BackgroundThe majority of missense variants in clinical genetic tests are classified as variants of uncertain significance. Prior research shows that the deleterious effects and the subsequent molecular consequences of variants are often conserved among paralogous protein sequences within a gene family. Here, we systematically quantify on an exome-wide scale whether the existence of pathogenic variants in paralogous genes at a conserved position can serve as evidence for the pathogenicity of a new variant. For the gene family of voltage-gated sodium channels, where variants and expert-curated clinical phenotypes are available, we also assess whether phenotype patterns of multiple disorders for each gene are conserved across variant positions within the gene family.ResultsMapping 590,000 pathogenic and 1.9 million population variants onto 9928 genes grouped into 2054 paralogous families increases the number of residues with classifiable evidence 5.1-fold compared with gene-specific data alone. The presence of a pathogenic variant in a paralogous gene is associated with a positive likelihood ratio of 13.0 for variant pathogenicity. Across ten genes encoding voltage-gated sodium channels and 22 expert-curated disorders, we identify cross-paralog correlated phenotypes based on 3D structure spatial position. For example, multiple established loss-of-function related disorders across SCN1A, SCN2A, SCN5A, and SCN8A show overlapping spatial variant clusters. Finally, we show that phenotype integration in paralog variant selection improves variant classification.ConclusionConserved pathogenic missense variants in paralogous genes provide robust, quantifiable support for clinical variant interpretation, and phenotype-informed mapping further improves predictions.

Neural tube defects (NTDs) are congenital malformations arising when the neural tube (NT), precursor of the brain and spine, fails to properly close during neurulation. Etiology is multifactorial, with environmental and genetic factors variably contributing on a case-by-case basis. Molecular genetic studies of murine NTD genes have been precious in the identification of predisposing NTD genes in humans, highlighting the peculiar role of the planar cell polarity (PCP) pathway in a fraction of human NTD patients. Seventy-eight patients with NTD treated at a pediatric tertiary care center were selected for genetic analysis. A custom next-generation sequencing (NGS) panel of 29 genes encoding for components of the core PCP pathway or for family members and paralogs of proteins (including SHROOM and GRHL) underlying NTDs in well-known animal models was used to re-sequence patients with NTD. A gene-burden analysis was also performed to assess potential enrichment of rare damaging variants in the NTD cohort compared to ethnically matched controls. Thirty-nine of 78 individuals (50%) presented with at least one putatively damaging rare variant, most of which (87%) were missense substitutions. Rare variants of GRHL1 and WNT5A, and among gene families GRHL and SHROOM, were significantly enriched in the patients' cohort compared to controls. This study supports the involvement of human orthologues of mouse genes in human NTD phenotypes. Further re-sequencing or, even better, whole-exome sequencing of a large group of cases will give the clues for a better understanding of NTD etiology, ameliorating the clinical management of patients and their families.

The fidelity of assembly of multiprotein complexes is essential for the formation of stable and functional protein complexes that are critical for cell growth and survival. In this context, TBP-associated factor (TAF) subunits maintain tight specificity for their integration into TFIID and SAGA complexes. In this work, using affinity purification-coupled mass spectrometry of epitope-tagged TFIID subunits TBP and TAF11, and the SAGA subunit TAF12L we identified components of the C. albicans TFIID and SAGA complexes. Whereas TAF12 is a subunit of TFIID, the paralogous TAF12L is a subunit of the SAGA complex, and we further identified each of the TFIID and SAGA complex subunits with high confidence. We found that the steady-state levels of the H2B-H2A-like histone fold domain containing pairs, TAF12-TAF4 and TAF12L-Ada1 proteins, are mutually dependent on the stable expression of each other. Using RNA coimmunoprecipitation from polysome-containing extracts, we found that nascent TAF4 and Ada1 proteins interact with TAF12 and TAF12L, respectively, by a cotranslational mechanism in an ordered, sequential mode of assembly. Thus, our results indicate that heterodimerization of the TAF12 paralogs with cognate partners occur by sequential cotranslational assembly thereby ensuring both selectivity and stability of the H2A-H2B heterodimers in fungal pathogen C. albicans.

RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Polypyrimidine tract binding proteins, PTBP1 and PTBP2, are paralogous RNA binding proteins that play a critical role in the process of neuronal differentiation and maturation; changes in the concentration of PTB proteins during neuronal development direct splicing changes in many transcripts that code for proteins critical for neuronal differentiation. PTBP1 can compensate for the loss of PTBP2 in some developmental contexts but not others signifying the paralogs have distinct functions. How two highly structurally similar proteins regulate different sets of neuronal exons is unclear and if known, will reveal how gene families evolved to achieve tissue-specific splicing and in turn, gene expression patterns. Here, we incubated PTBP1 and PTBP2 under splicing reaction conditions containing neuronal WERI retinoblastoma nuclear extract and probed for interacting partner proteins and chemical modifications via mass spectrometry. Our results reveal key differences in the kinds of proteins and processes the paralogs associate with under these conditions. Our data also highlight the presence of novel and distinct chemical modifications on the paralogs when incubated with neuronal nuclear extracts. Collectively, our study suggests a role for chemical modifications in regulating PTBP function in neuronal vs non-neuronal cells.

In a classical view, each paralogous ribosomal protein (RP) is equally synthesized and integrated into the ribosome. Therefore, RP-paralog mRNAs are generally believed to be similarly regulated on their transcription and/or stability. In this paper, we report that two Rps7p/eS7 paralogs of Saccharomyces cerevisiae are differently regulated; deletion of RPS7A upregulates RPS7B paralogous mRNA expression but not vice versa. Their 3'-UTR sequences critically regulated the stabilities of both RPS7A and RPS7B mRNAs. Alterations in these sequences led to a diminished expression of RPS7A and RPS7B mRNAs in a transcript-dependent manner, suggesting that RPS7-paralog mRNAs have different properties for their expression and/or stability. The C-terminal tagging of the ORF and mutation analyses in the 3'-UTR indicate that both RPS7-paralog mRNAs critically rely on their 3'-UTRs for mRNA expressions and/or stabilities. We also found that activities of both RPS7A and RPS7B promoters are regulated by abundance of Rps7Ap and that Fhl1p, a key transcriptional regulator of RP genes, is essential for transcription of RPS7B but not RPS7A while simultaneous deletion of a consensus sequence for Fhl1p in the RPS7A promoter region and the FHL1 gene completely abolishes the promoter activity. These results indicate that yeast has a distinct buffering system for Rps7p production between the two RPS7-paralogs, which is sensitive to variation on their 3'-UTRs and is partially mediated in a transcription-dependent manner.

Membrane contact sites are molecular bridges between organelles that are sustained by tethering proteins and enable organelle communication. The endoplasmic reticulum (ER) membrane harbors many distinct families of tether proteins that enable the formation of contacts with all other organelles. One such example is the LAM (Lipid transfer protein Anchored at Membrane contact sites) family in yeast, which is composed of six members, each containing a putative lipid binding and transfer domain and an ER-embedded transmembrane segment. The family is divided into three homologous pairs each unique in their molecular architecture and localization to different ER subdomains. However, what determines the distinct localization of the different LAMs and which specific roles they carry out in each contact are still open questions. To address these, we utilized a labeling approach to profile the proximal protein landscape of the entire family. Focusing on unique, candidate interactors we could support that Lam5 resides at the ER-mitochondria contact site and demonstrate a role for it in sustaining mitochondrial activity. Capturing shared, putative interactors of multiple LAMs, we show how the Lam1/3 and Lam2/4 paralogous pairs could be associated specifically with the plasma membrane. Overall, our work provides new insights into the regulation and function of the LAM family members. More globally it demonstrates how proximity labeling can help identify the shared or unique functions of paralogous proteins.

Ku is essential in non-homologous end-joining (NHEJ) across prokaryotes and eukaryotes, primarily in double-stranded breaks (DSBs) repair. It often presents as a multi-domain protein in eukaryotes, unlike their prokaryotic single-domain homologs. We systematically searched for Ku proteins across different domains of life. To elucidate the evolutionary history of the Ku protein, we constructed a maximum likelihood phylogenetic tree using Ku protein sequences from 100 representative eukaryotic, prokaryotic, and viral species. The resulting tree revealed a common node for eukaryotic Ku proteins, while viral and prokaryotic species clustered into a distinct clade. Our phylogenetic analysis reveals that the common ancestry of Ku70 and Ku80 likely resulted from a gene duplication event in the ancestral eukaryote. This inference is supported by BLASTp results, which indicate a close resemblance between archaeal Ku and eukaryotic Ku, particularly Ku70. The presence of both Ku protein paralogs in the Discoba group further supports the hypothesis that the gene duplication occurred early in eukaryotic evolution. It is plausible that archaea, which may have acted as intermediaries for Ku transfer, subsequently lost the Ku protein. Nonetheless, the extensive horizontal transfer of Ku among prokaryotes and its relatively higher prevalence in bacteria complicates our understanding of how Ku protein was inherited by early-branching eukaryotes.

