Eukaryotic Proteomes Research Articles

Conserved degronome features governing quality control associated proteolysis.

The eukaryotic proteome undergoes constant surveillance by quality control systems that either sequester, refold, or eliminate aberrant proteins by ubiquitin-dependent mechanisms. Ubiquitin-conjugation necessitates the recognition of degradation determinants, termed degrons, by their cognate E3 ubiquitin-protein ligases. To learn about the distinctive properties of quality control degrons, we performed an unbiased peptidome stability screen in yeast. The search identify a large cohort of proteome-derived degrons, some of which exhibited broad E3 ligase specificity. Consequent application of a machine-learning algorithm establishes constraints governing degron potency, including the amino acid composition and secondary structure propensities. According to the set criteria, degrons with transmembrane domain-like characteristics are the most probable sequences to act as degrons. Similar quality control degrons are present in viral and human proteins, suggesting conserved degradation mechanisms. Altogether, the emerging data indicate that transmembrane domain-like degron features have been preserved in evolution as key quality control determinants of protein half-life.

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs.

New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein-protein and protein-lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenous─as opposed to overexpressed─membrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane-protein complexes from Chaetomium thermophilum in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane-protein complexes. Mass spectrometry results revealed ∼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane-protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes.

Structural basis for sequence-independent substrate selection by eukaryotic wobble base tRNA deaminase ADAT2/3

The essential deamination of adenosine A34 to inosine at the wobble base is the individual tRNA modification with the greatest effects on mRNA decoding, empowering a single tRNA to translate three different codons. To date, many aspects of how eukaryotic deaminases specifically select their multiple substrates remain unclear. Here, using cryo-EM, we present the structure of a eukaryotic ADAT2/3 deaminase bound to a full-length tRNA, revealing that the enzyme distorts the anticodon loop, but in contrast to the bacterial enzymes, selects its substrate via sequence-independent contacts of eukaryote-acquired flexible or intrinsically unfolded motifs distal from the conserved catalytic core. A gating mechanism for substrate entry to the active site is identified. Our multi-step tRNA recognition model yields insights into how RNA editing by A34 deamination evolved, shaped the genetic code, and directly impacts the eukaryotic proteome.

The Difference in Structural States between Canonical Proteins and Their Isoforms Established by Proteome-Wide Bioinformatics Analysis

Alternative splicing is an important means of generating the protein diversity necessary for cellular functions. Hence, there is a growing interest in assessing the structural and functional impact of alternative protein isoforms. Typically, experimental studies are used to determine the structures of the canonical proteins ignoring the other isoforms. Therefore, there is still a large gap between abundant sequence information and meager structural data on these isoforms. During the last decade, significant progress has been achieved in the development of bioinformatics tools for structural and functional annotations of proteins. Moreover, the appearance of the AlphaFold program opened up the possibility to model a large number of high-confidence structures of the isoforms. In this study, using state-of-the-art tools, we performed in silico analysis of 58 eukaryotic proteomes. The evaluated structural states included structured domains, intrinsically disordered regions, aggregation-prone regions, and tandem repeats. Among other things, we found that the isoforms have fewer signal peptides, transmembrane regions, or tandem repeat regions in comparison with their canonical counterparts. This could change protein function and/or cellular localization. The AlphaFold modeling demonstrated that frequently isoforms, having differences with the canonical sequences, still can fold in similar structures though with significant structural rearrangements which can lead to changes of their functions. Based on the modeling, we suggested classification of the structural differences between canonical proteins and isoforms. Altogether, we can conclude that a majority of isoforms, similarly to the canonical proteins are under selective pressure for the functional roles.

Intramembrane client recognition potentiates the chaperone functions of calnexin

One‐third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar‐binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin‐based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.

