Minor structural changes, major functional impacts: posttranslational modifications and drug targets.

Background:Clostridioides difficile (CD) is an enteric multi-drug resistant pathogenic bacterium. CD-associated infections are the leading cause of nosocomial diarrhea that can further lead to pseudomembranous colitis, toxic mega-colon or sepsis with greater mortality and morbidity risks. CD infection possesses higher rates of recurrence due to its greater resistance to antibiotics. Considering its higher rates of recurrence, it has become a major burden on healthcare facilities. Therefore, there is a dire need to identify novel drug targets to combat antibiotic resistance of Clostridioides difficile.Objective:To identify and propose new and novel drug targets against the Clostridioides difficile.Methods:In the current study, a computational subtractive genomics approach was applied to obtain a set of potential drug targets that exist in the multi-drug resistant strain of Clostridioides difficile. Here, the uncharacterized proteins were studied as potential drug targets. The methodology involved several bioinformatics databases and tools. The druggable proteins sequences were retrieved based on non-homology with host proteome and essentiality for the survival of the pathogen. The uncharacterized proteins were functionally characterized using different computational tools, and sub-cellular localization was also predicted. The metabolic pathways were analyzed using the KEGG database. Eventually, the druggable proteome has been fetched using sequence similarity with the already available drug targets present in the DrugBank database. These druggable proteins were further explored for the structural details to identify drug candidates.Results :A priority list of potential drug targets was provided with the help of the applied method on the complete proteome set of the C. difficile. Moreover, the drug-like compounds have been screened against the potential drug targets to prioritize potential drug candidates. To facilitate the need for drug targets and therapies, the study proposed five potential protein drug targets, out of which three proposed drug targets were subjected to homology modeling to explore their structural and functional activitiesConclusion:In conclusion, we proposed three unique, unexplored drug targets against C. difficile. The structure-based methods were applied and resulted in a list of top-scoring compounds as potential inhibitors to proposed drug targets.

Alzheimer's disease (AD) is a neurodegenerative disease, which is mainly characterized by the abnormal deposition of β‐amyloid peptide (Aβ) and Tau. Since Tau aggregation is more closely associated with synaptic loss, neurodegeneration, and cognitive decline than Aβ, the correlation between Tau and cognitive function in AD has gradually gained attention. The posttranslational modifications (PTMs) of Tau are key factors contributing to its pathological changes, which include phosphorylation, acetylation, ubiquitination, glycosylation, glycation, small ubiquitin‐like modifier mediated modification (SUMOylation), methylation, succinylation, etc. These modifications change the structure of Tau, regulating Tau microtubule interactions, localization, degradation, and aggregation, thereby affecting its propensity to aggregate and leading to neuronal injury and cognitive impairments. Among numerous PTMs, drug development based on phosphorylation, acetylation, ubiquitination, and SUMOylation primarily involves enzymatic reactions, affecting either the phosphorylation or degradation processes of Tau. Meanwhile, methylation, glycosylation, and succinylation are associated with maintaining the structural stability of Tau. Current research is more extensive on phosphorylation, acetylation, ubiquitination, and methylation, with related drugs already developed, particularly focusing on phosphorylation and ubiquitination. In contrast, there is less research on SUMOylation, glycosylation, and succinylation, requiring further basic research, with the potential to become novel drug targets. In conclusion, this review summarized the latest research on PTMs of Tau and related drugs, highlighting the potential of targeting specific PTMs for developing novel therapeutic strategies in AD.

The effects of high-pressure/temperature treatment and pulsed electric field treatment on native peanut Ara h 2, 6 and apple Mal d 3 and Mal d 1b prepared by heterologous expression were examined. Changes in secondary structure and aggregation state of the treated proteins were characterized by circular dichroism spectroscopy and gel-filtration chromatography. Pulsed electric field treatment did not induce any significant changes in the structure of any of the allergens. High-pressure/temperature at 20 °C did not change the structure of the Ara h 2, 6 or Mal d 3 and resulted in only minor changes in structure of Mal d 1b. Ara h 2, 6 was stable to HPP at 80 °C, whereas changes in circular dichroism spectra were observed for both apple allergens. However, these changes were attributable to aggregation and adiabatic heating during HPP. An ELISA assay of temperature treated Mal d 3 showed the antibody reactivity correlated well with the loss of structure. In conclusion, novel-processing techniques had little effect on purified allergen structure. Further studies will demonstrate if these stability properties are retained in foodmatrices.

