Leptospirosis, a widespread zoonotic disease caused by Leptospira interrogans, poses a major public health challenge, particularly in tropical regions. Despite progress in genomic studies, many putative virulence factors (VFs) remain uncharacterized. This study focuses on LIC10280, a conserved hypothetical protein identified as a potential VF through yeast-based screening. In silico analysis predicted that LIC10280 possesses a cleavable signal peptide and a β-barrel fold in its mature region, features common to secreted bacterial proteins. BlastP analysis showed that LIC10280 is conserved in pathogenic Leptospira but absent in saprophytic species, suggesting a role in pathogenesis. Growth assay in budding yeast, Saccharomyces cerevisiae revealed that only the full-length LIC10280 inhibited yeast growth and reduced cell viability as confirmed in FUN-1 staining. Fluorescence microscopy study revealed that mature part of LIC10280 was involved in localisation to yeast nucleus without vesicular trafficking. A yeast genetic screen showed that deletion of SCH9, a component of the TORC1 signalling pathway, partially suppressed LIC10280-induced toxicity, implicating a possible link to nutrient-sensing or stress-response pathways. Together, these findings provide new insights into the potential function of LIC10280 and highlight the value of yeast as a model system for studying putative virulence factors of L. interrogans.

This study reports the design and characterization of a novel dimer stabilizer targeting the 14-3-3σ protein, a signaling protein with purported tumor-suppressive roles. Using an in silico design strategy based on the derivatization of GCP-Lys-OMe, a known stabilizer, a series of analogues was evaluated, leading to the identification of 2-N,5-N-dicarbamimidoyl-1H-pyrrole-2,5-dicarboxamide (Compound 14) as the most promising candidate. Furthermore, molecular docking studies and three independent molecular dynamics simulations suggested that the charged state of the guanidinocarbonyl-pyrrole moiety substantially contributed to binding at the dimer interface. Compound 14 was synthesized via a novel route and characterized by NMR and LC-MS/MS, and its binding to recombinantly expressed 14-3-3σ was validated using multiple orthogonal biophysical techniques. 1H CPMG NMR indicated interaction at the dimer interface and enhanced the binding of 14-3-3σ to an exoenzyme S-derived peptide. Meanwhile, UV-Vis titrations yielded an estimated dissociation constant of 1.21 ± 0.15 μM, while thermal-ramp dynamic light scattering showed an increase in aggregation temperature consistent with dimer stabilization. Together, these results identify Compound 14 as a new 14-3-3σ dimer stabilizer and provide a basis for future analogue development.

Ixotrophy is a unique epibiotic predation strategy employed by bacteria in the family Saprospiraceae, involving the capture of swimming prey via adhesive surface substances—analogous to “flypaper”—followed by prey lysis mediated by the Type VI Secretion System (T6SS). While ixotrophy predation mechanisms have been elucidated, the behavioral and molecular adaptation of these facultative predators under varying nutrient conditions remains poorly understood. This study investigates the regulation of the killing apparatus and the secretion of degradative enzymes by Saprospira sp. CCB-QB6, a potent predator of the aquaculture pathogen Vibrio parahaemolyticus that causes Acute Hepatopancreatic Necrosis Disease (AHPND). Microscopic observations revealed that CCB-QB6 utilizes contact-dependent predation, leading to rapid prey lysis and the release of cytoplasmic contents. Genomic analysis confirmed the presence of a T6SS subtype iv, phylogenetically related to the T6SS of the Aureispira sp. CCB-QB1. On the plaque assays, predatory activity was significantly suppressed by increasing concentrations of nutrients, such as tryptone or casamino acids. Gene expression analysis using RT-qPCR demonstrated that the expression of cis8, encoding T6SS spike protein, was specifically induced by the presence of prey under nutrient limitation, but this response was abolished under nutrient-rich conditions. Furthermore, LC-MS/MS-based proteomic analysis of the secretome identified a more diverse secretion of hydrolytic enzymes under starvation than in nutrient-replete conditions. Many of these enzymes possess Type IX Secretion System (T9SS) sorting domains, highlighting the essential role of the T9SS in the saprotrophic and predatory lifestyle of CCB-QB6. Collectively, these findings provide novel insights into the foraging strategies employed by ixotrophic bacteria in response to environmental nutrient fluctuations.

Regulation of cell proliferation is important for maintaining healthy and functional cellular homeostasis. Normal cells proliferate in the presence of growth factors, but when cells experience genomic instability and oxidative stress, cell cycle arrest is triggered, resulting in cellular senescence and ageing. We identified the involvement of type 2 C protein phosphatases (PP2Cs) in the regulation of cell proliferation in budding yeast by cloning and expressing Ptc1-7, and measuring their proliferation using a 96-well microplate-based assay. Interestingly, loss of PP2C could also promote cell proliferation. Besides, mutations of Ptc4 at D48 or D83 or both could still promote cell proliferation and did not affect the cell proliferation regulated by Ptc4. Moreover, a proliferation assay using gene deletion strains reveals that Ptc4 is potentially involved in the regulation of Rim15, which is a key regulator of cell cycle arrest to promote cell proliferation. We investigated the roles of PP2Cs in proliferation and discovered that its role in promoting cell proliferation activity is potentially by modulating Rim15, mediated through the regulation of the HOG or TORC1/Sch9 pathways. This study also highlights the challenge of investigating genes with isoforms as stochastic modulation mechanisms can allow cells to bypass their predetermined signal transduction network to adapt to cellular changes, giving rise to new phenotypes.

Nasopharyngeal carcinoma (NPC) is a distinctive epithelial cancer closely associated with Epstein-Barr Virus (EBV) infection, posing significant challenges in diagnosis and treatment due to its resistance to conventional therapies and high recurrence rates. Current therapies, including radiotherapy and chemotherapy, exhibit limited efficacy, particularly in recurrent or metastatic cases, highlighting the urgent need for novel therapeutic strategies. Targeting EBV oncoproteins, such as Epstein-Barr Virus encoded Nuclear Antigen 1 (EBNA1), Latent Membrane Protein 1 (LMP1), and Latent Membrane Protein 2 (LMP2), presents a promising therapeutic avenue in NPC treatment. This review discusses the latest advancements in drug discovery targeting EBV oncoproteins, emphasizing the identification of inhibitors for specific functional regions of oncoproteins EBNA1, LMP1, and LMP2. Particular attention is given to the molecular mechanisms of these inhibitors and their preclinical or clinical potential in treating EBV-positive NPC. These developments highlight a promising future for targeted therapies in improving outcomes for NPC patients.

Pectic acid (polygalacturonic acid, PGA) and pectin oligosaccharides (POS) have shown selective anticancer activities with cytotoxicity against various cancer cells, including breast adenocarcinoma MCF-7. While promising, their anticancer activities against the nasopharyngeal carcinoma (NPC) have yet to be determined. To this end, the antiproliferation activities of pectic acid and enzymatically derived unsaturated POS trimer were investigated against the EBV-positive NPC cell line, C666-1, as well as on MCF-7 (as positive control) and NIH-3T3 cells (as non-tumorigenic cell model). Our study shows that both POS trimer and PGA can inhibit C666-1 cells’ proliferation at concentrations above 20 mg/mL and 2.5 mg/mL, respectively. Previous studies had also demonstrated that one of the key biological targets for these compounds is the human galectin-3 (hGal3). However, the binding mode of pectic acid and the unsaturated POS trimer to human galectin-3 is still lacking. To investigate this, in silico molecular docking and all-atom molecular dynamics simulations were performed. Analysis of the MD trajectories revealed that the unsaturated POS trimer prefers binding to hGal3 oligomer over hGal3 monomer with a ligand: protein monomer ratio of 1:2 and 1:3. On the other hand, both hGal3 monomer and dimer were found to bind stably to the POS 15-mer (as PGA model) along its linear chain throughout the simulation. In conclusion, both unsaturated POS trimer and PGA have the potential to be novel anticancer agents against EBV-positive NPC, though further development to increase their potency and/or selectivity would be necessary.