Twin cyclic mononucleotide cyclase and phosphodiesterase domain architecture as a common feature in complex plant proteins

The majority of proteins in both prokaryote and eukaryote proteomes consist of two or more functional centers, which allows for intramolecular tuning of protein functions. Such architecture, as opposed to animal orthologs, applies to the plant cyclases (CNC) and phosphodiesterases (PDEs), the vast majority of which are part of larger multifunctional proteins. In plants, until recently, only two cases of combinations of CNC-PDE in one protein were reported. Here we propose that in plants, multifunctional proteins in which the PDE motif has been identified, the presence of the additional CNC center is common. Searching the Arabidopsis thaliana proteome with a combined PDE-CNC motif allowed the creation of a database of proteins with both activities. One such example is methylenetetrahydrofolate dehydrogenase, in which we determined the activities of adenylate cyclase (AC) and PDE. Based on biochemical and mutagenesis analyses we assessed the impact of the AC and PDE catalytic centers on the dehydrogenase activity. This allowed us to propose additional regulatory mechanism that govern folate metabolism by cAMP. It is therefore conceivable that the combined CNC-PDE architecture is a common regulatory configuration, where control of the level of cyclic nucleotides (cNMP) influences other catalytic activities of the protein.

The sequence context in poly-alanine regions: structure, function and conservation.

Poly-alanine (polyA) regions are protein stretches mostly composed of alanines. Despite their abundance in eukaryotic proteomes and their association to nine inherited human diseases, the structural and functional roles exerted by polyA stretches remain poorly understood. In this work we study how the amino acid context in which polyA regions are settled in proteins influences their structure and function. We identified glycine and proline as the most abundant amino acids within polyA and in the flanking regions of polyA tracts, in human proteins as well as in 17 additional eukaryotic species. Our analyses indicate that the non-structuring nature of these two amino acids influences the α-helical conformations predicted for polyA, suggesting a relevant role in reducing the inherent aggregation propensity of long polyA. Then, we show how polyA position in protein N-termini relates with their function as transit peptides. PolyA placed just after the initial methionine is often predicted as part of mitochondrial transit peptides, whereas when placed in downstream positions, polyA are part of signal peptides. A few examples from known structures suggest that short polyA can emerge by alanine substitutions in α-helices; but evolution by insertion is observed for longer polyA. Our results showcase the importance of studying the sequence context of homorepeats as a mechanism to shape their structure-function relationships. The datasets used and/or analyzed during the current study are available from the corresponding author onreasonable request. Supplementary data are available at Bioinformatics online.

FOXA1 regulates alternative splicing in prostate cancer.

SummaryDysregulation of alternative splicing in prostate cancer is linked to transcriptional programs activated by AR, ERG, FOXA1, and MYC. Here, we show that FOXA1 functions as the primary orchestrator of alternative splicing dysregulation across 500 primary and metastatic prostate cancer transcriptomes. We demonstrate that FOXA1 binds to the regulatory regions of splicing-related genes, including HNRNPK and SRSF1. By controlling trans-acting factor expression, FOXA1 exploits an “exon definition” mechanism calibrating alternative splicing toward dominant isoform production. This regulation especially impacts splicing factors themselves and leads to a reduction of nonsense-mediated decay (NMD)-targeted isoforms. Inclusion of the NMD-determinant FLNA exon 30 by FOXA1-controlled oncogene SRSF1 promotes cell growth in vitro and predicts disease recurrence. Overall, we report a role for FOXA1 in rewiring the alternative splicing landscape in prostate cancer through a cascade of events from chromatin access, to splicing factor regulation, and, finally, to alternative splicing of exons influencing patient survival.

The Endoplasmic Reticulum and the Fidelity of Nascent Protein Localization.