Protein degradation has become an area of focus for modern research due to the technological advancement of targeted protein degradation as a highly specific and selective therapeutic approach [1]. However, as can be said of most scientific discoveries, the cell itself has long utilized and fine-tuned this regulatory process to create intricate layers of regulation surrounding protein degradation to attune the proteome to intracellular and extracellular cues. Thus far, the answer to this question has indicated that post-translational modifications (PTMs) on target proteins are responsible for tuning protein degradation to the cell's needs [2]. As our understanding of protein degradation delves further into the mechanistic and biological implications of the relationship between the cellular environment, PTMs, and protein degradation, further questions are uncovered regarding why PTMs have evolved to play these central roles in protein degradation. This hypothesis by Hwang et al., [3] takes a refreshing perspective on this next step: Can the ubiquitination machinery itself be directly subject to environmental cues to add an additional regulatory layer to protein degradation? Protein degradation largely occurs through the ubiquitin-proteasome system (UPS). The UPS utilizes a three-step enzymatic cascade to processively attach ubiquitin (Ub) onto surface lysine residues of proteins targeted for degradation. The ubiquitination process is highly coordinated, thereby allowing for regulation at almost every step before, during, and after the ubiquitination cascade. The most common regulatory step occurs at ubiquitin attachment, wherein E3 ligase complexes function to coordinate transfer of the ubiquitin from an E2 ubiquitin-conjugating enzyme onto the target protein. These complexes are often made up of multiple adaptor and scaffold proteins, again indicating the level of regulation required for ubiquitination. Cullin-RING ligases (CRLs), the majority of known E3 ligases, consist of a Cullin scaffold protein and a RING adaptor protein which functions to bind the E2-Ub complex. Families of CRLs are characterized based on the other proteins that make up the complex. Skp1-cullin1-Fbox (SCF) are a subset of CRLs that utilize adaptor protein Skp1 to bind an F-box protein (FBP) bound to the target protein [4]. Intuitively, the recruitment of the target protein to the SCF complex via FBPs is crucial for protein ubiquitination via SCF complexes. This article posits the hypothesis that FBPs function as environmental sensors to control stress response mechanisms, particularly in response to heavy metals [3]. FBPs are well-evidenced to respond to various PTMs (including but not limited to phosphorylation, methylation, and acetylation), which are arguably all ways in which the cell can transmit environmental signals to protein degradation. The authors emphasis of hydroxylation [5] within this context is, therefore, an expansion on well-known regulatory processes dictating how PTMs allow FBP-target protein interactions to remain sensitive to environmental perturbations. The author's expansion into heavy metal sensing, however, is an exciting new hypothesis that is so novel that it is potentially under-evidenced. Canonically, the FBP is made up of two domains: (1) an F-box domain highly conserved for the interaction to adaptor protein Skp1 and (2) a protein interaction domain that binds the protein targeted for degradation. The authors hypothesize that the F-box domain may also sense heavy metals with functional implications to protein degradation, in addition to being an anchor to Skp1 and the rest of the SCF complex. The foundation for this hypothesis is found in the examples of yeast FBPs outlined within the article [6, 7], leading to the suggestion that human FBPs may also contain these functions. However, initial phenotypic verification of this hypothesis is not currently available. Correlative studies demonstrating statistically significant links between SCF complex activity and/or stability in the presence of various metals via cell-wide techniques (i.e., proteomics) could address this issue. In summary, the authors present an intriguing next step for the field of protein homeostasis: the integration of findings from other species to delve into domains and mechanisms long believed to contain just one simple function. This hypothesis builds on a strong foundation of FBPs as conditionally sensitive via PTM-mediated or -hindered protein-protein interactions with proteins targeted for degradation and looks ahead to new possibilities for the regulation of protein degradation. This article comments on the hypothesis paper by Jiwon Hwang, Linda Lauinger, and Peter Kaiser https://doi.org/10.1002/bies.202400249. The authors declare no conflicts of interest.

