Protein Superfamily Research Articles

N6-methyladenosine-modified TRIM37 augments sunitinib resistance by promoting the ubiquitin-degradation of SmARCC2 and activating the Wnt signaling pathway in renal cell carcinoma

Tripartite motif-containing 37 (TRIM37) is reportedly a key member of the superfamily of TRIM proteins. Emerging evidence underscores the close association between dysregulated TRIM37 expression and the progression of various human malignancies. However, the precise biological functions and regulatory mechanisms of TRIM37 remain elusive. This study aimed to elucidate the impact of TRIM37 on the chemotherapy sensitivity of renal cell carcinoma (RCC) and uncover its specific molecular regulatory role. Using RT-qPCR and western blot assays, we assessed TRIM37 expression in both RCC patients and RCC cells. Through in vitro and in vivo experiments, we investigated the effects of TRIM37 silencing and overexpression on RCC cell proliferation, stemness capacity, and chemotherapy sensitivity using colony formation and sphere formation assays. Additionally, a co-immunoprecipitation (Co-IP) experiment was conducted to explore putative interacting proteins. Our results revealed elevated TRIM37 expression in both RCC patient tumor tissues and RCC cells. Functional experiments consistently demonstrated that TRIM37 silencing reduced proliferation and stemness capacity while enhancing chemotherapy sensitivity in RCC cells. Furthermore, we discovered that TRIM37 mediates the degradation of SMARCC2 via ubiquitin-proteasome pathways, thereby further activating the Wnt signaling pathway. In conclusion, this study not only sheds light on the biological role of TRIM37 in RCC progression but also identifies a potential molecular target for therapeutic intervention in RCC patients.

Trem2 acts as a non-classical receptor of interleukin-4 to promote diabetic wound healing.

The immunoglobulin superfamily protein Trem2 (triggering receptor expressed on myeloid cells 2) is primarily expressed on myeloid cells where it functions to regulate macrophage-related immune response induction. While macrophages are essential mediators of diabetic wound healing, the specific regulatory role that Trem2 plays in this setting remains to be established. This study was developed to explore the potential importance of Trem2 signalling in diabetic wound healing and to clarify the underlying mechanisms through which it functions. Following wound induction, diabetic model mice exhibited pronounced upregulation of Trem2 expression, which was primarily evident in macrophages. No cutaneous defects were evident in mice bearing a macrophage-specific knockout of Trem2 (T2-cKO), but they induced more pronounced inflammatory responses and failed to effectively repair cutaneous wounds, with lower levels of neovascularization, slower rates of wound closure, decreased collagen deposition following wounding. Mechanistically, we showed that interleukin (IL)-4 binds directly to Trem2, inactivating MAPK/AP-1 signalling to suppress the expression of inflammatory and chemoattractant factors. Co-culture of fibroblasts and macrophages showed that macrophages from T2-cKO mice suppressed the in vitro activation and proliferation of dermal fibroblasts through upregulation of leukaemia inhibitory factor (Lif). Injecting soluble Trem2 in vivo was also sufficient to significantly curtail inflammatory responses and to promote diabetic wound healing. These analyses offer novel insight into the role of IL-4/Trem2 signalling as a mediator of myeloid cell-fibroblast crosstalk that may represent a viable therapeutic target for efforts to enhance diabetic wound healing.

Testing the Capability of Embedding-Based Alignments on the GST Superfamily Classification: The Role of Protein Length.

In order to shed light on the usage of protein language model-based alignment procedures, we attempted the classification of Glutathione S-transferases (GST; EC 2.5.1.18) and compared our results with the ARBA/UNI rule-based annotation in UniProt. GST is a protein superfamily involved in cellular detoxification from harmful xenobiotics and endobiotics, widely distributed in prokaryotes and eukaryotes. What is particularly interesting is that the superfamily is characterized by different classes, comprising proteins from different taxa that can act in different cell locations (cytosolic, mitochondrial and microsomal compartments) with different folds and different levels of sequence identity with remote homologs. For this reason, GST functional annotation in a specific class is problematic: unless a structure is released, the protein can be classified only on the basis of sequence similarity, which excludes the annotation of remote homologs. Here, we adopt an embedding-based alignment to classify 15,061 GST proteins automatically annotated by the UniProt-ARBA/UNI rules. Embedding is based on the Meta ESM2-15b protein language. The embedding-based alignment reaches more than a 99% rate of perfect matching with the UniProt automatic procedure. Data analysis indicates that 46% of the UniProt automatically classified proteins do not conserve the typical length of canonical GSTs, whose structure is known. Therefore, 46% of the classified proteins do not conserve the template/s structure required for their family classification. Our approach finds that 41% of 64,207 GST UniProt proteins not yet assigned to any class can be classified consistently with the structural template length.

Identification and functional validation of P450 genes associated with pyrethroid resistance in the malaria vector Anopheles sinensis (Diptera Culicidae)

Cytochrome P450 monooxygenases (P450s), a multifunctional protein superfamily, are one of three major classes of detoxification enzymes. However, the diversity and functions of P450 genes from pyrethroid-resistant populations of Anopheles sinensis have not been fully explored. In this study, P450 genes associated with pyrethroid resistance were systematically screened using RNA-seq in three field pyrethroid-resistant populations (AH-FR, CQ-FR, YN-FR) and one laboratory resistant strain (WX-LR) at developmental stages, tissues, and post blood-meal in comparison to the laboratory susceptible strain (WX-LS) in An. sinensis. Importantly, the expression of significantly upregulated P450s was verified using RT-qPCR, and the function of selected P450s in pyrethroid detoxification was determined with RNA interference using four laboratory pyrethroid-resistant strains (WX-LR, AH-LR, CQ-LR, YN-LR). Sixteen P450 genes were significantly upregulated in at least one field-resistant population, and 44 were significantly upregulated in different developmental stages, tissues or post blood-meal. A total of 19 P450s were selected to verify their association with pyrethroid resistance, and four of them (AsCYP6P3v1, AsCYP6P3v2, AsCYP9J10, and AsCYP9K1) demonstrated significant upregulation in laboratory pyrethroid-resistant strains using RT-qPCR. Knockdown of these four genes all significantly reduced pyrethroid resistance and increased the mortality by 57.19% (AsCYP6P3v1 and AsCYP6P3v2 knockdown group), 38.39% (AsCYP9K1 knockdown group) and 48.87% (AsCYP9J10 knockdown group) in An. sinensis by RNAi, which determined the pyrethroid detoxification function of these four genes. This study revealed the diversity of P450 genes and provided functional evidence for four P450s in pyrethroid detoxification in An. sinensis for the first time, which increases our understanding of the pyrethroid resistance mechanism, and is of potential value for pyrethroid resistance detection and surveillance.

Mechanistic model for epigenetic maintenance by methyl-CpG-binding domain proteins.

DNA methylation is a fundamental element of epigenetic regulation that is governed by the MBD protein superfamily, a group of "readers" that share a highly conserved methyl-CpG-binding domain (MBD) and mediate chromatin remodeler recruitment, transcription regulation, and coordination of DNA and histone modification. Previous work has characterized the binding affinity and sequence selectivity of MBD-containing proteins toward palindromes of 5-methylcytosine (5mC) containing 5mCpG dinucleotides, often referred to as single symmetrically methylated CpG sites. However, little is known about how MBD binding is influenced by the prototypical local clustering of methylated CpG sites and the presence of DNA structural motifs encountered, e.g., during DNA replication and transcription. Here, we use Single-Molecule Kinetics through Equilibrium Poisson Sampling (SiMKEPS) to measure precise binding and dissociation rate constants of the MBD of human protein MBD1 to DNAs with varying patterns of multiple methylated CpG sites and diverse structural motifs. MBD binding is promoted by two major properties of its DNA substrates: 1) tandem (consecutive) symmetrically methylated CpG sites in double-stranded DNA and secondary structures in single-stranded DNA; and 2) DNA forks. Based on our findings, we propose a mechanistic model for how MBD proteins contribute to epigenetic boundary maintenance between transcriptionally silenced and active genome regions.

EsxA, a type VII secretion system-dependent effector, reveals a novel function in the sporulation of Bacillus cereus ATCC14579

BackgroundBacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA’s roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished.MethodsWe used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar.ResultsThrough LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3’ end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567.ConclusionB. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA.

Impact of AbaI mutation on virulence, biofilm development, and antibiotic susceptibility in Acinetobacter baumannii

The quorum sensing (QS) system mediated by the abaI gene in Acinetobacter baumannii is crucial for various physiological and pathogenic processes. In this study, we constructed a stable markerless abaI knockout mutant (ΔabaI) strain using a pEXKm5-based allele replacement method to investigate the impact of abaI on A. baumannii. Proteomic analysis revealed significant alterations in protein expression between the wild type (WT) and ΔabaI mutant strains, particularly in proteins associated with membrane structure, antibiotic resistance, and virulence. Notably, the downregulation of key outer membrane proteins such as SurA, OmpA, OmpW, and BamA suggests potential vulnerabilities in outer membrane integrity, which correlate with structural abnormalities in the ΔabaI mutant strain, including irregular cell shapes and compromised membrane integrity, observed by scanning and transmission electron microscopy. Furthermore, diminished expression of regulatory proteins such as OmpR and GacA-GacS highlights the broader regulatory networks affected by abaI deletion. Functional assays revealed impaired biofilm formation and surface-associated motility in the mutant strain, indicative of altered colonization capabilities. Interestingly, the mutant showed a complex antibiotic susceptibility profile. While it demonstrated increased susceptibility to membrane-targeting antibiotics, its response to beta-lactams was more nuanced. Despite increased expression of metallo-beta-lactamase (MBL) superfamily proteins and DcaP-like protein, the mutant unexpectedly showed lower MICs for carbapenems (imipenem and meropenem) compared to the wild-type strain. This suggests that abaI deletion affects antibiotic susceptibility through multiple, potentially competing mechanisms. Further investigation is needed to fully elucidate the interplay between quorum sensing, antibiotic resistance genes, and overall antibiotic susceptibility in A. baumannii. Our findings underscore the multifaceted role of the abaI gene in modulating various cellular processes and highlight its significance in A. baumannii physiology, pathogenesis, and antibiotic resistance. Targeting the abaI QS system may offer novel therapeutic strategies for this clinically significant pathogen.

Copper Complexes with Protein-Based N-Donor Ligands as cis-Selective Nascent Cyclopropanases.

In this study, we aimed to develop protein-based metal ligands to catalyze cis-selective cyclopropanation using the TM1459 cupin protein superfamily. Copper complexes with TM1459 mutants containing the 3-His metal-binding site exhibited excellent diastereoselectivity in cyclopropanation reactions with styrene and ethyl diazoacetate. Further mutations in the secondary coordination sphere increased the cis-preference with t-butyl diazoacetate as the substrate with up to 80 : 20 (cis:trans ratio) and high enantioselectivity (90 % ee).

BHLHE41, a transcriptional repressor involved in physiological processes and tumor development.

BHLHE41 is a nuclear transcriptional repressor that belongs to the basic helix-loop-helix protein superfamily. BHLHE41 expression tends to be restricted to specific tissues and is regulated by environmental cues and biological events. BHLHE41 homodimerizes or heterodimerizes with various partners, influencing its transcription factor function. BHLHE41 is involved in the regulation of many physiological processes implicated in tissue/organ homeostasis, such as myogenesis, adipogenesis, circadian rhythms and DNA repair. At cellular level, BHLHE41 is involved in the regulation of mesenchymal stem cell properties, tissue-specific macrophage functions and lymphoid lineage physiology. In several cancer types, BHLHE41 modulates the expression of different transcriptional programs influencing cell cycle control, apoptosis, invasiveness, epithelial to mesenchymal transition and hypoxia response in the tumor environment. Depending on the cancer cell type, BHLHE41 can act as a tumor suppressor or an oncogene, and could be a target for innovative therapies. This review summarizes the available knowledge on BHLHE41 structure, biological functions, regulation and potential partners, as well as its role in physiological processes, and its implication in major cancer steps.

