Molecular Evolution of Tubulins in Diatoms

Microtubules are formed by α- and β-tubulin heterodimers nucleated with γ-tubulin. Tubulins are conserved eukaryotic proteins. Previously, it was shown that microtubules are involved in diatom silica frustule morphogenesis. Diatom frustules are varied, and their morphology is species-specific. Despite the attractiveness of the problem of elucidating the molecular mechanisms of genetically programmed morphogenesis, the structure and evolution of diatom tubulins have not been studied previously. Based on available genomic and transcriptome data, we analyzed the phylogeny of the predicted amino acid sequences of diatom α-, β- and γ-tubulins and identified five groups for α-tubulins, six for β-tubulins and four for γ-tubulins. We identified characteristic amino acids of each of these groups and also analyzed possible posttranslational modification sites of diatom tubulins. According to our results, we assumed what changes occurred in the diatom tubulin structures during their evolution. We also identified which tubulin groups are inherent in large diatom taxa. The similarity between the evolution of diatom tubulins and the evolution of diatoms suggests that molecular changes in α-, β- and γ-tubulins could be one of the factors in the formation of a high morphological diversity of diatoms.

Monitoring Protein Import into the Endoplasmic Reticulum in Living Cells with Proximity Labeling

The proper trafficking of eukaryotic proteins is essential to cellular function. Genetic, environmental, and other stresses can induce protein mistargeting, and in turn threaten cellular protein homeostasis. Current methods for measuring protein mistargeting are difficult to translate to living cells, and thus the role of cellular signaling networks in stress-dependent protein mistargeting processes, such as ER pre-emptive quality control (ER pQC), are difficult to parse. Herein, we use genetically encoded peroxidases to characterize protein import into the endoplasmic reticulum (ER). We show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for identifying protein mistargeting, using the known ER pQC substrate transthyretin (TTR). Although ERHRP labeling induces formation of detergent-resistant TTR aggregates, this is minimized by using low ERHRP expression, without loss of labeling efficiency. cytAPEX labeling recovers TTR that is mistargeted as a consequence of Sec61 inhibition or ER stress-induced ER pQC. Furthermore, we demonstrate that stress-free activation of the ER stress-associated transcription factor ATF6 recapitulates the TTR import deficiency of ER pQC. Hence, proximity labeling is an effective strategy for characterizing factors that influence ER protein import in living cells.

Improvement of eukaryotic proteins prediction from soil metagenomes

Background | During the last decades, shotgun metagenomics and metabarcoding have highlighted the diversity of microorganisms from environmental or host-associated samples. Most assembled metagenome public repositories use annotation pipelines tailored for prokaryotes regardless of the taxonomic origin of contigs and metagenome-assembled genomes (MAGs). Consequently, eukaryotic contigs and MAGs, with intrinsically different gene features, are not optimally annotated, resulting in an incorrect representation of the eukaryotic component of biodiversity, despite their biological relevance. Results | Using an automated analysis pipeline, we have filtered eukaryotic contigs from 6,873 soil metagenomes from the IMG/M database of the Joint Genome Institute. We have re-annotated genes using eukaryote-tailored methods, yielding 5,6 million eukaryotic proteins. Our pipeline improves eukaryotic proteins completeness, contiguity and quality. Moreover, the better quality of eukaryotic proteins combined with a more comprehensive assignment method improves the taxonomic annotation as well. Conclusions | Using public soil metagenomic data, we provide a dataset of eukaryotic soil proteins with improved completeness and quality as well as a more reliable taxonomic annotation. This unique resource is of interest for any scientist aiming at studying the composition, biological functions and gene flux in soil communities involving eukaryotes.