Identification of novel drug targets is a critical step in drug development. Many recent studies have produced multiple types of data, which provides an opportunity to mine the relationships among them to predict drug targets. In this study, we present a novel integrative approach that combines ontology reasoning with network-assisted gene ranking to predict new drug targets. We utilized colorectal cancer (CRC) as a proof-of-concept use case to illustrate the approach. Starting from FDA-approved CRC drugs and the relationships among disease, drug, gene, pathway, and SNP in an ontology representing PharmGKB data, we inferred 113 potential CRC drug targets. We further prioritized these genes based on their relationships with CRC disease genes in the context of human protein–protein interaction networks. Thus, among the 113 potential drug targets, 15 were selected as the promising drug targets, including some genes that are supported by previous studies. Among them, EGFR, TOP1 and VEGFA are known targets of FDA-approved drugs. Additionally, CCND1 (cyclin D1), and PTGS2 (prostaglandin-endoperoxide synthase 2) have reported to be relevant to CRC or as potential drug targets based on the literature search. These results indicate that our approach is promising for drug target prediction for CRC treatment, which might be useful for other cancer therapeutics.

BackgroundOne of the major issues in the fight against infectious diseases is the notable increase in multiple drug resistance in pathogenic species. For that reason, newly acquired high-throughput data on virulent microbial agents attract the attention of many researchers seeking potential new drug targets. Many approaches have been used to evaluate proteins from infectious pathogens, including, but not limited to, similarity analysis, reverse docking, statistical 3D structure analysis, machine learning, topological properties of interaction networks or a combination of the aforementioned methods. From a biological perspective, most essential proteins (knockout lethal for bacteria) or highly conserved proteins (broad spectrum activity) are potential drug targets. Ribosomal proteins comprise such an example. Many of them are well-known drug targets in bacteria. It is intuitive that we should learn from nature how to design good drugs. Firstly, known antibiotics are mainly originating from natural products of microorganisms targeting other microorganisms. Secondly, paleontological data suggests that antibiotics have been used by microorganisms for million years. Thus, we have hypothesized that good drug targets are evolutionary constrained and are subject of evolutionary selection. This means that mutations in such proteins are deleterious and removed by selection, which makes them less susceptible to random development of resistance. Analysis of the speed of evolution seems to be good approach to test this hypothesis.ResultsIn this study we show that pN/pS ratio of genes coding for known drug targets is significantly lower than the genome average and also lower than that for essential genes identified by experimental methods. Similar results are observed in the case of dN/dS analysis. Both analyzes suggest that drug targets tend to evolve slowly and that the rate of evolution is a better predictor of drugability than essentiality.ConclusionsEvolutionary rate can be used to score and find potential drug targets. The results presented here may become a useful addition to a repertoire of drug target prediction methods. As a proof of concept, we analyzed GO enrichment among the slowest evolving genes. These may become the starting point in the search for antibiotics with a novel mechanism.

Post-translational protein modifications are covalent modifications of amino acid side chains that give rise to a high number of proteins deriving from a relatively small number of genes. The biological functions of post-translational modifications in cells are as diverse as their nature and play a significant and widespread role in the regulation of parasitic proteins. Parasites belonging to the phylum Apicomplexa are the causative agents of fatal diseases including malaria, toxoplasmosis, and cryptosporidiosis. In apicomplexan parasites, post-translational modifications are crucial for various processes, such as invasion into and egress from host cells (phosphorylation, prenylation, and palmitoylation), intracellular signaling (S-nitrosylation), trafficking (ubiquitination, prenylation, palmitoylation), hemoglobin degradation (proteolytic processing), regulation of gene expression (methylation and acetylation), cell cycle regulation (ubiquitination), and many more. Due to their essential functions, several post-translational modifications attract considerable attention with regard to antiparasitic drug development. A range of enzymes that mediate certain post-translational modifications are studied as highly interesting antiparasitic drug targets, including prenyltransferases, proteases, methyltransferases, histone acetytransferases and histone deacetylases, and deubiquitinating enzymes. In this article, we aim to summarize the current knowledge on post-translational modifications, their regulation, and their functions in apicomplexan parasites.

Unlocking the potential of chemical probes for methyl-lysine reader proteins.

Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.