The cytoplasmic polyadenylation element-binding proteins (CPEBs) are a family of translational regulators involved in multiple biological processes, including memory-related synaptic plasticity. In vertebrates, four paralogous genes (CPEB1-4) encode proteins with phylogenetically conserved C-terminal RNA-binding domains and variable N-terminal regions (NTRs). The CPEB NTRs are characterized by low-complexity regions (LCRs), including homopolymeric amino acid repeats (AARs), and have been identified as mediators of liquid-liquid phase separation (LLPS) and prion-like aggregation. After their appearance following gene duplication, the four paralogous CPEB proteins functionally diverged in terms of activation mechanisms and modes of mRNA binding. The paralog-specific NTRs may have contributed substantially to such functional diversification but their evolutionary history remains largely unexplored. Here, we traced the evolution of vertebrate CPEBs and their LCRs/AARs focusing on primary sequence composition, complexity, repetitiveness, and their possible functional impact on LLPS propensity and prion-likeness. We initially defined these composition- and function-related quantitative parameters for the four human CPEB paralogs and then systematically analyzed their evolutionary variation across more than 500 species belonging to nine major clades of different stem age, from Chondrichthyes to Euarchontoglires, along the vertebrate lineage. We found that the four CPEB proteins display highly divergent, paralog-specific evolutionary trends in composition- and function-related parameters, primarily driven by variation in their LCRs/AARs and largely related to clade stem ages. These findings shed new light on the molecular and functional evolution of LCRs in the CPEB protein family, in both quantitative and qualitative terms, highlighting the emergence of CPEB2 as a proline-rich prion-like protein in younger vertebrate clades, including Primates.

While members of large paralogous protein families share structural features, their functional niches often diverge significantly. Serine protease inhibitors (SERPINs), whose members typically function as covalent inhibitors of serine proteases, are one such family. Plasminogen activator inhibitor-1 (PAI-1) is a prototypic SERPIN, which canonically inhibits tissue- and urokinase-type plasminogen activators (tPA and uPA) to regulate fibrinolysis. PAI-1 has been shown to also inhibit other serine proteases, including coagulation factor XIIa (FXIIa) and transmembrane serine protease 2 (TMPRSS2). The structural determinants of PAI-1 inhibitory function toward these non-canonical protease targets, and the biological significance of these functions, are unknown. We applied deep mutational scanning (DMS) to assess the effects of ~80% of all possible single-amino acid substitutions in PAI-1 on its ability to inhibit three putative serine protease targets (uPA, FXIIa, and TMPRSS2). Selection with each target protease generated a unique PAI-1 mutational landscape, with the determinants of protease specificity distributed throughout PAI-1's primary sequence. Next, we conducted a comparative analysis of extant orthologous sequences, demonstrating that key residues modulating PAI-1 inhibition of uPA and FXIIa, but not TMPRSS2, are maintained by purifying selection (also referred to as "negative selection"). PAI-1's activity toward FXIIa may reflect how protease evolutionary relationships predict SERPIN functional divergence, which we support via a cophylogenetic analysis of secreted SERPINs and their cognate serine proteases. This work provides insight into the functional diversification of SERPINs and lays the framework for extending these studies to other proteases and their regulators.

The movement and pathogenicity of trypanosomatid species, the causative agents of trypanosomiasis and leishmaniasis, are dependent on a flagellum that contains an axoneme of dynein-bound doublet microtubules (DMTs). In this work, we present cryo-electron microscopy structures of DMTs from two trypanosomatid species, Leishmania tarentolae and Crithidia fasciculata, at resolutions up to 2.7 angstrom. The structures revealed 27 trypanosomatid-specific microtubule inner proteins, a specialized dynein-docking complex, and the presence of paralogous proteins that enable higher-order periodicities or proximal-distal patterning. Leveraging the genetic tractability of trypanosomatid species, we quantified the location and contribution of each structure-identified protein to swimming behavior. Our study shows that proper B-tubule closure is critical for flagellar motility, exemplifying how integrating structural identification with systematic gene deletion can dissect individual protein contributions to flagellar motility.