High-fidelity protein localization is essential to define the identities and functions of different organelles and to maintain cellular homeostasis. Accurate localization of nascent proteins requires specific protein targeting pathways as well as quality control (QC) mechanisms to remove mislocalized proteins. The endoplasmic reticulum (ER) is the first destination for approximately one-third of the eukaryotic proteome and a major site of protein biosynthesis and QC. In mammalian cells, trafficking from the ER provides nascent proteins access to the extracellular space and essentially every cellular membrane and organelle except for mitochondria and possibly peroxisomes. Here, we discuss the biosynthetic mechanisms that deliver nascent proteins to the ER and the QC mechanisms that interface with the ER to correct or degrade mislocalized proteins.

Intrinsically disordered protein, DNA binding with one finger transcription factor (OsDOF27) implicates thermotolerance in yeast and rice.

Intrinsically disorder regions or proteins (IDRs or IDPs) constitute a large subset of the eukaryotic proteome, which challenges the protein structure–function paradigm. These IDPs lack a stable tertiary structure, yet they play a crucial role in the diverse biological process of plants. This study represents the intrinsically disordered nature of a plant-specific DNA binding with one finger transcription factor (DOF-TF). Here, we have investigated the role of OsDOF27 and characterized it as an intrinsically disordered protein. Furthermore, the molecular role of OsDOF27 in thermal stress tolerance has been elucidated. The qRT-PCR analysis revealed that OsDOF27 was significantly upregulated under different abiotic stress treatments in rice, particularly under heat stress. The stress-responsive transcript induction of OsDOF27 was further correlated with enriched abiotic stress-related cis-regulatory elements present in its promoter region. The in vivo functional analysis of the potential role of OsDOF27 in thermotolerance was further studied in yeast and in planta. Ectopic expression of OsDOF27 in yeast implicates thermotolerance response. Furthermore, the rice transgenic lines with overexpressing OsDOF27 revealed a positive role in mitigating heat stress tolerance. Collectively, our results evidently show the intrinsically disorderedness in OsDOF27 and its role in thermal stress response in rice.

The WD40-Protein PfWLP1 Ensures Stability of the PfCCp-Based Adhesion Protein Complex in Plasmodium falciparum Gametocytes.

Members of the WD40-repeat protein family can be found in all eukaryotic proteomes where they usually serve as interaction platforms for the assembly of large protein complexes and are therefore essential for the integrity of these complexes. In the malaria parasite Plasmodium falciparum, the WD40-repeat protein PfWLP1 has been shown to interact with members of distinct adhesion protein complexes in the asexual blood stages and gametocyte stages. In this study, we demonstrate that the presence of PfWLP1 is crucial for both the stability of these gametocyte-specific adhesion complexes as well as for gametocyte maturation and gametogenesis. Using reverse genetics, we generated a PfWLP1-knockdown parasite line for functional characterization of the protein. Knockdown of PfWLP1 resulted in a slight reduction of gametocyte numbers and significantly the impaired ability of the gametocytes to exflagellate. PfWLP1-knockdown further led to reduced protein levels of the Limulus coagulation factor C-like (LCCL)-domain proteins PfCCp1 and PfCCp2, which are key components of the adhesion complexes. These findings suggest that the interaction of PfWLP1 with members of the PfCCp-based adhesion complex ensures complex stability and thereby contributes to gametocyte viability and exflagellation.

The free energy folding penalty accompanying binding of intrinsically disordered α-helical motifs.

Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes and preform critical roles in many cellular processes, most often through the association with globular proteins. Despite lacking a stable three‐dimensional structure by themselves, they may acquire a defined conformation upon binding globular targets. The most common type of secondary structure acquired by these binding motifs entails formation of an α‐helix. It has been hypothesized that such disorder‐to‐order transitions are associated with a significant free energy penalty due to IDP folding, which reduces the overall IDP‐target affinity. However, the exact magnitude of IDP folding penalty in α‐helical binding motifs has not been systematically estimated. Here, we report the folding penalty contributions for 30 IDPs undergoing folding‐upon‐binding and find that the average IDP folding penalty is +2.0 kcal/mol and ranges from 0.7 to 3.5 kcal/mol. We observe that the folding penalty scales approximately linearly with the change in IDP helicity upon binding, which provides a simple empirical way to estimate folding penalty. We analyze to what extent do pre‐structuring and target‐bound IDP dynamics (fuzziness) reduce the folding penalty and find that these effects combined, on average, reduce the folding cost by around half. Taken together, the presented analysis provides a quantitative basis for understanding the role of folding penalty in IDP‐target interactions and introduces a method estimate this quantity. Estimation and reduction of IDP folding penalty may prove useful in the rational design of helix‐stabilized inhibitors of IDP‐target interactions.StatementThe α‐helical binding motifs are ubiquitous among the intrinsically disordered proteins (IDPs). Upon binding their targets, they undergo a disorder‐to‐order transition, which is accompanied by a significant folding penalty whose magnitude is generally not known. Here, we use recently developed statistical‐thermodynamic model to estimate the folding penalties for 30 IDPs and clarify the roles of IDP pre‐folding and bound‐state dynamics in reducing the folding penalty.

Cooperativity and Folding Kinetics in a Multidomain Protein with Interwoven Chain Topology.

Although a large percentage of eukaryotic proteomes consist of proteins with multiple domains, not much is known about their assembly mechanism, especially those with intricate native state architectures. Some have a complex topology in which the structural elements along the sequence are interwoven in such a manner that the domains cannot be separated by cutting at any location along the sequence. Such proteins are multiply connected multidomain proteins (MMPs) with the three-domain (NMP, LID, and CORE) phosphotransferase enzyme adenylate kinase (ADK) being an example. We devised a coarse-grained model to simulate ADK folding initiated by changing either the temperature or guanidinium chloride (GdmCl) concentration. The simulations reproduce the experimentally measured melting temperatures (associated with two equilibrium transitions), FRET efficiency as a function of GdmCl concentration, and the folding times quantitatively. Although the NMP domain orders independently, cooperative interactions between the LID and the CORE domains are required for complete assembly of the enzyme. Kinetic simulations show that, on the collapse time scale, multiple interconnected metastable states are populated, attesting to the folding heterogeneity. The network of kinetically connected states reveals that the CORE domain folds only after the NMP and LID domains, reflecting the interwoven nature of the chain topology.

Comparative Analysis of Structural Features in SLiMs from Eukaryotes, Bacteria, and Viruses with Importance for Host-Pathogen Interactions.

Protein-protein interactions drive functions in eukaryotes that can be described by short linear motifs (SLiMs). Conservation of SLiMs help illuminate functional SLiMs in eukaryotic protein families. However, the simplicity of eukaryotic SLiMs makes them appear by chance due to mutational processes not only in eukaryotes but also in pathogenic bacteria and viruses. Further, functional eukaryotic SLiMs are often found in disordered regions. Although proteomes from pathogenic bacteria and viruses have less disorder than eukaryotic proteomes, their proteins can successfully mimic eukaryotic SLiMs and disrupt host cellular function. Identifying important SLiMs in pathogens is difficult but essential for understanding potential host-pathogen interactions. We performed a comparative analysis of structural features for experimentally verified SLiMs from the Eukaryotic Linear Motif (ELM) database across viruses, bacteria, and eukaryotes. Our results revealed that many viral SLiMs and specific motifs found across viruses and eukaryotes, such as some glycosylation motifs, have less disorder. Analyzing the disorder and coil properties of equivalent SLiMs from pathogens and eukaryotes revealed that some motifs are more structured in pathogens than their eukaryotic counterparts and vice versa. These results support a varying mechanism of interaction between pathogens and their eukaryotic hosts for some of the same motifs.

