Peptide deformylase (PDF) belongs to a conserved enzyme family critical for N-terminal methionine excision (NME), an essential protein maturation process in prokaryotes and eukaryotic organelles (chloroplasts, mitochondria). To explore the potential functions of OsPDFs in Oryza sativa, this study employed bioinformatics approaches and experimental validation to systematically identify and analyze the OsPDF gene family. Three OsPDF genes (OsPDF1A, OsPDF1B, OsPDF1B2) were identified in rice. These genes are exclusively distributed on chromosome 1. The biophysical properties of these proteins showed that OsPDF1A and OsPDF1B are alkaline proteins, while OsPDF1B2 is acidic, and all are hydrophilic with moderate thermostability potential. Synteny analysis revealed closer evolutionary relationships between Oryza sativa and the monocot Triticum aestivum than with dicots, reflecting conserved PDF function in gramineous plants. Analysis of cis-acting elements in the 2000 bp upstream region of OsPDF gene promoters revealed numerous elements associated with abiotic stress response and hormone regulation. Furthermore, quantitative real-time PCR (qRT-PCR) data supported these findings, indicating that OsPDF1A and OsPDF1B were upregulated under low-temperature stress, and all three OsPDF genes were transcriptionally activated by heat, salt and UV-B stresses, indicating their active involvement in rice growth, development, and abiotic stress tolerance. In summary, OsPDFs exhibit significant functions in rice's stress adaptation, growth, and development. This study not only enhances our understanding of the OsPDF gene family's genomic, evolutionary, and functional characteristics, but also provides new perspectives and foundational data for further exploring their regulatory mechanisms in protein maturation and abiotic stress responses, as well as their potential applications in rice stress tolerance breeding.

Proteostasis relies on the coordinated control of protein synthesis, folding, modification and degradation, and an increasingly clear picture is emerging that many important decisions governing protein fate occur co-translationally. As nascent chains first appear at the ribosome exit tunnel, they encounter a suite of ribosome-associated enzymes that begin to shape whether proteins fold productively, acquire the correct N-terminal imprinting modifications, or require surveillance and removal. This review focuses on two major facets of co-translational control that determine protein and proteome stability, with particular attention to recent advances in plants. First, N-terminal (Nt-) methionine excision, Nt- acetylation and Nt-myristoylation are examined as early imprinting steps that define the chemical identity and regulatory trajectories of newly synthesized proteins, including how they influence targeting to N-degron pathways of proteolysis. Second, ribosome-associated quality control (RQC) pathways that sense ribosome stalling or collision are outlined, along with their roles in directing aberrant nascent chains towards ubiquitylation, extraction and degradation before they can accumulate and trigger proteotoxic stress. Together, these modification and surveillance mechanisms form an integrated decision-making network that establishes protein stability at the earliest stages of synthesis, contributing to proteostasis and impacting plant growth, development, and stress adaptation.

N-terminal acetylation (NTA) is the most common protein modification in eukaryotes, playing a crucial role in proteostasis. Almost 40% of the human proteome is acetylated co-translationally by the NatA complex, which requires prior N-terminal methionine excision (NME). Recently, NatA was shown to form multi-enzyme complexes with MAP1/NAC or MAP2, combining the capabilities of NME and NTA into a single complex. Here, we show that NatA can also form ribosome-independent assemblies with several ribosome associated factors (RAFs). At the ribosome, NatA can form a ternary complex with the abundant pseudoenzyme Ebp1 or a second copy of NatA, which can be coordinated from a different binding site with closer access to a potential substrate. Further, we identify a conserved binding site on NatA, which can be accessed by four RAFs - Ebp1, NAC, Naa10 and HypK, allowing the formation of different multi-factor complexes at the ribosomal tunnel exit. Therefore, our data suggest that NatA constitutes an interaction hub, and contributes to the coordination of co-translational protein maturation.

Expression and purification of methionine aminopeptidases and N-terminal acetyltransferases.

Nascent chains undergo co-translational enzymatic processing as soon as their N-terminus becomes accessible at the ribosomal polypeptide tunnel exit (PTE). In eukaryotes, N-terminal methionine excision (NME) by Methionine Aminopeptidases (MAP1 and MAP2), and N-terminal acetylation (NTA) by N-Acetyl-Transferase A (NatA), is the most common combination of subsequent modifications carried out on the 80S ribosome. How these enzymatic processes are coordinated in the context of a rapidly translating ribosome has remained elusive. Here, we report two cryo-EM structures of multi-enzyme complexes assembled on vacant human 80S ribosomes, indicating two routes for NME-NTA. Both assemblies form on the 80S independent of nascent chain substrates. Irrespective of the route, NatA occupies a non-intrusive ‘distal’ binding site on the ribosome which does not interfere with MAP1 or MAP2 binding nor with most other ribosome-associated factors (RAFs). NatA can partake in a coordinated, dynamic assembly with MAP1 through the hydra-like chaperoning function of the abundant Nascent Polypeptide-Associated Complex (NAC). In contrast to MAP1, MAP2 completely covers the PTE and is thus incompatible with NAC and MAP1 recruitment. Together, our data provide the structural framework for the coordinated orchestration of NME and NTA in protein biogenesis.

