Paralogous Proteins Research Articles

Evolutionary adaptations of doublet microtubules in trypanosomatid parasites.

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.

Building a case for 'functional identity fraud' in eRpL22 paralogue-specific ribosomes in Drosophila germline development.

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'.

Stressosome-independent but RsbT-dependent environmental stress sensing in Bacillus subtilis

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.

Bacterial Genome Annotation Of Escherichia Coli

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.

Subtractive genome mining in Xanthomonas citri pv. citri strain 306 for identifying novel drug target proteins coupled with in-depth protein-protein interaction and coevolution analysis - A leap towards prospective drug design.

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)

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

From spots to stripes: Evolution of pigmentation patterns in monkeyflowers via modulation of a reaction-diffusion system and its prepatterns.

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.

Molecular Evolution of Paralogous Cold Shock Proteins in E. coli: A Study of Asymmetric Divergence and Protein Functional Networks.

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

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

Rubisco packaging and stoichiometric composition of the native β-carboxysome in Synechococcus elongatus PCC7942.

Carboxysomes are anabolic bacterial microcompartments that play an essential role in CO2 fixation in cyanobacteria. This self-assembling proteinaceous organelle uses a polyhedral shell constructed by hundreds of shell protein paralogs to encapsulate the key CO2-fixing enzymes Rubisco and carbonic anhydrase. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding carboxysome formation and overall functionality. Here, we employed cryoelectron tomography and subtomogram averaging to delineate the 3D packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also detected Rubisco binding with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using Quantification conCATamer-based quantitative MS, we determined the absolute stoichiometric composition of the entire β-carboxysome. This study provides insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.

Trypanosoma cruzi eIF4E3- and eIF4E4-containing complexes bind different mRNAs and may sequester inactive mRNAs during nutritional stress.

Many eIF4F and poly(A)-binding protein (PABP) paralogues are found in trypanosomes: six eIF4E, five eIF4G, one eIF4A and two PABPs. They are expressed simultaneously and assemble into different complexes, contrasting the situation in metazoans that use distinct complexes in different cell types/developmental stages. Each eIF4F complex has its own proteins, messenger RNAs (mRNAs) and, consequently, a distinct function. We set out to study the function and regulation of the two eIF4F complexes of the parasite Trypanosoma cruzi and identified the associated proteins and mRNAs of eIF4E3 and eIF4E4 in cells in exponential growth and in nutritional stress, an inducer of differentiation to an infective stage. Upon stress, eIF4G and PABP remain associated with the eIF4E, but the associations with other 43S pre-initiation factors decrease, indicating ribosome attachment is impaired. Most eIF4E3-associated mRNAs encode for proteins involved in anabolic metabolism, while eIF4E4 associate with mRNAs encoding ribosomal proteins as in Trypanosoma brucei. Interestingly, for both eIF4E3/4, more mRNAs were associated in stressed cells than in non-stressed cells, even though these have lower translational efficiencies in stress. In summary, trypanosomes have two co-existing eIF4F complexes associating to different mRNAs, but not stress/differentiation-associated mRNAs. Under stress, both complexes exit translation but remain bound to their mRNA targets.

Molecular principles of the assembly and construction of a carboxysome shell.

Intracellular compartmentalization enhances biological reactions, crucial for cellular function and survival. An example is the carboxysome, a bacterial microcompartment for CO2 fixation. The carboxysome uses a polyhedral protein shell made of hexamers, pentamers, and trimers to encapsulate Rubisco, increasing CO2 levels near Rubisco to enhance carboxylation. Despite their role in the global carbon cycle, the molecular mechanisms behind carboxysome shell assembly remain unclear. Here, we present a structural characterization of α-carboxysome shells generated from recombinant systems, which contain all shell proteins and the scaffolding protein CsoS2. Atomic-resolution cryo-electron microscopy of the shell assemblies, with a maximal size of 54 nm, unveil diverse assembly interfaces between shell proteins, detailed interactions of CsoS2 with shell proteins to drive shell assembly, and the formation of heterohexamers and heteropentamers by different shell protein paralogs, facilitating the assembly of larger empty shells. Our findings provide mechanistic insights into the construction principles of α-carboxysome shells and the role of CsoS2 in governing α-carboxysome assembly and functionality.