PelQ1 from Saccharobesus litoralis is a Ca2+-dependent pectate lyase belonging to the polysaccharide lyase family 1 (PL1). Although being an endolytic enzyme, it degraded polygalacturonate into predominantly unsaturated trimer in an exolytic manner with delayed production of dimer, tetramer and pentamer. The enzyme harbours a C -terminal domain from the carbohydrate-binding module family 13 (CBM13), whose presence facilitated the production of dimer. PelQ1's homology model showed that it possessed a well-conserved catalytic cleft, with R232 acting as the general base and R203 as the general acid. Structural comparison with DcPelC, a similar trimer-generating pectate lyase from Dickeya chrysanthemi EC16, implied that both enzymes' catalytic clefts encompassed at least eight subsites, i.e. -5 to +3. The unequal distribution of the subsites between the reducing and non-reducing ends of the cleavage site might be responsible for the exolytic generation of the trimer. As all but the -1, +1 and + 2 subsites could accommodate methylated galacturonate, this subclass of PL1 pectate lyases may function to help break up methylated pectin.

mTOR regulates cell growth by forming the mTORC1 and mTORC2 complexes. DEPTOR partially inhibits mTORC1, which in turn phosphorylates and inactivates it. Despite the mTORC1–DEPTOR structures, the exact mechanism remains unclear largely because functionally flexible key elements, DEPTOR’s linker in particular, are unresolved. By taking DEPTOR’s dimerisation into consideration, our modelling of these missing loops suggests that monomeric DEPTOR bound to mTORC1 in a non-inhibitory mode, while the domain-swapped dimeric DEPTOR could interact with mTORC1’s FRB domain and block the kinase’s catalytic site with its linker. These two states indicate that linker phosphorylation inactivates DEPTOR possibly by disrupting its dimerisation, which could tether the linker to the kinase domain to enhance mTORC1 inhibition. In addition to DEPTOR, mTOR’s kα9b–kα10 loop, which harbours the S2481 autophosphorylation site, and mSIN1’s flexible domains in mTORC2 might act as inhibitory elements too.

14-3-3 sigma protein is one of the seven isoforms from the highly conserved eukaryotic 14-3-3 protein family. Downregulation of 14-3-3 sigma expression has been observed in various tumors. TRIM25 is responsible for the proteolytic degradation of 14-3-3 sigma, in which abrogation of TRIM25 suppressed tumor growth through 14-3-3 sigma upregulation. However, to date, the exact 14-3-3 sigma interacting residues of TRIM25 have yet to be resolved. Thus, this study attempts to identify the peptide binding sequence of TRIM25 on 14-3-3 sigma via both bioinformatics and biophysical techniques. Multiple sequence alignment of the CC domain of TRIM25 revealed five potential peptide binding sequences (Peptide 1-5). Nuclear magnetic resonance (NMR) assay (H-1 CPMG) identified Peptide 1 as an important sequence for binding to 14-3-3 sigma. Competition NMR assay suggested that Peptide 1 binds to the amphipathic pocket of 14-3-3 sigma with an estimated K-D of 116.4 mu M by isothermal titration calorimetry. Further in silico docking and molecular dynamics simulations studies proposed that Peptide 1 is likely to interact with Lys49, Arg56, Arg129, and Tyr130 residues at the amphipathic pocket of 14-3-3 sigma. These results suggest that Peptide 1 may serve as a biological probe or a template to design inhibitors of TRIM25-14-3-3 sigma interaction as a potentially novel class of anticancer agents.Communicated by Ramaswamy H. Sarma

Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified βαββαβα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na + and K + ) is crucial for nuclease activity. An active site with a unique D 11 (X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D 11 (X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2.

this dataset is related to "In silico prediction and biophysical validation of novel 14-3-3σ homodimer stabilizers" Aljabal G., Teh, A.-H., Yap B.K.

Abstract Among the enzymes required for the efficient utilisation of pectin is polygalacturonase. Saccharobesus litoralis harbours two polygalacturonases belonging to glycoside hydrolase family 28 (GH28). One of them, PGQ1, cleaved polygalacturonate exolytically at the non-reducing end into monomeric units. It was most active at 60 °C and pH 8, with K m and k cat values of 2.3 mg/ml and 6.4 s−1 respectively. Its homology model of a right-handed parallel β-helix core consisted of Asp297 as the general acid and either Asp276 or Asp298 as the general base. By inferring the substrate binding modes at the −1 and +1 subsites from known crystal structures, a hexagalacturonate could be docked into the highly electropositive binding cleft. Interestingly, while no residues were present in the vicinity to make up the +2 and +4 subsites, Arg361 and Arg430 could readily bind to the carboxyl groups of the galacturonates at the +3 and +5 subsites respectively. Structural comparison suggested that this binding pattern with missing subsites might be unique to closely related exopolygalacturonases. As S. litoralis grew much more slowly on extracellular galacturonate due to the lack of a transporter for the monosaccharide, PGQ1 probably functioned in the periplasm to help degrade oligopectates completely. Communicated by Ramaswamy H. Sarma