Approximately 40% of the mammalian proteome undergoes N-terminal methionine excision and acetylation, mediated sequentially by methionine aminopeptidase (MetAP) and N-acetyltransferase A (NatA), respectively1. Both modifications are strictly cotranslational and essential in higher eukaryotic organisms1. The interaction, activity and regulation of these enzymes on translating ribosomes are poorly understood. Here we perform biochemical, structural and in vivo studies to demonstrate that the nascent polypeptide-associated complex2,3 (NAC) orchestrates the action of these enzymes. NAC assembles a multienzyme complex with MetAP1 and NatA early during translation and pre-positions the active sites of both enzymes for timely sequential processing of the nascent protein. NAC further releases the inhibitory interactions from the NatA regulatory protein huntingtin yeast two-hybrid protein K4,5 (HYPK) to activate NatA on the ribosome, enforcing cotranslational N-terminal acetylation. Our results provide a mechanistic model for the cotranslational processing of proteins in eukaryotic cells.

PurposeImbalances 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. MethodsHere, 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. ResultsOur investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities. ConclusionBiallelic 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.

Background: Ribosome biogenesis is a recurrent process in which nascent ribosomes are synthesized by pre-existing ribosomes and is essential for the self-replication of life. Hence, the reconstitution of ribosome biogenesis is important for understanding the self-replication of life and creating self-replicating artificial cells. Previously, we have successfully reconstituted E. coli ribosome biogenesis in vitro[1]. In this study, we tried to analyze the characteristics of in vitro ribosome biogenesis. Method: For the reconstitution of ribosome biogenesis in vitro, we expressed a ribosomal RNA (rRNA) operon gene and ribosomal protein (r-protein) genes in a cell-free transcription and translation system based on an optimized S150 E. coli cell extract. To identify the optimal condition, we comprehensively explored the concentration ratio of rRNA operon and r-protein genes. To investigate whether alleviating the imbalance of ribosome small subunits (SSU) and large subunits (LSU) improves in vitro ribosome biogenesis, we added extra LSU or SSU to the in vitro SSU or LSU biogenesis. To analyze the post-transcriptional modifications of nascent rRNA, we purified nascent ribosome using 16S and 23S rRNA with streptavidin-binding aptamer (Sb-aptamer)[2][3] and performed mass spectrometric analysis. To analyze the post-translational modifications of the nascent r-proteins, we labeled nascent r-proteins with stable isotope-labeled l-arginine (13C6, 15N4) and l-lysine (13C6, 15N2) and performed mass spectrometric analysis. Results and Discussion: Comprehensive exploration of the expression patterns of the rRNA operon gene and r-protein genes in an optimized S150 E. coli cell extract resulted in the identification of optimal conditions which enable in vitro ribosome biogenesis. We analyzed the characteristics of the in vitro ribosome biogenesis. First, we confirmed that alleviating the imbalance between SSU and LSU by adding extra LSU or SSU did not improve the efficiency of in vitro ribosome biogenesis. Second, we detected some of the post-transcriptional modifications that native ribosomes have in nascent rRNA. Specifically, we detected post-transcriptional modifications including m62A, m1G, Ψ, and m5C in the nascent 16S and 23S rRNA. We also detected some of the post-translational modifications that native ribosomes have in nascent r-proteins including N-terminal methionine excision in the uS8, uS9, and uS12 and methylation of glutamine in the uL3. This research will facilitate the understanding of the self-replication of life and the bottom-up creation of self-replicating artificial cells.