Structural basis for C. elegans pairing center DNA binding specificity by the ZIM/HIM-8 family proteins

Pairing center (PC) on each chromosome of Caenorhabditis elegans is crucial for homolog pairing and initiating synapsis. Within each PC, clusters of 11/12 bp DNA motif recruit one of four paralogous meiosis-specific proteins: ZIM-1, ZIM-2, ZIM-3, or HIM-8. However, the mechanistic basis underlying the specificity of ZIM/HIM-8-PC DNA interaction remains elusive. Here, we describe crystal structures of HIM-8, ZIM-1 and ZIM-2 DNA binding domains (ZF1, ZF2 and CTD) in complex with their cognate PC DNA motifs, respectively. These structures demonstrated the ZF1-2-CTD folds as an integrated structural unit crucial for its DNA binding specificity. Base-specific DNA-contacting residues are exclusively distributed on ZF1-2 and highly conserved. Furthermore, the CTD potentially contributes to the conformational diversity of ZF1-2, imparting binding specificity to distinct PC DNA motifs. These findings shed light on the mechanism governing PC DNA motif recognition by ZIM/HIM-8 proteins, suggesting a co-evolution relationship between PC DNA motifs and ZF1-2-CTD in shaping the specific recognition.

Ancient duplication and functional differentiation of phytochelatin synthases is conserved in plant genomes.

Despite the paramount importance in metal(loid) detoxification by phytochelatin synthase (PCS) genes, no comprehensive analysis of their evolutionary patterns has been carried out in land plants in general and in crops in particular. A phylogenetic large-scale analysis of gene duplication in angiosperms was carried out followed by in vitro recombinant protein assays as well as complementation analysis (growth, thiol-peptides, elements) of Arabidopsis cad1-3 mutant with four representative PCS genes from two model crop species, Malus domestica and Medicago truncatula. We uncovered a so far undetected ancient tandem duplication (D duplication) spanning the whole core eudicotyledon radiation. Complementation with PCS genes from both D-subclades from M. domestica and M. truncatula displayed clear in vivo conservation of the differences between D1 and D2 paralogous proteins in plant growth, phytochelatin, and glutathione pools, as well as element contents under metal(loid) stress. In vitro recombinant PCS analysis identified analogous patterns of differentiation, showing a higher activity of D2 PCS genes, so far largely overlooked, compared to their paralogs from the D1 clade. This suggests that in many other crop species where the duplication is present, the D2 copy might play a significant role in metal(loid) detoxification. The retention of both PCS paralogs and of their functional features for such long divergence time suggests that PCS copy number could be constrained by functional specialization and/or gene dosage sensitivity. These results uncover the patterns of PCS evolution in plant genomes and of functional specialization of their paralogs in the genomes of two important model crops.

ISWI1 complex proteins facilitate developmental genome editing in Paramecium.

One of the most extensive forms of natural genome editing occurs in ciliates, a group of microbial eukaryotes. Ciliate germline and somatic genomes are contained in distinct nuclei within the same cell. During the massive reorganization process of somatic genome development, ciliates eliminate tens of thousands of DNA sequences from a germline genome copy. Recently, we showed that the chromatin remodeler ISWI1 is required for somatic genome development in the ciliate Paramecium tetraurelia Here, we describe two high similarity paralogous proteins, ICOPa and ICOPb, essential for their genome editing. ICOPa and ICOPb are highly divergent from known proteins; the only domain detected showed distant homology with the WSD (WHIM2 + WHIM3) motif. We show that both ICOPa and ICOPb interact with the chromatin remodeler ISWI1. Upon ICOP knockdown, changes in alternative DNA excision boundaries and nucleosome densities are similar to those observed for ISWI1 knockdown. We thus propose that a complex comprising ISWI1 and either or both ICOPa and ICOPb are needed for Paramecium's precise genome editing.

Activation of plant immunity through conversion of a helper NLR homodimer into a resistosome.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins can engage in complex interactions to detect pathogens and execute a robust immune response via downstream helper NLRs. However, the biochemical mechanisms of helper NLR activation by upstream sensor NLRs remain poorly understood. Here, we show that the coiled-coil helper NLR NRC2 from Nicotiana benthamiana accumulates in vivo as a homodimer that converts into a higher-order oligomer upon activation by its upstream virus disease resistance protein Rx. The cryo-EM structure of NbNRC2 in its resting state revealed intermolecular interactions that mediate homodimer formation and contribute to immune receptor autoinhibition. These dimerization interfaces have diverged between paralogous NRC proteins to insulate critical network nodes and enable redundant immune pathways, possibly to minimise undesired cross-activation and evade pathogen suppression of immunity. Our results expand the molecular mechanisms of NLR activation pointing to transition from homodimers to higher-order oligomeric resistosomes.

Conserved role for spliceosomal component PRPF40A in microexon splicing.

Microexons (exons ≤30 nt) are important features of neuronal transcriptomes, but pose mechanistic challenges to the splicing machinery. We previously showed that PRP-40, a component of the U1 spliceosome, is globally required for microexon splicing in Caenorhabditis elegans Here we show that the homologous PRPF40A is also globally required for microexon splicing in mouse neuroblastoma cells. We find that PRPF40A coregulates microexons along with SRRM4, a neuron-specific regulator of microexon splicing. The relationship between exon size and dependence on PRPF40A/SRRM4 is distinct, with SRRM4-dependence exhibiting a size threshold (∼30 nt) and PRPF40A-dependence exhibiting a graded decrease as exon size increases. Finally, we show that PRPF40A knockdown causes an increase in productive splicing of its spliceosomal binding partner Luc7l by the skipping of a small "poison exon." Similar homeostatic cross-regulation is often observed across paralogous RNA-binding proteins. Here we find this concept likewise applies across evolutionarily unrelated but functionally and physically coupled spliceosomal components.

N-terminal domain homologs of the orange carotenoid protein increase quenching of cyanobacterial phycobilisomes.

Stress exerted by excess captured light energy in cyanobacteria is prevented by the photoprotective activity of the orange carotenoid protein (OCP). Under high light, the OCP converts from an orange, inactive form (OCPO) into the red form (OCPR) that binds to and quenches the phycobilisome (PBS). Structurally, the OCP consists of two domains: the N-terminal effector domain and a C-terminal regulatory domain. Structural analysis of the OCP-PBS complex showed that the N-terminal domains of an OCP dimer interact with the PBS core. These N-terminal OCP domains have single domain protein paralogs known as Helical Carotenoid Proteins (HCPs). Using phycobilisome quenching assays, we show that the HCP4 and HCP5 homologs efficiently quench PBS fluorescence in vitro, surpassing the quenching ability of the OCP. This is consistent with computational quantum mechanics/molecular mechanics results. Interestingly, when using a maximum quenching concentration of OCP with phycobilisomes, HCP5 addition further increases phycobilisome quenching. Our results provide mechanistic insight into the quenching capacity and roles of HCP4 and HCP5 in cyanobacteria, suggesting that they are more than simply functionally redundant to the OCP.

Conserved role for spliceosomal component PRPF40A in microexon splicing.

Microexons (exons ≤30 nts) are important features of neuronal transcriptomes, but pose mechanistic challenges to the splicing machinery. We previously showed that PRP-40, a component of the U1 spliceosome, is globally required for microexon splicing in C. elegans. Here we show that the homologous PRPF40A is also globally required for microexon splicing in mouse neuroblastoma cells. We find that PRPF40A co-regulates microexons along with SRRM4, a neuron-specific regulator of microexon splicing. The relationship between exon size and dependence on PRPF40A/SRRM4 is distinct, with SRRM4-dependence exhibiting a size threshold (~30 nts) and PRPF40A-dependence exhibiting a graded decrease as exon size increases. Finally, we show that PRPF40A knockdown causes an increase in productive splicing of its spliceosomal binding partner Luc7l by skipping of a small "poison exon." Similar homeostatic cross-regulation is often observed across paralogous RNA binding proteins. Here we find this concept likewise applies across evolutionarily unrelated but functionally and physically coupled spliceosomal components.

Rubisco packaging and stoichiometric composition of a native β-carboxysome.

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO2-fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire β-carboxysome. This study and recent findings on the β-carboxysome structure provide insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.