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., with global implications primarily in tropical countries. However, the mechanisms of leptospiral pathogenesis are still not fully known and not all virulence factors (VFs) have been identified. Budding yeast, Saccharomyces cerevisiae is a popular eukaryotic model which has been used to identify bacterial VFs that target the conserved eukaryotic cellular processes. In this study, we screened for putative VFs of L. interrogans, one of the dominant species causing leptospirosis, by expressing candidate VFs in budding yeast and examining their impact on yeast growth in a high-throughput format. From an initial selection of 288 L. interrogans ORFs, we screened 226 candidate VFs in a yeast growth inhibition assay and identified nine putative VFs in four categories (adhesion, enzymatic, host structure interaction, and immunogenicity). Notably, LIC10280 was highly toxic even when expressed at low copies. We also observed specific subcellular localization for several putative VFs. This study shows that there are still potential L. interrogans VFs that await discovery. KEY POINTS: • High-throughput cloning and expression of leptospiral proteins in yeast. • Heterologous expression of nine leptospiral proteins inhibited yeast growth. • An uncharacterized protein LIC10280 maybe a putative VF for further validation.

The RelEB3 toxin-antitoxin (TA) system of Salmonella enterica subsp. enterica serovar Typhimurium consists of a RelE3 toxin which suppresses bacterial growth, but its RelB3 antitoxin does not neutralise the toxin. The relEB3 operon is widespread in Proteobacteria and is related to higBA2 from Vibrio cholerae. In contrast to the ribosome-dependent HigB2 toxin, however, the RelE3 toxin degraded free RNA independently of the ribosome. A basic loop possibly involved in HigB2's binding to the ribosome is shortened in RelE3, which instead contains a uniquely conserved R51 important for RelE3's toxicity. The RelB3 antitoxin, meanwhile, specifically recognised the CACCTGGTG palindromic motif in the promoter site. RelB3 contains a unique P14 which is conserved as Ala in most homologues, and mutating P14 to Ala enabled the antitoxin to bind to RelE3 and restored bacterial growth. The P14 RelB3 variant, which most likely arose by a point mutation in a recent ancestor of S. Typhimurium and closely related serovars, could have possibly provided the bacteria with a faster response to stress, and might have spread to other serovars through homologous recombination.

Yeast cell death is triggered when essential nutrients such as potassium and lipid are limited but ammonium is in excess. When ammonium and glucose were maintained at 100% of the normal concentration while all the other essential nutrients in yeast nitrogen base (YNB) were reduced to 2%, yeast growth was halted by ammonium toxicity. Yeast started to grow again when either ammonium was also reduced to 2% or gluconate was added, but simultaneously adding gluconate as well as reducing all the nutrients except glucose 50-fold revived yeast growth to a greater extent, i.e. a quarter of the normal growth. Gluconate, as well as formate and alginate, stimulated yeast growth by buffering the drop in pH. Yeast cells were seemingly more susceptible to low pH under the nutrient-limited conditions, entering the stationary phase at pH higher than that of the normal condition. Carboxylate salts may prove a cost-efficient replacement for large proportions of the essential nutrients as yeast cells, in the presence of 2 mg ml-1 gluconate, could still achieve nearly 90% of the normal growth when cultured in only 10% of the normal YNB concentration.

Ageing-related proteins play various roles such as regulating cellular ageing, countering oxidative stress, and modulating signal transduction pathways amongst many others. Hundreds of ageing-related proteins have been identified, however the functions of most of these ageing-related proteins are not known. Here, we report the identification of proteins that extended yeast chronological life span (CLS) from a screen of ageing-related proteins. Three of the CLS-extending proteins, Ptc4, Zwf1, and Sme1, contributed to an overall higher survival percentage and shorter doubling time of yeast growth compared to the control. The CLS-extending proteins contributed to thermal and oxidative stress responses differently, suggesting different mechanisms of actions. The overexpression of Ptc4 or Zwf1 also promoted rapid cell proliferation during yeast growth, suggesting their involvement in cell division or growth pathways.