Ribosomal protein S18 acetyltransferase RimI is responsible for the acetylation of elongation factor Tu

N-terminal acetylation is widespread in the eukaryotic proteome but in bacteria is restricted to a small number of proteins mainly involved in translation. It was long known that elongation factor Tu (EF-Tu) is N-terminally acetylated, whereas the enzyme responsible for this process was unclear. Here, we report that RimI acetyltransferase, known to modify ribosomal protein S18, is likewise responsible for N-acetylation of the EF-Tu. With the help of inducible tufA expression plasmid, we demonstrated that the acetylation does not alter the stability of EF-Tu. Binding of aminoacyl tRNA to the recombinant EF-Tu in vitro was found to be unaffected by the acetylation. At the same time, with the help of fast kinetics methods, we demonstrate that an acetylated variant of EF-Tu more efficiently accelerates A-site occupation by aminoacyl-tRNA, thus increasing the efficiency of in vitro translation. Finally, we show that a strain devoid of RimI has a reduced growth rate, expanded to an evolutionary timescale, and might potentially promote conservation of the acetylation mechanism of S18 and EF-Tu. This study increased our understanding of the modification of bacterial translation apparatus.

The evolution of microtubule associated proteins \u2013 a reference proteomic perspective

Microtubule associated proteins (MAPs), defined as proteins that bind microtubules but are not molecular motors or severing enzymes, play a key role in regulating microtubule stability in neurons. Existing studies of the evolutionary relationships between these proteins are limited to genomic data from a small number of species. We therefore used a large collection of publicly available reference-quality eukaryotic proteomes to carry out a phylogenetic analysis of microtubule associated proteins in both vertebrates and invertebrates. Complete or near-complete reference quality proteomes were obtained from Uniprot. Microtubule associated proteins were identified using InterProtScan, aligned using MUSCLE and then phylogenetic trees constructed using the WAG algorithm. We identified 889 proteins with tubulin binding domains, of which 663 were in eukaryotes, including 168 vertebrates and 64 invertebrates. The vertebrate proteins separated into three families, resembling human MAP 2, MAP4 and MAPT, respectively, while invertebrate MAPs clustered separately. We found significant variation in number of microtubule associated proteins and number of microtubule binding domains between taxa, with fish and mollusks having an unexpectedly high number of MAPs and binding domains, respectively. Our findings represent a novel analysis of the evolution of microtubule associated proteins based on publicly available proteomics data sets. We were able to confirm the phylogeny of MAPs identified based on more limited genomic analyses, and in addition, derived several novel insights on the structure and function of MAPs.

GATD3A, a mitochondrial deglycase with evolutionary origins from gammaproteobacteria, restricts the formation of advanced glycation end products

BackgroundFunctional complexity of the eukaryotic mitochondrial proteome is augmented by independent gene acquisition from bacteria since its endosymbiotic origins. Mammalian homologs of many ancestral mitochondrial proteins have uncharacterized catalytic activities. Recent forward genetic approaches attributed functions to proteins in established metabolic pathways, thereby limiting the possibility of identifying novel biology relevant to human disease. We undertook a bottom-up biochemistry approach to discern evolutionarily conserved mitochondrial proteins with catalytic potential.ResultsHere, we identify a Parkinson-associated DJ-1/PARK7-like protein—glutamine amidotransferase-like class 1 domain-containing 3A (GATD3A), with bacterial evolutionary affinities although not from alphaproteobacteria. We demonstrate that GATD3A localizes to the mitochondrial matrix and functions as a deglycase. Through its amidolysis domain, GATD3A removes non-enzymatic chemical modifications produced during the Maillard reaction between dicarbonyls and amines of nucleotides and amino acids. GATD3A interacts with factors involved in mitochondrial mRNA processing and translation, suggestive of a role in maintaining integrity of important biomolecules through its deglycase activity. The loss of GATD3A in mice is associated with accumulation of advanced glycation end products (AGEs) and altered mitochondrial dynamics.ConclusionsAn evolutionary perspective helped us prioritize a previously uncharacterized but predicted mitochondrial protein GATD3A, which mediates the removal of early glycation intermediates. GATD3A restricts the formation of AGEs in mitochondria and is a relevant target for diseases where AGE deposition is a pathological hallmark.

