Mapping Degradation Signals and Pathways in a Eukaryotic N-terminome.

Most eukaryotic proteins are N-terminally acetylated. This modification can be recognized as a signal for selective protein degradation (degron) by the N-end rule pathways. However, the prevalence and specificity of such degrons in the proteome are unclear. Here, by systematically examining how protein turnover is affected by N-terminal sequences, we perform a comprehensive survey of degrons in the yeast N-terminome. We find that approximately 26% of nascent protein N termini encode cryptic degrons. These degrons exhibit high hydrophobicity and are frequently recognized by the E3 ubiquitin ligase Doa10, suggesting a role in protein quality control. In contrast, N-terminal acetylation rarely functions as a degron. Surprisingly, we identify two pathways where N-terminal acetylation has the opposite function and blocks protein degradation through the E3 ubiquitin ligase Ubr1. Our analysis highlights the complexity of N-terminal degrons and argues that hydrophobicity, not N-terminal acetylation, is the predominant feature of N-terminal degrons in nascent proteins.

Biallelic USP14 variants cause a syndromic neurodevelopmental disorder.

PURPOSE: Imbalances in protein homeostasis affect human brain development, with the ubiquitin-proteasome system (UPS) and autophagy playing crucial roles in neurodevelopmental disorders (NDD). This study explores the impact of biallelic USP14 variants on neurodevelopment, focusing on its role as a key hub connecting UPS and autophagy. METHODS: Here, we identified biallelic USP14 variants in 4 individuals from 3 unrelated families: 1 fetus, a newborn with a syndromic NDD and 2 siblings affected by a progressive neurological disease. Specifically, the 2 siblings from the latter family carried 2 compound heterozygous variants c.8T>C p.(Leu3Pro) and c.988C>T p.(Arg330∗), whereas the fetus had a homozygous frameshift c.899_902del p.(Lys300Serfs∗24) variant, and the newborn patient harbored a homozygous frameshift c.233_236del p.(Leu78Glnfs∗11) variant. Functional studies were conducted using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, western blotting, and mass spectrometry analyses in both patient-derived and CRISPR-Cas9-generated cells. RESULTS: Our investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities. CONCLUSION: Biallelic USP14 variants in NDD patients perturbed protein degradation pathways, potentially contributing to disorder etiology. Altered UPS, autophagy, and mitophagy activities underscore the intricate interplay, elucidating their significance in maintaining proper protein homeostasis during brain development.

N-Terminally arginylated ubiquitin is attached to histone H2A by RING1B E3 ligase in human cells.

Ubiquitin (Ub) is highly conserved in all eukaryotic organisms and begins at the N-terminus with Met and Gln. Our recent research demonstrates that N-terminally (Nt-) arginylated Ub can be produced in the yeast Saccharomyces cerevisiae. However, the existence of Nt-arginylated Ub in multicellular organisms remains unknown. Here we explore the mechanism for creating Nt-arginylated Ub using human embryonic kidney HEK293 cells that express various Nt-modified Ubs. We found that Gln-starting Q-Ub was converted into Glu-starting E-Ub by NTAQ1 Nt-deamidase and subsequently Nt-arginylated by ATE1 arginyltransferase in HEK293 cells. We also found that the resulting Arg-Glu-starting RE-Ub was mainly deposited on the Lys119 residue of histone H2A. Furthermore, RING1B E3 Ub ligase mediated the attachment of RE-Ub to H2A. These findings reveal a previously unknown type of histone ubiquitylation which greatly increases the combinatorial complexity of histone and ubiquitin codes.

Anti-Tuberculosis Potential of OJT008 against Active and Multi-Drug-Resistant Mycobacterium Tuberculosis: In Silico and In Vitro Inhibition of Methionine Aminopeptidase.