DEPTOR is an inhibitor of the mTOR kinase which controls cell growth. DEPTOR consists of two DEP domains and a PDZ domain connected by an unstructured linker, and its stability is tightly regulated through post-translational modifications of its linker region that contains the (286)SSGYFS(291) degron. Based on the mTORC1 complex, our modelling suggests a possible spatial arrangement of DEPTOR which is characterised to form a dimer. Our model shows that the two PDZ domains of a DEPTOR dimer bind separately to the dimeric mTOR's FAT domains similar to 130 angstrom apart, while each of the two extended linkers is sufficiently long to span from the FAT domain to the kinase domain of mTOR and beyond to join a shared dimer of the DEP domains. This places the linker's 5299 closest to the kinase's catalytic site, indicating that phosphorylation would start with it and successively upstream towards DEPTOR's degron. The CK1 alpha kinase is reportedly responsible for the phosphorylation of the degron, and our docking analysis further reveals that CK1 alpha contains sites to bind DEPTOR's pS286, pS287 and pT295, which may act as priming phosphates for the phosphorylation of the degron's 5291. DEPTOR's linker can also be ubiquitylated by the UbcH5A-SCF beta-TrCP complex without its PDZ dissociating from mTOR according to the modelling. As the catalytic cleft of mTOR's kinase is restricted, interactions between the kinase's unstructured segment surrounding the cleft and DEPTOR's linker, which may involve 5293 and 5299, may be critical to controlling DEPTOR's access to the catalytic cleft and hence its phosphorylation by mTOR in a manner dependent on mTOR's activation.

The alginate lyase AlyQ from Persicobacter sp. CCB-QB2 is a three-domained enzyme with a carbohydrate-binding module (CBM) from family 32. The CBM32 domain, AlyQB, binds enzymatically cleaved but not intact alginate. Co-crystallisation of AlyQB with the cleaved alginate reveals that it binds to the 4,5-unsaturated mannuronic acid of the non-reducing end. The binding pocket contains a conserved R248 that interacts with the sugar's carboxyl group, as well as an invariant W303 that stacks against the unsaturated pyranose ring. Targeting specifically the non-reducing end is more efficient than the reducing end since the latter consists of a mixture of mannuronic acid and guluronic acid. AlyQB also seems unable to bind these two saturated sugars as they contain OH groups that will clash with the pocket. Docking analysis of YeCBM32, which binds oligogalacturonic acid, shows that the stacking of the pyranose ring is shifted in order to accommodate the sugar's axial C1-OH, and its R69 is accordingly elevated to bind the sugar's carboxyl group. Unlike AlyQB, YeCBM32's binding pocket is able to accommodate both saturated and unsaturated galacturonic acid.

Group 21 and 5 allergens are homologous house dust mite proteins known as mid-tier allergens. To reveal the biological function of group 21 allergens and to understand better the allergenicity of the rDer f 21 allergen, we determined the 1.5 Å crystal structure of rDer f 21 allergen from Dermatophagoides farinae. The rDer f 21 protein consists of a three helical bundle, similar to available structures of group 21 and homologous group 5 allergens. The rDer f 21 dimer forms a hydrophobic binding pocket similar to the one in the Der p 5 allergen, which indicates that both of the homologous groups could share a similar function. By performing structure-guided mutagenesis, we mutated all 38 surface-exposed polar residues of the rDer f 21 allergen and carried out immuno-dot blot assays using 24 atopic sera. Six residues, K10, K26, K42, E43, K46, and K48, which are located in the region between the N-terminus and the loop 1 of rDer f 21 were identified as the major IgE epitopes of rDer f 21. Epitope mapping of all potential IgE epitopes on the surface of the rDer f 21 crystal structure revealed heterogeneity in the sIgE recognition of the allergen epitopes in atopic individuals. The higher the allergen-sIgE level of an individual, the higher the number of epitope residues that are found in the allergen. The results illustrate the clear correlation between the number of specific major epitope residues in an allergen and the sIgE level of the atopic population.