NGOME-Lite: Proteome-wide prediction of spontaneous protein deamidation highlights differences between taxa.

Asparagines in proteins deamidate spontaneously, which changes the chemical structure of a protein and often affects its function. Current prediction algorithms for asparagine deamidation require a structure as an input or are too slow to be applied at a proteomic scale. We present NGOME-Lite, a new version of our sequence-based predictor for spontaneous asparagine deamidation that is faster by over two orders of magnitude at a similar degree of accuracy. The algorithm takes into account intrinsic sequence propensities and slowing down of deamidation by local structure. NGOME-Lite can run in a proteomic analysis mode that provides the half-time of the intact form of each protein, predicted by taking into account sequence propensities and structural protection or sequence propensities only, and a structure protection factor. The detailed analysis mode also provides graphical output for all Asn residues in the query sequence. We applied NGOME-Lite to over 257,000 sequences in 38 proteomes and found that different taxa differ in their predicted deamidation dynamics. Spontaneous protein deamidation is faster in Eukarya than in Bacteria because of a higher degree of structural protection in the latter. Predicted protein deamidation half-lifes correlate with protein turnover in human, mouse, rat, C. elegans and budding yeast but not in two plants and two bacteria. NGOME-Lite is implemented in a docker container available at https://ngome.proteinphysiologylab.org.

Enhanced protein glutaminase production from Chryseobacterium proteolyticum combining physico-chemical mutagenesis and resistance screening and its application to soybean protein isolates.

BACKGROUND: Protein glutaminase (PG) is a novel protein modification biotechnology that is increasingly being used in the food industry. However, the current level of fermentation of PG-producing strains still does not meet the requirements of industrial production. To obtain the mutant strains with high PG production, the atmospheric and room temperature plasma (ARTP) combined with LiCl chemical mutagen were used in mutagenesis of a PG producing Chryseobacterium proteolyticum 1003. RESULTS: A mutant strain (WG15) was successfully obtained based on malonic acid resistance screening after compound mutagenesis of the starting strain C. proteolyticum 1003 using ARTP with LiCl, and it was confirmed to be genetically stable in PG synthesis after 15 generations. The protein glutaminase production of WG15 was 2.91 U mL-1 after optimization of fermentation conditions, which is 48.69% higher than the original strain C. proteolyticum 1003. The PG obtained from fermentation showed good activities in deamidation of soy protein isolate. The solubility and foaming properties of the PG-treated soy protein isolate were significantly increased by 36.50% and 10.03%, respectively, when PG was added at the amount of 100 U mL-1 . In addition, the emulsifying activity and emulsion stability of the treated soy protein isolate were improved by 12.44% and 10.34%, respectively, on the addition of 10 U mL-1 PG. The secondary structure of the soy protein isolate changed after PG treatment, with an increased proportion of glutamate. CONCLUSION: The results of the present study indicate that the PG produced by this mutant strain could improve the functional properties of soybean protein isolate and the C. proteolyticum mutant WG15 has great potential in food industry. © 2023 Society of Chemical Industry.

Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif.

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.

Insertion-trigger residues differentially modulate endosomal escape by cytotoxic necrotizing factor toxins.

The cellular specificity, potency, and modular nature of bacterial protein toxins enable their application for targeted cytosolic delivery of therapeutic cargo. Efficient endosomal escape is a critical step in the design of bacterial toxin-inspired drug delivery (BTIDD) vehicles to avoid lysosomal degradation and promote optimal cargo delivery. The cytotoxic necrotizing factor (CNF) family of modular toxins represents a useful model for investigating cargo-delivery mechanisms due to the availability of many homologs with high sequence identity, their flexibility in swapping domains, and their differential activity profiles. Previously, we found that CNFy is more sensitive to endosomal acidification inhibitors than CNF1 and CNF2. Here, we report that CNF3 is even less sensitive than CNF1/2. We identified two amino acid residues within the putative translocation domain (E374 and E412 in CNFy, Q373 and S411 in CNF3) that differentiate between these two toxins. Swapping these corresponding residues in each toxin changed the sensitivity to endosomal acidification and efficiency of cargo-delivery to be more similar to the other toxin. Results suggested that trafficking to the more acidic late endosome is required for cargo delivery by CNFy but not CNF3. This model was supported by results from toxin treatment of cells in the presence of NH4Cl, which blocks endosomal acidification, and of small-molecule inhibitors EGA, which blocks trafficking to late endosomes, and ABMA, which blocks endosomal escape and trafficking to the lysosomal degradative pathway. These findings suggest that it is possible to fine-tune endosomal escape and cytosolic cargo delivery efficiency in designing BTIDD platforms.