Lessons learned during the journey of data: from experiment to model for predicting kinase affinity, selectivity, polypharmacology, and resistance.

Recent advances in machine learning (ML) are reshaping drug discovery. Structure-based ML methods use physically-inspired models to predict binding affinities from protein:ligand complexes. These methods promise to enable the integration of data for many related targets, which addresses issues related to data scarcity for single targets and could enable generalizable predictions for a broad range of targets, including mutants. In this work, we report our experiences in building KinoML, a novel framework for ML in target-based small molecule drug discovery with an emphasis on structure-enabled methods. KinoML focuses currently on kinases as the relative structural conservation of this protein superfamily, particularly in the kinase domain, means it is possible to leverage data from the entire superfamily to make structure-informed predictions about binding affinities, selectivities, and drug resistance. Some key lessons learned in building KinoML include: the importance of reproducible data collection and deposition, the harmonization of molecular data and featurization, and the choice of the right data format to ensure reusability and reproducibility of ML models. As a result, KinoML allows users to easily achieve three tasks: accessing and curating molecular data; featurizing this data with representations suitable for ML applications; and running reproducible ML experiments that require access to ligand, protein, and assay information to predict ligand affinity. Despite KinoML focusing on kinases, this framework can be applied to other proteins. The lessons reported here can help guide the development of platforms for structure-enabled ML in other areas of drug discovery.

Melastatin subfamily Transient Receptor Potential channels support spermatogenesis in planarian flatworms.

The Transient Receptor Potential superfamily of proteins (TRPs) form cation channels that are abundant in animal sensory systems. Amongst TRPs, the Melastatin-related subfamily (TRPMs) is composed of members that respond to temperature, pH, sex hormones, and various other stimuli. Some TRPMs exhibit enriched expression in gonads of vertebrate and invertebrate species, but their contributions to germline development remain to be determined. We identified twenty-one potential TRPMs in the planarian flatworm Schmidtea mediterranea and analyzed their anatomical distribution of expression by whole-mount in situ hybridization. Enriched expression of two TRPMs (Smed-TRPM-c and Smed-TRPM-l) was detected in testis, whereas eight TRPM genes had detectable expression in patterns representative of neuronal and/or sensory cell types. Functional analysis of TRPM homologs by RNA-interference (RNAi) revealed that disruption of Smed-TRPM-c expression results in reduced sperm development, indicating a role for this receptor in supporting spermatogenesis. Smed-TRPM-l RNAi did not result in a detectable phenotype, but it increased sperm development deficiencies when combined with Smed-TRPM-c RNAi. Fluorescence in situ hybridization revealed expression of Smed-TRPM-c in early spermatogenic cells within testes, suggesting cell-autonomous regulatory functions in germ cells for this gene. In addition, Smed-TRPM-c RNAi resulted in reduced numbers of presumptive germline stem cell clusters in asexual planarians, suggesting that Smed-TRPM-c supports establishment, maintenance, and/or expansion of spermatogonial germline stem cells. While further research is needed to identify the factors that trigger Smed-TRPM-c activity, these findings reveal one of few known examples for TRPM function in direct regulation of sperm development.

KIFC3 promotes the proliferation, migration and invasion of non-small cell lung cancer through the PI3K/AKT signaling pathway

KIFC3 is a member of the Kinesin superfamily proteins (KIFs). The role of KIFC3 in non-small cell lung cancer (NSCLC) is unknown. This study aimed to elucidate the function of KIFC3 in NSCLC and the underlying mechanism. Immunohistochemistry indicated that KIFC3 was highly expressed in NSCLC tissues and correlated with the degree of differentiation, tumor size, lymph node metastasis and TNM stage. MTT, colony formation and Transwell assays demonstrated that KIFC3 overexpression promoted the proliferation, migration and invasion of NSCLC cells in vitro, while KIFC3 knockdown led to the opposite results. The protein expression levels of PI3Kp85α and p-Akt were increased after KIFC3 overexpression, meanwhile the downstream protein expression levels such as cyclin D1, CDK4, CDK6, RhoA, RhoC and MMP2 were increased. This promotion effect could be inhibited by a specific inhibitor of the PI3K/Akt pathway, LY294002. Co-immunoprecipitation assays confirmed the interaction between endogenous/exogenous KIFC3 and PI3Kp85α. Tumor formation experiments in nude mice confirmed that KIFC3 overexpression promoted the proliferation, migration and invasion of NSCLC cells in vivo and performed its biological function through the PI3K/Akt signaling pathway.In conclusion, KIFC3 promotes the malignant behavior of NSCLC cells through the PI3K/Akt signaling pathway.

Plasmodium berghei HMGB1 controls the host immune responses and splenic clearance by regulating the expression of pir genes

High mobility group box (HMGB) proteins belong to high mobility group (HMG) superfamily of non-histone nuclear proteins that are involved in chromatin remodeling, regulation of gene expression and DNA repair. When extracellular, HMGBs serve as alarmins inducing inflammation and this is attributed to the proinflammatory activity of box B. Here, we show that Plasmodium HMGB1 has key amino acid changes in box B resulting in the loss of TNF-α stimulatory activity. Site-directed mutagenesis of the critical amino acids in box B with respect to mouse HMGB1 renders recombinant Plasmodium berghei (Pb) HMGB1 capable of inducing TNF-α release. Targeted deletion of PbHMGB1 and a detailed in vivo phenotyping show that PbHMGB1 knockout (KO) parasites can undergo asexual stage development. Interestingly, Balb/c mice-infected with PbHMGB1KO parasites display a protective phenotype with subsequent clearance of blood parasitemia, and develop long-lasting protective immunity against the challenges performed with Pb wildtype parasites. The characterization of splenic responses show prominent germinal centres leading to effective humoral responses and enhanced T follicular helper cells. There is also a complete protection from experimental cerebral malaria in CBA/CaJ mice susceptible for cerebral pathogenesis with subsequent parasite clearance. Transcriptomic studies suggest the involvement of PbHMGB1 in pir expression. Our findings highlight the gene regulatory function of parasite HMGB1 and its in vivo significance in modulating the host immune responses. Further, clearance of asexual stages in PbHMGB1KO-infected mice underscores the important role of parasite HMGB1 in host immune evasion. These findings have implications in developing attenuated blood-stage vaccine for malaria.

Unraveling the Role of PCDH9 in Breast Cancer and Identifying Therapeutic Strategies for PCDH9-Deficient Tumors.

Protocadherin 9 (PCDH9), a member of the cadherin superfamily of transmembrane proteins, plays a role in cell adhesion and neural development. Recent studies suggest that PCDH9 may function as a tumor suppressor in certain cancers, though its specific role in breast cancer remains unclear. UALCAN database to retrieve information on PCDH9 expression in breast cancer tissues compared with that in normal tissues. The biological effects of PCDH9 in breast cancer cells were analyzed using the DepMap database. Stable knockdown or overexpression of PCDH9 in breast cancer cell lines and subsequently assessed tumor cell proliferation and migration. Synthetic lethal screening was conducted for breast cancer cells with low PCDH9 expression or deficiency. In this study, we observed significant downregulation of PCDH9 in breast cancer tissues, with its expression negatively correlated with progression-free survival. Further investigations revealed that decreased PCDH9 expression promotes breast cancer cell proliferation and migration, while overexpression of PCDH9 has the opposite effect. Subsequently, we identified the TAS-102, an approved drug for metastatic colorectal cancer, exhibited selective cytotoxicity against breast cancer cells with low PCDH9 expression. In summary, our study identified PCDH9 as a tumor suppressor in breast cancer and highlighted TAS-102 as a potential therapeutic option for tumors with low PCDH9 expression or deficiency. The specific interaction between TAS-102 and PCDH9 warrants further exploration, providing deeper insights into its mode of action in treating PCDH9-deficient breast cancer.

GTPBP8 mitigates nonalcoholic steatohepatitis (NASH) by depressing hepatic oxidative stress and mitochondrial dysfunction via PGC-1α signaling

Nonalcoholic steatohepatitis (NASH) is emerging as a major cause of liver transplantation and hepatocellular carcinoma (HCC). Regrettably, its pathological mechanisms are still not fully comprehended. GTP-binding protein 8 (GTPBP8), belonging to the GTP-binding protein superfamily, assumes a crucial role in RNA metabolism, cell proliferation, differentiation, and signal transduction. Its aberrant expression is associated with oxidative stress and mitochondrial dysfunctions. Nevertheless, its specific functions and mechanisms of action, particularly in NASH, remain elusive. In our current study, we initially discovered that human hepatocytes L02 displayed evident mitochondrial respiratory anomaly, mitochondrial damage, and dysfunction upon treatment with palmitic acids and oleic acids (PO), accompanied by significantly reduced GTPBP8 expression levels through RNA-Seq, RT-qPCR, western blotting, and immunofluorescence assays. We then demonstrated that GTPBP8 overexpression mediated by adenovirus vector (Ad-GTPBP8) markedly attenuate lipid accumulation, inflammatory response, and mitochondrial impair and dysfunction in hepatocytes stimulated by PO. Conversely, adenovirus vector-mediated GTPBP8 knockdown (Ad-shGTPBP8) significantly accelerated lipid deposition, inflammation and mitochondrial damage in PO-treated hepatocytes in vitro. Furthermore, we constructed an in vivo NASH murine model by giving a 16-week high fat high cholesterol diet (HFHC) diet to hepatocyte specific GTPBP8-knockout (GTPBP8HKO) mice. We firstly found that HFHC feeding led to metabolic disorder in mice, including high body weight, blood glucose and insulin levels, and liver dysfunctions, which were accelerated in these NASH mice with GTPBP8 deficiency in hepatocytes. Consistently, GTPBP8HKO remarkably exacerbated the progression of NASH phenotypes induced by HFHC, as proved by the anabatic lipid accumulation, inflammation, fibrosis and reactive oxygen species (ROS) production in liver tissues, which could be largely attributed to the severe mitochondrial damage and dysfunction. Mechanistically, we further identified that GTPBP8 interacted with peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in hepatocytes. Importantly, the hepaprotective effects of GTPBP8 against mitochondrial dysfunction, oxidative stress and inflammation was largely dependent on PGC-1α expression. Collectively, GTPBP8 may exert a protective role in the progression of NASH, and targeting the GTPBP8/PGC-1α axis may represent a potential strategy for NASH treatment by improving mitochondrial functions.

Intrinsic Disorder and Other Malleable Arsenals of Evolved Protein Multifunctionality.

Microscopic evolution at the functional biomolecular level is an ongoing process. Leveraging functional and high-throughput assays, along with computational data mining, has led to a remarkable expansion of our understanding of multifunctional protein (and gene) families over the past few decades. Various molecular and intermolecular mechanisms are now known that collectively meet the cumulative multifunctional demands in higher organisms along an evolutionary path. This multitasking ability is attributed to a certain degree of intrinsic or adapted flexibility at the structure-function level. Evolutionary diversification of structure-function relationships in proteins highlights the functional importance of intrinsically disordered proteins/regions (IDPs/IDRs) which are highly dynamic biological soft matter. Multifunctionality is favorably supported by the fluid-like shapes of IDPs/IDRs, enabling them to undergo disorder-to-order transitions upon binding to different molecular partners. Other new malleable members of the protein superfamily, such as those involved in fold-switching, also undergo structural transitions. This new insight diverges from all traditional notions of functional singularity in enzyme classes and emphasizes a far more complex, multi-layered diversification of protein functionality. However, a thorough review in this line, focusing on flexibility and function-driven structural transitions related to evolved multifunctionality in proteins, is currently missing. This review attempts to address this gap while broadening the scope of multifunctionality beyond single protein sequences. It argues that protein intrinsic disorder is likely the most striking mechanism for expressing multifunctionality in proteins. A phenomenological analogy has also been drawn to illustrate the increasingly complex nature of modern digital life, driven by the need for multitasking, particularly involving media.

Seqrutinator: scrutiny of large protein superfamily sequence datasets for the identification and elimination of non-functional homologues

Seqrutinator is an objective, flexible pipeline that removes sequences with sequencing and/or gene model errors and sequences from pseudogenes from complex, eukaryotic protein superfamilies. Testing Seqrutinator on major superfamilies BAHD, CYP, and UGT removes only 1.94% of SwissProt entries, 14% of entries from the model plant Arabidopsis thaliana, but 80% of entries from Pinus taeda’s recent complete proteome. Application of Seqrutinator on crude BAHDomes, CYPomes, and UGTomes obtained from 16 plant proteomes shows convergence of the numbers of paralogues. MSAs, phylogenies, and particularly functional clustering improve drastically upon Seqrutinator application, indicating good performance.

Protein disulfide isomerase PDI8 is indispensable for parasite growth and associated with secretory protein processing in Toxoplasma gondii.

Apicomplexans, a phylum of intracellular parasites, encompass various zoonotic pathogens, including Plasmodium, Cryptosporidium, Toxoplasma, and Babesia, causing a significant economic burden on human populations. These parasites exhibit hypersensitivity to disruptions in endoplasmic reticulum (ER) redox homeostasis, necessitating the presence of ER-localized thioredoxin (Trx) superfamily proteins, particularly protein disulfide isomerase (PDI), for proper oxidative folding. However, the functional characteristics of ER-localized PDI proteins in Toxoplasma gondii remain largely unexplored. In this study, we identified two ER-localized proteins, namely, TgPDI8 and TgPDI6, and demonstrated the indispensable role of TgPDI8 in parasite survival. Through a comprehensive multi-omics analysis, we elucidated the crucial role of TgPDI8 in the processing of secretory proteins in T. gondii. Additionally, we introduced a novel ER-anchored TurboID method to label and identify canonical secretory proteins in T. gondii. This research opens up new avenues for understanding oxidative folding and the secretory pathway in apicomplexan parasites, laying the groundwork for future advancements in antiparasitic drug development.

Antiviral Mx proteins have an ancient origin and widespread distribution among eukaryotes.

First identified in mammals, Mx proteins are potent antivirals against a broad swathe of viruses. Mx proteins arose within the Dynamin superfamily of proteins (DSP), mediating critical cellular processes, such as endocytosis and mitochondrial, plastid, and peroxisomal dynamics. And yet, the evolutionary origins of Mx proteins are poorly understood. Using a series of phylogenomic analyses with stepwise increments in taxonomic coverage, we show that Mx proteins predate the interferon signaling system in vertebrates. Our analyses find an ancient monophyletic DSP lineage in eukaryotes that groups vertebrate and invertebrate Mx proteins with previously undescribed fungal MxF proteins, the relatively uncharacterized plant and algal Dynamin 4A/4C proteins, and representatives from several early-branching eukaryotic lineages. Thus, Mx-like proteins date back close to the origin of Eukarya. Our phylogenetic analyses also reveal that host-encoded and NCLDV (nucleocytoplasmic large DNA viruses)-encoded DSPs are interspersed in four distinct DSP lineages, indicating recurrent viral theft of host DSPs. Our analyses thus reveal an ancient history of viral and antiviral functions encoded by the Dynamin superfamily in eukaryotes.

RNA or DNA? Revisiting the Chemical Nature of the Cenancestral Genome

One of the central issues in the understanding of early cellular evolution is the characterisation of the cenancestor. This includes the description of the chemical nature of its genome. The disagreements on this question comprise several proposals, including the possibility that AlkB-mediated methylation repair of alkylated RNA molecules may be interpreted as evidence of a cenancestral RNA genome. We present here an evolutionary analysis of the cupin-like protein superfamily based on tertiary structure-based phylogenies that includes the oxygen-dependent AlkB and its homologs. Our results suggest that the repair of methylated RNA molecules is the outcome of the enzyme substrate ambiguity, and doesn´t necessarily indicates that the last common ancestor was endowed with an RNA genome.