Despite the recent progress in the diagnosis of tuberculosis (TB), the chemotherapeutic management of TB continues to be challenging. Mycobacterium tuberculosis (Mtb), the etiological agent of TB, is classified as the 13th leading cause of death globally. In addition, 450,000 people were reported to develop multi-drug-resistant TB globally. The current project focuses on targeting methionine aminopeptidase (MetAP), an essential protein for the viability of Mtb. MetAP is a metalloprotease that catalyzes the excision of the N-terminal methionine (NME) during protein synthesis, allowing the enzyme to be an auspicious target for the development of novel therapeutic agents for the treatment of TB. Mtb possesses two MetAP1 isoforms, MtMetAP1a and MtMetAP1c, which are vital for Mtb viability and, hence, a promising chemotherapeutic target for Mtb therapy. In this study, we cloned and overexpressed recombinant MtMetAP1c. We investigated the in vitro inhibitory effect of the novel MetAP inhibitor, OJT008, on the cobalt ion- and nickel ion-activated MtMetAP1c, and the mechanism of action was elucidated through an in silico approach. The compound's potency against replicating and multi-drug-resistant (MDR) Mtb strains was also investigated. The induction of the overexpressed recombinant MtMetAP1c was optimized at 8 h with a final concentration of 1 mM Isopropyl ß-D-1-thiogalactopyranoside. The average yield from 1 L of Escherichia coli culture for MtMetAP1c was 4.65 mg. A preliminary MtMetAP1c metal dependency screen showed optimum activation with nickel and cobalt ions occurred at 100 µM. The half-maximal inhibitory concentration (IC50) values of OJT008 against MtMetAP1c activated with CoCl2 and NiCl2 were 11 µM and 40 µM, respectively. The in silico study showed OJT008 strongly binds to both metal-activated MtMetAP1c, as evidenced by strong molecular interactions and a higher binding score, thereby corroborating our result. This in silico study validated the pharmacophore's metal specificity. The potency of OJT008 against both active and MDR Mtb was <0.063 µg/mL. Our study reports OJT008 as an inhibitor of MtMetAP1c, which is potent at low micromolar concentrations against both active susceptible and MDR Mtb. These results suggest OJT008 is a potential lead compound for the development of novel small molecules for the therapeutic management of TB.

N-terminal methionine excision of proteins creates tertiary destabilizing N-degrons of the Arg/N-end rule pathway.

All organisms begin protein synthesis with methionine (Met). The resulting initiator Met of nascent proteins is irreversibly processed by Met aminopeptidases (MetAPs). N-terminal (Nt) Met excision (NME) is an evolutionarily conserved and essential process operating on up to two-thirds of proteins. However, the universal function of NME remains largely unknown. MetAPs have a well-known processing preference for Nt-Met with Ala, Ser, Gly, Thr, Cys, Pro, or Val at position 2, but using CHX-chase assays to assess protein degradation in yeast cells, as well as protein-binding and RT-qPCR assays, we demonstrate here that NME also occurs on nascent proteins bearing Met-Asn or Met-Gln at their N termini. We found that the NME at these termini exposes the tertiary destabilizing Nt residues (Asn or Gln) of the Arg/N-end rule pathway, which degrades proteins according to the composition of their Nt residues. We also identified a yeast DNA repair protein, MQ-Rad16, bearing a Met-Gln N terminus, as well as a human tropomyosin-receptor kinase-fused gene (TFG) protein, MN-TFG, bearing a Met-Asn N terminus as physiological, MetAP-processed Arg/N-end rule substrates. Furthermore, we show that the loss of the components of the Arg/N-end rule pathway substantially suppresses the growth defects of naa20Δ yeast cells lacking the catalytic subunit of NatB Nt acetylase at 37 °C. Collectively, the results of our study reveal that NME is a key upstream step for the creation of the Arg/N-end rule substrates bearing tertiary destabilizing residues in vivo.

Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts.

Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genomes. While various hypotheses dealing with the occurrence of these intriguing sequences have been made, no further characterization and functional studies have been described thus far. In this study, we characterize the bacteriophage Vp16 PDF enzyme, as representative member of the newly identified C-terminally truncated viral PDFs. We show here that conditions classically used for bacterial PDFs lead to an enzyme exhibiting weak activity. Nonetheless, our integrated biophysical and biochemical approaches reveal specific effects of pH and metals on Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 PDF specific activity to values similar to those of bacterial PDFs. We next show that Vp16 PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.

An improved workflow for quantitative N-terminal charge-based fractional diagonal chromatography (ChaFRADIC) to study proteolytic events in Arabidopsis thaliana.

We applied an extended charge-based fractional diagonal chromatography (ChaFRADIC) workflow to analyze the N-terminal proteome of Arabidopsis thaliana seedlings. Using iTRAQ protein labeling and a multi-enzyme digestion approach including trypsin, GluC, and subtilisin, a total of 200 µg per enzyme, and measuring only one third of each ChaFRADIC-enriched fraction by LC-MS, we quantified a total of 2791 unique N-terminal peptides corresponding to 2249 different unique N-termini from 1270 Arabidopsis proteins. Our data indicate the power, reproducibility, and sensitivity of the applied strategy that might be applicable to quantify proteolytic events from as little as 20 µg of protein per condition across up to eight different samples. Furthermore, our data clearly reflect the methionine excision dogma as well as the N-end rule degradation pathway (NERP) discriminating into a stabilizing or destabilizing function of N-terminal amino acid residues. We found bona fide NERP destabilizing residues underrepresented, and the list of neo N-termini from wild type samples may represent a helpful resource during the evaluation of NERP substrate candidates. All MS data have been deposited in the ProteomeXchange with identifier PXD001855 (http://proteomecentral.proteomexchange.org/dataset/PXD001855).

Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart.

Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of the N-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeable in vivo and display similar properties in vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

N-Terminal Modifications of Ubiquitin via Methionine Excision, Deamination, and Arginylation Expand the Ubiquitin Code.

Ubiquitin (Ub) is post-translationally modified by Ub itself or Ub-like proteins, phosphorylation, and acetylation, among others, which elicits a variety of Ub topologies and cellular functions. However, N-terminal (Nt) modifications of Ub remain unknown, except the linear head-to-tail ubiquitylation via Nt-Met. Here, using the yeast Saccharomyces cerevisiae and an Nt-arginylated Ub-specific antibody, we found that the detectable level of Ub undergoes Nt-Met excision, Nt-deamination, and Nt-arginylation. The resulting Nt-arginylated Ub and its conjugated proteins are upregulated in the stationary-growth phase or by oxidative stress. We further proved the existence of Nt-arginylated Ub in vivo and identified Nt-arginylated Ub-protein conjugates using stable isotope labeling by amino acids in cell culture (SILAC)-based tandem mass spectrometry. In silico structural modeling of Nt-arginylated Ub predicted that Nt-Arg flexibly protrudes from the surface of the Ub, thereby most likely providing a docking site for the factors that recognize it. Collectively, these results reveal unprecedented Nt-arginylated Ub and the pathway by which it is produced, which greatly expands the known complexity of the Ub code.

Structural insights into the interplay of protein biogenesis factors with the 70S ribosome.

Bacterial co-translational N-terminal methionine excision, an early event of nascent polypeptide chain processing, is mediated by two enzymes: peptide deformylase (PDF) and methionine aminopeptidase (MetAP). Trigger factor (TF), the only ribosome-associated bacterial chaperone, offers co-translational chaperoning assistance. Here, we present two high-resolution cryoelectron microscopy structures of tRNA-bound E. coli ribosome complexes showing simultaneous binding of PDF and TF, in the absence (3.4 Å) and presence of MetAP (4.1 Å). These structures establish molecular details of the interactions of the factors with the ribosome, and thereby reveal the structural basis of nascent chain processing. Our results suggest that simultaneous binding of all three factors is not a functionally favorable mechanism of nascent chain processing. Strikingly, an unusual structural distortion of the 70S ribosome, potentially driven by binding of multiple copies of MetAP, is observed when MetAP is incubated with a pre-formed PDF-TF-bound ribosome complex.

Characterization of N-terminal protein modifications in Pseudomonas aeruginosa PA14.

Even though protein initiator methionine excision (NME) and N-terminal acetylation (NTA) have been relatively well investigated in eukaryotic proteomes, few studies were dedicated to these modifications in bacteria up to now. In this work, we investigated, for the first time, the N-terminal proteome of the bacterium Pseudomonas aeruginosa PA14 by studying the NME and NTA processes using proteomic approaches. For NME, most of proteins had their initiator Met cleaved (63%) and the nature of the penultimate residue seems to be essential for this cleavage. Concerning NTA, two methods were applied (protein fractionation and peptide enrichment). This allowed us to identify 117 Nα-acetylated proteins, among them 113 have not yet been described as modified in bacteria. Most often, the non-acetylated form was over-represented compared to the acetylated form, arguing that this latter was a minor part of the total abundance of a given protein. Furthermore, some proteins with acetylated initiator methionine were observed. The present work significantly enlarges the number of N-terminally modified proteins in bacteria and confirms that these modifications are a general and fundamental process, not only restricted to eukaryotes. BIOLOGICAL SIGNIFICANCE: Protein modifications in prokaryotes have been detected more recently than in eukaryotes. Methionine cleavage and N-terminal acetylation are two common protein N-terminal modifications. Despite their importance in bacterial processes, they are less investigated. The characterization of N-terminal acetylation in bacteria is a challenge because no antibody exists and it is a less frequent modification than in eukaryotes. We used proteomic approaches (enrichment, fractionation, nanoLC-MS/MS, and bioinformatic analyses) to investigate the N-terminal methionine excision and to profile the N-terminal acetylome of P. aeruginosa strain PA14. From our results, around 60% of the proteins had their iMet cleaved. In total, 117 proteins were identified constituting the largest dataset in prokaryotes. Among them, proteins kept their initiator methionine and were acetylated. These results may facilitate the design of experiments to better understand the role of acetylation at the protein N-terminus of P. aeruginosa PA14.