The N-termini of proteins can regulate their degradation, and the same protein with different N-termini may have distinct dynamics. Recently, it was found that N-terminal glycine can serve as a degron recognized by two E3 ligases, but N-terminal glycine was also reported to stabilize proteins. Here we developed a chemoenzymatic method for selective enrichment of proteoforms with N-terminal glycine and integrated dual protease cleavage to further improve the enrichment specificity. Over 2000 unique peptides with protein N-terminal glycine were analyzed from >1000 proteins, and most of them are previously unknown, indicating the effectiveness of the current method to capture low-abundance proteoforms with N-terminal glycine. The degradation rates of proteoforms with N-terminal glycine were quantified along with those of proteins from the whole proteome. Bioinformatic analyses reveal that proteoforms with N-terminal glycine with the fastest and slowest degradation rates have different functions and localizations. Membrane proteins with N-terminal glycine and proteins with N-terminal glycine from the N-terminal methionine excision degrade more rapidly. Furthermore, the secondary structures, adjacent amino acid residues, and protease specificities for N-terminal glycine are also vital for protein degradation. The results advance our understanding of the effects of N-terminal glycine on protein properties and functions.

Tuberculosis (TB) is a chronic systemic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). Methionine aminopeptidase 1 (MtMET-AP1) is a hydrolase that mediates the necessary post-translational N-terminal methionine excision (NME) of peptides during protein synthesis, which is necessary for bacterial proliferation and is a potential target for the treatment of tuberculosis. Based on the functional characteristics of MtMET-AP1, we developed an enzymatic activated near-infrared fluorescent probe DDAN-MT for rapid, highly selective, and real-time monitoring of endogenous MtMET-AP1 activity in M. tuberculosis. Using the probe DDAN-MT, a visually high-throughput screening technique was established, which obtained three potential inhibitors (GSK-J4 hydrochchloride, JX06, and lavendustin C) against MtMET-AP1 from a 2560 compounds library. More importantly, these inhibitors could inhibit the growth of M. tuberculosis H37Ra especially (MICs < 5 μM), with low toxicities on intestinal bacteria strains and human cells. Therefore, the visual sensing of MtMET-AP1 was successfully performed by DDAN-MT, and MtMET-AP1 inhibitors were discovered as potential antituberculosis agents.

N-terminal methionine excision from newly synthesized proteins, catalyzed cotranslationally by methionine aminopeptidases (METAPs), is an essential and universally conserved process that plays a key role in cell homeostasis and protein biogenesis. However, how METAPs interact with ribosomes and how their cleavage specificity is ensured is unknown. We discovered that in eukaryotes the nascent polypeptide-associated complex (NAC) controls ribosome binding of METAP1. NAC recruits METAP1 using a long, flexible tail and provides a platform for the formation of an active methionine excision complex at the ribosomal tunnel exit. This mode of interaction ensures the efficient excision of methionine from cytosolic proteins, whereas proteins targeted to the endoplasmic reticulum are spared. Our results suggest a broader mechanism for how access of protein biogenesis factors to translating ribosomes is controlled.

<b>Abstract ID 26564</b> <b>Poster Board 94</b> Despite recent progress in the diagnosis of Tuberculosis (TB), the chemotherapeutic management of TB is still challenging. <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) is the etiological agent of TB, and TB is classified as the 13th leading cause of death globally. It is estimated that 558,000 people were reported to develop multi-drug resistant TB globally. Our research focuses on targeting Methionine Aminopeptidase (MetAP), an essential protein for the viability of <i>Mtb</i>. MetAP is a metalloprotease that catalyzes the N-terminal methionine excision (NME) during protein synthesis. This essential role of MetAPs allows this enzyme an auspicious target for the development of novel therapeutic agents for the treatment of TB. <i>Mtb</i> possesses 2 MetAP1 isoforms: MtMetAP1a and MtMetAP1c. In our study, we cloned, overexpressed recombinant MtMetAP1c, and investigated the <i>in&nbsp;vitro</i> inhibitory effect of OJT008 on cobalt and nickel ion activated MtMetAP1c and the mechanism of action was elucidated through an <i>in-silico</i> approach. The compound’s potency against replicating and multidrug-resistant (MDR) <i>Mtb</i> strains was also investigated. The induction of the overexpressed recombinant MtMetAP1c was optimized at 8 hours with a final concentration of 1mM Isopropyl β-D-1-thiogalactopyranoside. The average yield for MtMetAP1c was 4.65mg mg/L of <i>Escherichia coli</i> culture. A preliminary MtMetAP1c metal dependency screen Providedoptimum activation with nickel and cobalt ions at 100μM. The half maximal inhibitory concentration (IC₅₀) values of OJT008 against MtMetAP1c activated with CoCl₂ and NiCl₂ were 11μM and 40 μM respectively. The <i>in-silico</i> study showed OJT008 strongly binds to both metals activated MtMetAP1c, as evidenced by strong molecular interactions and higher binding score thereby corroborating our result. This <i>in-silico</i> study validated the pharmacophore’s metal specificity. The potency of OJT008 against drug sensitive and MDR <i>Mtb</i> was &lt; 0.063 μg/mL. Our study reports OJT008 as an inhibitor of MtMetAP1c which is potent at low micromolar concentrations against both drug-sensitive and MDR-<i>Mtb</i>. These results suggest OJT008 is a potential lead compound for the development of novel small molecules for the therapeutic management of TB. This project is sponsored by NIH-RCMI (U54MD007605) Grants.