Enterobacter cloacae is a clinically significant Gram-negative, facultatively-anaerobic, rod-shaped bacterium belonging to the family of Enterobacteriaceae. It has emerged as a prevalent nosocomial pathogen due to high level resistance to disinfec- tants and antimicrobial agents. The availability of complete genome sequence of E. cloacae has paved the new way to identify the novel drug targets. In the present work comparative analysis of the metabolic pathways of the pathogen and host was performed to identify the novel drug targets involved in pathogen but absent in Homo sapiens. All enzymes involved in the metabolic pathways of E. cloacae were searched against the proteome of H. sapiens using the BLASTp program. The threshold of percentage identity was set to as 0.001, and the query coverage < 50. Using these parameters approximately 44 unique putative targets were identified. Out of those non-homologous targets, 22 coding genes for putative targets were identified as essential genes from the DEG database. Based on extensive literature search, 8 targets such as UDP-N-acetylmuramoyl-L-alanyl-D-gluta- mate synthetase, UDP-N-acetylmuramate-L-alanine ligase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2, 6-diaminopimelate ligase, UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase, UDP-N-acetylmuramate-L-alanine ligase (MurC) α-isopro- pylmalate synthase, UDP-N-Acetylglucosamine Enolpyruvyl Transferase, UDP-N-acetylenolpyruvylglucosamine reductase and Aspartate beta-semialdehyde dehydrogenase were identified as potential drug targets. Among these drug targets, UDP-N-acetylmu - ramate-L-alanine ligase (MurC) was chosen as potential therapeutic drug target. The homology modeling of MurC was performed using SWISS-MODEL and HHPred servers respectively. Subsequently, all the predicted models were evaluated using the SAVES server. The stereochemical parameters of all 3D models suggest that the best model was predicted by HHPred server. In order the refine the modeled structure, energy minimization was also performed using Deep View tool. The refined 3D structure was further validated by ProSA server. In future, the 3D structure of MurC in E. cloacae might be exploited for the discovery of novel inhibitors that could potentially inhibit this nosocomial pathogen.

Neisseria gonorrhoeae is responsible for causing gonorrhea, one of the most common sexually transmitted diseases prevailing globally. Although extensive researches are in progress in order to control the transmission of the disease and to develop drug(s) against the pathogen, till date no effective vaccine or specific drug could be developed and only antibiotic treatment is in use. Perhaps, due to excess use of antibiotics, several resistant strains have been found. In the present study, metabolic pathways-related candidate drug and vaccine targets have been identified in N. gonorrhoeae virulent strain FA 1090 using an in silico subtractive genomics approach. 106 putative drug targets out of 537 essential genes have been predicted. 67 cytoplasmic and 9 membrane enzymes, along with 10 membrane transporters are found to be the potential drug targets from the host-pathogen common metabolic pathways. Among these targets, competence lipoproteins (NGO0277) and cysW have been identified as candidate vaccine targets. 20 drug targets have been identified from pathogen specific unique metabolic pathways. Out of these, 6 enzymes are involved in dual metabolic pathways and 2 are expressed in cell wall and fimbrium. These gonococci-specific proteins are expected to be better possible drug targets. Screening of the functional inhibitors against these novel targets may result in discovery of novel therapeutic compounds that can be effective against antibiotic resistant strains.

Alternative UPS drug targets upstream the 26S proteasome

MON-213 Impact of post-translational modifications in chronic kidney disease and cardiovascular disease

Post-translational modifications (PTMs) represent an important regulatory instrument that modulates structure, dynamics and function of proteins. The large number of PTM sites in the Hsp90 proteins that are scattered throughout different domains indicated that synchronization of multiple PTMs through a combinatorial code can be invoked as an important mechanism to orchestrate diverse chaperone functions and recognize multiple client proteins. In this study, we have combined structural and coevolutionary analysis with molecular simulations and perturbation response scanning analysis of the Hsp90 structures to characterize functional role of PTM sites in allosteric regulation. The results reveal a small group of conserved PTMs that act as global mediators of collective dynamics and allosteric communications in the Hsp90 structures, while the majority of flexible PTM sites serve as sensors and carriers of the allosteric structural changes. This study provides a comprehensive structural, dynamic and network analysis of PTM sites across Hsp90 proteins, identifying specific role of regulatory PTM hotspots in the allosteric mechanism of the Hsp90 cycle. We argue that plasticity of a combinatorial PTM code in the Hsp90 may be enacted through allosteric coupling between effector and sensor PTM residues, which would allow for timely response to structural requirements of multiple modified enzymes.

The thoracic wall, pleurae, and diaphragm enclose the lung and are interconnected. A thoracic computed tomography (CT) examination usually encompasses all these thoracic structures and a standard protocol may be routinely adopted. CT scanning is excellent at detecting small amounts of both air and fluid and can often be used to identify the underlying intrathoracic causes. Thorough assessment of the pleura for minor changes, such as pleural tags, small nodules, or diffuse thickening, however, may require high-resolution computed tomography evaluation of the thorax. High-quality MDCT data can detect even slight pleural changes and minor structural changes in the diaphragm.