Citrullination was introduced into animals by horizontal gene transfer from cyanobacteria

Protein post-translational modifications (PTMs) add great sophistication to biological systems. Citrullination, a key regulatory mechanism in human physiology and pathophysiology, is enigmatic from an evolutionary perspective. Although the citrullinating enzymes peptidylarginine deiminases (PADIs) are ubiquitous across vertebrates, they are absent from yeast, worms and flies. Based on this distribution PADIs were proposed to have been horizontally transferred, but this has been contested. Here, we map the evolutionary trajectory of PADIs into the animal lineage. We present strong phylogenetic support for a clade encompassing animal and cyanobacterial PADIs that excludes fungal and other bacterial homologues. The animal and cyanobacterial PADI proteins share functionally relevant primary and tertiary synapomorphic sequences that are distinct from a second PADI type present in fungi and actinobacteria. Molecular clock calculations and sequence divergence analyses using the fossil record estimate the last common ancestor of the cyanobacterial and animal PADIs to be less than one billion years old. Additionally, under an assumption of vertical descent, PADI sequence change during this evolutionary time frame is anachronistically low, even when compared to products of likely endosymbiont gene transfer, mitochondrial proteins and some of the most highly conserved sequences in life. The consilience of evidence indicates that PADIs were introduced from cyanobacteria into animals by horizontal gene transfer (HGT). The ancestral cyanobacterial PADI is enzymatically active and can citrullinate eukaryotic proteins, suggesting that the PADI HGT event introduced a new catalytic capability into the regulatory repertoire of animals. This study reveals the unusual evolution of a pleiotropic protein modification.

MPDB: a unified multi-domain protein structure database integrating structural analogue detection

With the development of protein structure prediction methods and biological experimental determination techniques, the structure of single-domain proteins can be relatively easier to be modeled or experimentally solved. However, more than 80% of eukaryotic proteins and 67% of prokaryotic proteins contain multiple domains. Constructing a unified multi-domain protein structure database will promote the research of multi-domain proteins, especially in the modeling of multi-domain protein structures. In this work, we develop a unified multi-domain protein structure database (MPDB). Based on MPDB, we also develop a server with two functional modules: (1) the culling module, which filters the whole MPDB according to input criteria; (2) the detection module, which identifies structural analogues of the full-chain according to the structural similarity between input domain models and the protein in MPDB. The module can discover the potential analogue structures, which will contribute to high-quality multi-domain protein structure modeling.

Post-translational Modifications of the Protein Termini

Post-translational modifications (PTM) involve enzyme-mediated covalent addition of functional groups to proteins during or after synthesis. These modifications greatly increase biological complexity and are responsible for orders of magnitude change between the variety of proteins encoded in the genome and the variety of their biological functions. Many of these modifications occur at the protein termini, which contain reactive amino- and carboxy-groups of the polypeptide chain and often are pre-primed through the actions of cellular machinery to expose highly reactive residues. Such modifications have been known for decades, but only a few of them have been functionally characterized. The vast majority of eukaryotic proteins are N- and C-terminally modified by acetylation, arginylation, tyrosination, lipidation, and many others. Post-translational modifications of the protein termini have been linked to different normal and disease-related processes and constitute a rapidly emerging area of biological regulation. Here we highlight recent progress in our understanding of post-translational modifications of the protein termini and outline the role that these modifications play in vivo.

The evolution of autophagy proteins – diversification in eukaryotes and potential ancestors in prokaryotes

ABSTRACT Autophagy is a degradative pathway for cytoplasmic constituents, and is conserved across eukaryotes. Autophagy-related (ATG) genes have undergone extensive multiplications and losses in different eukaryotic lineages, resulting in functional diversification and specialization. Notably, even though bacteria and archaea do not possess an autophagy pathway, they do harbor some remote homologs of Atg proteins, suggesting that preexisting proteins were recruited when the autophagy pathway developed during eukaryogenesis. In this Review, we summarize our current knowledge on the distribution of Atg proteins within eukaryotes and outline the major multiplication and loss events within the eukaryotic tree. We also discuss the potential prokaryotic homologs of Atg proteins identified to date, emphasizing the evolutionary relationships and functional differences between prokaryotic and eukaryotic proteins.

Gene Replacement in Arabidopsis Reveals Manganese Transport as an Ancient Feature of Human, Plant and Cyanobacterial UPF0016 Proteins

The protein family 0016 (UPF0016) is conserved through evolution, and the few members characterized share a function in Mn2+ transport. So far, little is known about the history of these proteins in Eukaryotes. In Arabidopsis thaliana five such proteins, comprising four different subcellular localizations including chloroplasts, have been described, whereas non-photosynthetic Eukaryotes have only one. We used a phylogenetic approach to classify the eukaryotic proteins into two subgroups and performed gene-replacement studies to investigate UPF0016 genes of various origins. Replaceability can be scored readily in the Arabidopsis UPF0016 transporter mutant pam71, which exhibits a functional deficiency in photosystem II. The N-terminal region of the Arabidopsis PAM71 was used to direct selected proteins to chloroplast membranes. Transgenic pam71 lines overexpressing the closest plant homolog (CMT1), human TMEM165 or cyanobacterial MNX successfully restored photosystem II efficiency, manganese binding to photosystem II complexes and consequently plant growth rate and biomass production. Thus AtCMT1, HsTMEM165, and SynMNX can operate in the thylakoid membrane and substitute for PAM71 in a non-native environment, indicating that the manganese transport function of UPF0016 proteins is an ancient feature of the family. We propose that the two chloroplast-localized UPF0016 proteins, CMT1 and PAM71, in plants originated from the cyanobacterial endosymbiont that gave rise to the organelle.

Evolutionary velocity with protein language models

Predicting the order of biological homologs is a fundamental task in evolutionary biology. For protein evolution, this order is often determined by first arranging sequences into a phylogenetic tree, which has limiting assumptions and can suffer from substantial ambiguity. Here, we demonstrate how machine learning algorithms called language models can learn mutational likelihoods that predict the directionality of evolution, thereby enabling phylogenetic analysis that addresses key limitations of existing methods. Our main conceptual advance is to construct a "vector field" of protein evolution through local evolutionary predictions that we refer to as evolutionary velocity (evo-velocity). We show that evo-velocity can successfully predict evolutionary order at vastly different timescales, from viral proteins evolving over years to eukaryotic proteins evolving over geologic eons. Evo-velocity also yields new evolutionary insights, predicting strategies of viral-host immune escape, resolving conflicting theories on the evolution of serpins, and revealing a key role of horizontal gene transfer in the evolution of eukaryotic glycolysis. In doing so, our work suggests that language models can learn sufficient rules of natural protein evolution to enable evolutionary predictability.

Identification of NAA40 as a Potential Prognostic Marker for Aggressive Liver Cancer Subtypes

Liver hepatocellular carcinoma (LIHC) is a leading cause of cancer-related mortality. In this study we initially interrogated the Cancer Genome Atlas (TCGA) dataset to determine the implication of N-terminal acetyltransferases (NATs), a family of enzymes that modify the N-terminus of the majority of eukaryotic proteins, in LIHC. This examination unveiled NAA40 as the NAT family member with the most prominent upregulation and significant disease prognosis for this cancer. Focusing on this enzyme, which selectively targets histone proteins, we show that its upregulation occurs from early stages of LIHC and is not specifically correlated with any established risk factors such as viral infection, obesity or alcoholic disease. Notably, in silico analysis of TCGA and other LIHC datasets found that expression of this epigenetic enzyme is associated with high proliferating, poorly differentiating and more aggressive LIHC subtypes. In particular, NAA40 upregulation was preferentially linked to mutational or non-mutational P53 functional inactivation. Accordingly, we observed that high NAA40 expression was associated with worse survival specifically in liver cancer patients with inactivated P53. These findings define NAA40 as a NAT with potentially oncogenic functions in LIHC and uncover its prognostic value for aggressive LIHC subtypes.