify and decipher immune mechanisms of economic, ecological, and therapeutic importance. A subset of prokaryotic antiviral systems are conserved in eukaryotes and have crucial roles in immune pathways. This Essay introduces the concept of ancestral immunity, which refers to the set of immune modules (domains and proteins) conserved between prokaryotes and eukaryotes, discussing the topology of ancestral immunity and a putative evolutionary scenario for its existence. [ABSTRACT FROM AUTHOR]

Land plants (embryophytes) came about in a momentous evolutionary singularity: plant terrestrialization. This event marks not only the conquest of land by plants but also the massive radiation of embryophytes into a diverse array of novel forms and functions. The unique suite of traits present in the earliest land plants is thought to have been ushered in by a burst in genomic novelty. Here, we asked the question of how these bursts were possible. For this, we explored: (i) the initial emergence and (ii) the reshuffling of domains to give rise to hallmark environmental response genes of land plants. We pinpoint that a quarter of the embryophytic genes for stress physiology are specific to the lineage, yet a significant portion of this novelty arises not de novo but from reshuffling and recombining of pre-existing domains. Our data suggest that novel combinations of old genomic substrate shaped the plant terrestrialization toolkit, including hallmark processes in signalling, biotic interactions and specialized metabolism. [ABSTRACT FROM AUTHOR]

Rickettsia, a group of intracellular bacteria found in eukaryotes, exhibits diverse lifestyles, with some acting as vertebrate pathogens transmitted by arthropod vectors and others serving as maternally transmitted arthropod endosymbionts, some of which manipulate host reproduction for their own benefit. Two phenotypes, namely male-killing and parthenogenesis induction are known as Rickettsia-induced host reproductive manipulations, but it remains unknown whether Rickettsia can induce other types of host manipulation. In this study, we discovered that Rickettsia induced strong cytoplasmic incompatibility (CI), in which uninfected females produce no offspring when mated with infected males, in the predatory insect Nesidiocoris tenuis (Hemiptera: Miridae). Molecular phylogenetic analysis revealed that the Rickettsia strain was related to Rickettsia bellii, a common insect endosymbiont. Notably, this strain carried plasmid-encoded homologues of the CI-inducing factors (namely cifA-like and cifB-like genes), typically found in Wolbachia, which are well-known CI-inducing endosymbionts. Protein domain prediction revealed that the cifB-like gene encodes PD-(D/E)XK nuclease and deubiquitinase domains, which are responsible for Wolbachia-induced CI, as well as ovarian tumour-like (OTU-like) cysteine protease and ankyrin repeat domains. These findings suggest that Rickettsia and Wolbachia endosymbionts share underlying mechanisms of CI and that CI-inducing ability was acquired by microbes through horizontal plasmid transfer. [ABSTRACT FROM AUTHOR]