Ribosomes are macromolecular complexes responsible for protein synthesis, comprising ribosomal proteins (RPs) and ribosomal RNA. While most RPs are present as single copies in higher eukaryotes, a handful of them have paralogues that emerged through duplication events. However, it is still unclear why a small subset of RP paralogues were preserved through evolution, and whether they can endow ribosomes with specialized functions. In this review, we focus on RP paralogue pairs present in humans, providing an overview of the most recent findings on RP paralogue functions and their roles in ribosome specialization.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.

Investigations of expression and function of eukaryotic-specific ribosomal protein paralogues, eRpL22 and eRpL22-like, within the Drosophila melanogaster male germline offer valuable insights supporting an emerging paradigm shift that ribosomes are now exempt from the traditional view of being homogeneous protein synthesis machines. Co-expression of these paralogues within the same cell contributes to structural and functional complexity-the latter demonstrated by differential translation specificities based on paralogue content. This commentary highlights some of the key findings related to the biology of specialized ribosomes containing paralogue eRpL22 or eRpL22-like in Drosophila spermatogenesis and raises several unresolved questions about eRpL22 family paralogue function and ribosome-mediated translation regulation within spermatogenesis. Our understanding of principles that govern specialized ribosome function is in nascent stages, and considerably more research is warranted to address the myriad of unresolved questions about specialized ribosomes and the impact on reproductive physiology.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.

Bacillus subtilis uses cytoplasmic complexes called stressosomes to initiate the σB-mediated general stress response to environmental stress. Each stressosome comprises two types of proteins — RsbS and four paralogous RsbR proteins — that are thought to sequester the RsbT protein until stress causes RsbT release and subsequent σB activation. RsbR proteins have been assumed to sense stress, but evidence for their sensing function has been elusive, and the identity of the true sensor has remained unknown. Here, we conduct an alanine-scanning analysis of the putative sensing domain of one of the RsbR paralogs, RsbRA. We find that single substitutions impact but do not abolish the σB response, suggesting that RsbRA has a key role in σB response dynamics and is “tunable” and robust to substitution, but not directly supporting a sensing function. Surprisingly, deletion of the stressosome does not abolish environmental stress-inducible σB activity and instead leads to a stronger and longer-lived response than in strains with stressosomes. Finally, we show that RsbT is necessary for the stressosome-independent response and that its kinase activity is also important. RsbT thus has a previously unappreciated role in initiating stress responses and may itself be a stress sensor in the general stress response.

Annotating the bacterial genome is essential for comprehending the genetic foundations of an organism's functions and behaviors. Escherichia coli serves as a well-researched model organism, and its genome annotation has been thoroughly examined. The E. coli genome includes four thousand two hundred and eighty eight protein-coding genes, of which thirty eight percent lack a defined function. Comparative genomics uncovers widespread and narrowly situated gene families, along with paralogous protein families, including the eighty ABC transporters. The genome is structured according to replication direction and includes insertion sequence elements, remnants of phages, and areas of atypical composition that suggest genome plasticity. Sophisticated bioinformatics tools and pipelines, including Galaxy, Prokka, Beav, and coliBASE, have been created to aid in E. coli genome annotation, offering insights into gene functionality, genome evolution, and species variation. These tools allow scientists to investigate the intricate biology of E. coli and its related species, promoting progress in areas like microbiology, genetics, and biotechnology.

Citrus canker poses a serious threat to a highly significant citrus fruit crop, this disease caused by one of the most destructive bacterial plant pathogens Xanthomonas citri pv. citri (Xcc). Bacterial plant diseases significantly reduce crop yields worldwide, making it more difficult to supply the growing food demand. The high levels of antibiotic resistance in Xcc strains are diminishing the efficacy of current control measures, necessitating the exploration of novel therapeutic targets to address the escalating antimicrobial resistance trend. Genome subtraction approach along with protein-protein network and coevolution analysis were used to identify potential drug targets in Xcc stain 306. The study involved retrieving the Xcc proteome from the UniProt database, eliminating paralogous proteins using CD-HIT (80% identity cutoff), and selecting nonhomologous proteins through BLASTp (e-value <0.005). Essential proteins were identified using BLAST against the DEG (e-value cutoff 0.00001). 750 essential proteins were identified that are nonhomologous to citrus plant. Subsequent analyses included metabolic pathway assessment, subcellular localization prediction, and druggability analysis. Protein network analysis, coevolution analysis, protein active site identification was also performed. In conclusion, this study identified eight potential drug targets (GlmU, CheA, RmlD, GspE, FleQ, RpoN, Shk, SecB), highlighting RpoN, FleQ, and SecB as unprecedented targets for Xcc. These findings may contribute to the development of novel antimicrobial agents in the future that can efficiently control citrus canker disease.