Promiscuous Domains in Eukaryotes and HAT Proteins in FUNGI Have Followed Different Evolutionary Paths.

Diverse studies have shown that the content of genes present in sequenced genomes does not seem to correlate with the complexity of the organisms. However, various studies have shown that organism complexity and the size of the proteome has, indeed, a significant correlation. This characteristic allows us to postulate that some molecular mechanisms have permitted a greater functional diversity to some proteins to increase their participation in developing organisms with higher complexity. Among those mechanisms, the domain promiscuity, defined as the ability of the domains to organize in combination with other distinct domains, is of great importance for the evolution of organisms. Previous works have analyzed the degree of domain promiscuity of the proteomes showing how it seems to have paralleled the evolution of eukaryotic organisms. The latter has motivated the present study, where we analyzed the domain promiscuity in a collection of 84 eukaryotic proteomes representative of all the taxonomy groups of the tree of life. Using a grammar definition approach, we determined the architecture of 1,223,227 proteins, conformed by 2,296,371 domains, which established 839,184 bigram types. The phylogenetic reconstructions based on differences in the content of information from measures of proteome promiscuity confirm that the evolution of the promiscuity of domains in eukaryotic organisms resembles the evolutionary history of the species. However, a close analysis of the PHD and RING domains, the most promiscuous domains found in fungi and functional components of chromatin remodeling enzymes and important expression regulators, suggests an evolution according to their function.

Enhancing the Discovery of Functional Post-Translational Modification Sites with Machine Learning Models - Development, Validation, and Interpretation.

Protein posttranslational modifications (PTMs) are a rapidly expanding feature class of significant importance in cell biology. Due to a high burden of experimental proof, the number of functionals PTMs in the eukaryotic proteome is currently underestimated. Furthermore, not all PTMs are functionally equivalent. Computational approaches that can confidently recommend PTMs of probable function can improve the heuristics of PTM investigation and alleviate these problems. To address this need, we developed SAPH-ire: a multifeature heuristic neural network model that takes community wisdom into account by recommending experimental PTMs similar to those which have previously been established as having regulatory impact. Here, we describe the principle behind the SAPH-ire model, how it is developed, how we evaluate its performance, and important caveats to consider when building and interpreting such models. Finally, we discus current limitations of functional PTM prediction models and highlight potential mechanisms for their improvement.

Split-Chloramphenicol Acetyl Transferase Assay to Study Protein-Protein Interactions and Ubiquitylation in Escherichia coli.

Protein-protein interactions and protein modifications play central roles in all living organisms. Of the more than 200 types of post-translational modifications, ubiquitylation is the most abundant, and it profoundly regulates the functionality of the eukaryotic proteome. Various in vitro and in vivo methodologies to study protein interactions and modifications have been developed, each presenting distinctive benefits and caveats. Here, we present a comprehensive protocol for applying a split-Chloramphenicol Acetyl-Transferase (split-CAT) based system, to study protein-protein interactions and ubiquitylation in E. coli . Functional assembly of bait and prey proteins tethered to the split-CAT fragments result in antibiotic resistance and growth on selective media. We demonstrate assays for protein interactions, protein ubiquitylation, and the system response to small compound modulators. To facilitate data collection, we provide an updated Scanner Acquisition Manager Program for Laboratory Experiments (SAMPLE; https://github.com/PragLab/SAMPLE ) that can be employed to monitor the growth of various microorganisms, including E. coli and S. cerevisiae . The advantage posed by this system lies in its sensitivity to a wide range of chloramphenicol concentrations, which allows the detection of a large spectrum of protein-protein interactions, without the need for their purification. The tight linkage between binding or ubiquitylation and growth enables the estimation of apparent relative affinity, and represents the system's quantitative characteristics. Graphical abstract.