Ribosome-nascent Chain Interaction Regulates N-terminal Protein Modification

The advancement of mass spectrometry provides advantages for transgenic protein characterization in support of safety assessments of genetically modified crops. Here, we describe how matrix-assisted laser desorption ionization in-source decay (ISD) mass spectrometry (MS) in combination with intact mass and bottom-up analyses can be applied to achieve high confidence in the sequences of transgenic proteins expressed in plants and establish the biochemical equivalence of microbially produced protein surrogates. ISD confirmed 40-60 near terminal residues regardless of the protein size, including the improvement of the coverage of cysteine-rich proteins by the reduction/alkylation of disulfide bonds. Negative ISD significantly improved spectral quality and sequence coverage of acidic proteins. Various post-translational modifications, such as terminal truncations and N-terminal methionine excision and acetylation, were identified in plant-produced proteins by top-down MS. Finally, we demonstrated that a combination of top-down and bottom-up analyses provides high confidence in sequence equivalence of plant and microbially produced proteins.

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

Selection of newly‐synthesized proteins into correct protein biogenesis pathways is crucial for cellular homeostasis. The ubiquitous N‐terminal methionine excision (NME) process is mediated by peptide deformylase (PDF) and methionine aminopeptidase (MAP), two essential enzymes in bacteria. This reaction takes place near the nascent peptide exit site of the ribosome, where multiple ribosome‐associating protein biogenesis factors (RPBs) also compete for the access to the nascent chain. How NME achieves its efficiency and specificity at this crowded environment is unknown. Here, using kinetic measurements on purified ribosome‐nascent chain complexes, we show that the ribosome accelerates the MAP reaction for optimal substrates by 102–104 folds. Kinetic competition with translation elongation and selective regulation from other RPBs enhance the specificity of NME by narrowing the processing time window for reactions on suboptimal substrates. With the kinetic data, we constructed a mathematical model and accurately predicted the cotranslational NME efficiency in cytosol. Our data demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity. Moreover, the remodeled MAP activity prompted us to develop a cotranslational assay to screen for MAP inhibitors in the physiological context of translating ribosome. The results explain the discrepancy between traditional peptide‐based assays and cellular data, providing a powerful tool for the development of antibacterial agents targeting the NME machineries.Support or Funding InformationThis work was supported by National Institutes of Health grant GM078024 and a grant from the Weston Havens Foundation to S.‐o. Shan, and a Think Global Education Trust Fellowship from Taiwan to C.‐I. Yang.

The nascent polypeptide exit site of the ribosome is a crowded environment where multiple ribosome-associated protein biogenesis factors (RPBs) compete for the nascent polypeptide to influence their localization, folding, or quality control. Here we address how N-terminal methionine excision (NME), a ubiquitous process crucial for the maturation of over 50% of the bacterial proteome, occurs in a timely and selective manner in this crowded environment. In bacteria, NME is mediated by 2 essential enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). We show that the reaction of MAP on ribosome-bound nascent chains approaches diffusion-limited rates, allowing immediate methionine excision of optimal substrates after deformylation. Specificity is achieved by kinetic competition of NME with translation elongation and by regulation from other RPBs, which selectively narrow the processing time window for suboptimal substrates. A mathematical model derived from the data accurately predicts cotranslational NME efficiency in the cytosol. Our results demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity, and provides a platform to study other cotranslational protein biogenesis pathways.

Formylated N-terminal methionine is absent from the Mycoplasma hyopneumoniae proteome: Implications for translation initiation

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.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable platform for top-down proteomics. However, the scale of top-down proteomics using CZE-MS/MS is still limited due to the low loading capacity and narrow separation window of CZE. In this work, for the first time we systematically evaluated the dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction-based CZE-MS/MS approached a 1 μL loading capacity, 90 min separation window, and high peak capacity (∼280) for characterization of an Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for top-down characterization of complex proteomes. Single-shot CZE-MS/MS identified about 2800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins from the Escherichia coli proteome with spectrum-level false discovery rate (FDR) less than 1%. The number of identified proteoforms in this work is over three times higher than that in previous single-shot CZE-MS/MS studies. Truncations, N-terminal methionine excision, signal peptide removal, and some post-translational modifications including oxidation and acetylation were detected.