A genus-wide study on venom proteome variation and phospholipase A2 inhibition in Asian lance-headed pit vipers (genus: Trimeresurus)

Ribosomes, once considered uniform protein biosynthesis machines, are now recognized as heterogeneous and dynamic entities with specialized functions. In Saccharomyces cerevisiae, ribosomal heterogeneity arises from variability in ribosomal protein (RP) composition, rRNA sequence polymorphisms, post-transcriptional modifications, and associations with ribosome-associated factors and noncoding RNAs. RP gene (RPG) paralogs and their differential expression influence growth, stress resistance, and drug responses. Introns and untranslated regions in RPGs regulate expression under stress, while ribosome composition adjusts to environmental cues via altered RP stoichiometry and post-translational modifications, such as phosphorylation and ubiquitination. Additionally, ribosome-associated factors contribute to selective translation of specific mRNA subsets. Ribosomal RNA heterogeneity, though less studied in yeast, is evident through polymorphisms in rDNA arrays and post-transcriptional modifications like pseudouridylation and 2'-O-ribose methylation. Furthermore, transient associations with small noncoding RNAs (e.g. tRNA-, snoRNA-, and mRNA-derived fragments) modulate translation in a stress-dependent manner, supporting the concept of specialized ribosomes. Despite growing evidence, functional significance of ribosome specialization remains under debate. Future research aims to uncover the extent, regulation, and biological roles of ribosome heterogeneity across organisms and conditions. Emerging tools such as ribosome sequencing, single-molecule fluorescence resonance energy transfer, and single-molecule fluorescence resonance energy transfer offer promising avenues to resolve these questions and reveal how specialized ribosomes contribute to adaptive gene expression.

The reaction-diffusion (RD) system is widely assumed to account for many complex, self-organized pigmentation patterns in natural organisms. However, the specific configurations of such RD networks and how RD systems interact with positional information (i.e., prepatterns) that may specify the initiation conditions for the RD operation remain largely unknown. Here, we introduced a three-substance RD system underlying the formation of repetitive pigment spots and stripes in Mimulus flowers. It consists of an R2R3-MYB activator (NEGAN), an R3-MYB inhibitor (RTO), and a coactivator represented by two paralogous bHLH proteins. Through fine-scale genetic analyses, transgenic experiments, and computer simulations, we identified the causal loci contributing to the evolutionary transition from sparsely dispersed spots to longitudinal stripes. Genetic changes at these loci modulate the prepatterns of the activator and coactivator expression and the promoter activities of the inhibitor and one of the coactivator paralogs. Our findings highlight the importance of prepatterns towards a realistic description of RD systems in natural organisms, and reveal the genetic mechanism generating pattern variation through modulation of the kinetics of the RD system and its prepatterns.

Nine homologous Cold Shock Proteins (Csps) have been recognized in the E.coli Cold Shock Domain gene family. These Csps function as RNA chaperones. This study aims to establish the evolutionary relationships among these genes by identifying and classifying their paralogous counterparts. It focuses on the physicochemical, structural, and functional analysis of the genes to explore the phylogeny of the Csp gene family. Computational tools were employed for protein molecular modeling, conformational analysis, functional studies, and duplication-divergence assessments. The research also examined amino acid conservation, protein mutations, domain-motif patterns, and evolutionary residue communities to better understand residual interactions, evolutionary coupling, and co-evolution. H33, M5, W11 and F53 residues were highly conserved within the protein family. It was further seen that residues M5, G17, G58, G61, P62, A64, V67 were intolerant to any kind of mutation whereas G3, D40, G41, Y42, S44, T54, T68, S69 were most tolerable towards substitutions. The study of residue communities displayed that the strongest residue coupling was observed in N13, F18, S27, F31, and W11. It was observed that all the gene pairs except CspF/CspH had new motifs generated over time. It was ascertained that all the gene pairs underwent asymmetric expression divergence after duplication. The Ka/ Ks ratio also revealed that all residues undertook neutral and purifying selection pressure. New functions were seen to develop in gene pairs evident from generation of new motifs. The discovery of new motifs and functions in Csps highlights their adaptive versatility, crucial for E. coli's resilience to environmental stressors and valuable for understanding bacterial stress response mechanisms. These findings will pave the way for future investigations into Csp evolution, with potential applications in microbial ecology and antimicrobial strategy development.

Unique and Common Agonists Activate the Insect Juvenile Hormone Receptor and the Human AHR