Arabidopsis protein l-ISOASPARTYL METHYLTRANSFERASE repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance.

Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging by protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress-tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.

Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells.

Modular AB-type bacterial protein toxins target mammalian host cells with high specificity and deliver their toxic cargo into the cytosol. Hence, these toxins are being explored as agents for targeted cytosolic delivery in biomedical and research applications. The cytotoxic necrotizing factor (CNF) family is unique among these toxins in that their homologous sequences are found in a wide array of bacteria, and their activity domains are packaged in various delivery systems. Here, to study how CNF cargo and delivery modules can be assembled for efficient cytosolic delivery, we generated chimeric toxins by swapping functional domains among CNF1, CNF2, CNF3, and CNFy. Chimeras with a CNFy delivery vehicle were more stably expressed, but were less efficient at cargo delivery into HEK293-T cells. We also found that CNFy cargo is the most universally compatible and that CNF3 delivery vehicle is the most flexible and efficient at delivering cargo. These findings suggest that domains within proteins can be swapped and accommodate each other for efficient function and that an individual domain could be engineered for compatibility with multiple partner domains. We anticipate that our insights could help inform chemical biology approaches to develop toxin-based cargo-delivery platforms for cytosolic cargo delivery of therapeutics or molecular probes into mammalian cells.

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis.

Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed.

Extensive deamidation of RNase A inhibits its oligomerization through 3D domain swapping.

Bovine pancreatic ribonuclease A (RNase A) is the monomeric prototype of the so-called secretory 'pancreatic-type' RNase super-family. Like the naturally domain-swapped dimeric bovine seminal variant, BS-RNase, and its glycosylated RNase B isoform, RNase A forms N- and C-terminal 3D domain-swapped oligomers after lyophilization from acid solutions, or if subjected to thermal denaturation at high protein concentration. All mentioned RNases can undergo deamidation at Asn67, forming Asp or isoAsp derivatives that modify the protein net charge and consequently its enzymatic activity. In addition, deamidation slightly affects RNase B self-association through the 3D domain swapping (3D-DS) mechanism. We report here the influence of extensive deamidation on RNase A tendency to oligomerize through 3D-DS. In particular, deamidation of Asn67 alone slightly decreases the propensity of the protein to oligomerize, with the Asp derivative being less affected than the isoAsp one. Contrarily, the additional Asp and/or isoAsp conversion of residues other than N67 almost nullifies RNase A oligomerization capability. In addition, Gln deamidation, although less kinetically favorable, may affect RNase A self-association. Using 2D and 3D NMR we identified the Asn/Gln residues most prone to undergo deamidation. Together with CD spectroscopy, NMR also indicates that poly-deamidated RNase A generally maintains its native tertiary structure. Again, we investigated in silico the effect of the residues undergoing deamidation on RNase A dimers structures. Finally, the effect of deamidation on RNase A oligomerization is discussed in comparison with studies on deamidation-prone proteins involved in amyloid formation.

Transglutaminase 2-dependent deamidation of glyceraldehyde-3-phosphate dehydrogenase promotes trophoblastic cell fusion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein as well as a classic glycolytic enzyme, and its pleiotropic functions are achieved by various post-translational modifications and the resulting translocations to intracellular compartments. In the present study, GAPDH in the plasma membrane of BeWo choriocarcinoma cells displayed a striking acidic shift in two-dimensional electrophoresis after cell-cell fusion induction by forskolin. This post-translational modification was deamidation of multiple glutaminyl residues, as determined by molecular mass measurement and tandem mass spectrometry of acidic GAPDH isoforms. Transglutaminase (TG) inhibitors prevented this acidic shift and reduced cell fusion. Knockdown of the TG2 gene by short hairpin RNA reproduced these effects of TG inhibitors. Various GAPDH mutants with replacement of different numbers (one to seven) of Gln by Glu were expressed in BeWo cells. These deamidated mutants reversed the suppressive effect of wild-type GAPDH overexpression on cell fusion. Interestingly, the mutants accumulated in the plasma membrane, and this accumulation was increased according to the number of Gln/Glu substitutions. Considering that GAPDH binds F-actin via an electrostatic interaction and that the cytoskeleton is rearranged in trophoblastic cell fusion, TG2-dependent GAPDH deamidation was suggested to participate in actin cytoskeletal remodeling.

Oxidation-induced structural changes of ceruloplasmin foster NGR motif deamidation that promotes integrin binding and signaling.

Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface ((568)NGR) and the other buried in the tertiary structure ((962)NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. We observed that the exposed (568)NGR site can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden (962)NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidation-induced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion.