Archives of Biochemistry and Biophysics

[FeFe]-hydrogenases are very active biocatalysts for H2 conversion. However, their active site is vulnerable to irreversible degradation initiated by O2 binding at the catalytic iron ion (Fed) of the active center. CbA5H, the [FeFe]-hydrogenases from Clostridium beijerinckii exhibits stability towards oxygen (O2) due to its ability to reversibly enter an inactive state termed Hinact upon contact with O2. We previously proposed that the close distance of approximately 3.1 [A] between the thiol of a nearby cysteine (C367) and the Fed, based on a 2.9 [A] crystal structure of CbA5H in the Hinact state, enables their binding to each other. This binding therefore was suggested to shield the Fed from O2 damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of the Fed over the thiol of C367. In this study, we present the crystal structure of CbA5H in the Hinact state at an improved resolution of 2.15 [A]. The structure reveals a direct binding between the thiol of C367 and the Fed with a distance of approximated 2.77 [A] which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 [A] bond distance is strikingly long when compared with other iron-sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the Hinact state.

Thymosin {beta}4 (T{beta}4) is a conserved acidic polypeptide with 43-amino acids participating in multiple pathophysiological processes. In this study in vivo effects of T{beta}4 on liver regeneration are investigated in carbon-tetrachloride (CCL4) induced rodent animal liver jury models. Results illustrate that exogenous T{beta}4 treatment significantly reduced CCL4-rendered liver necrosis around central vein. At 48 hours after CCL4 insults hepatocytes proliferation occur mainly around the periportal area, while hepatocytes proliferation around the necrosis area is prominently increased by exogenous T{beta}4 treatment. The holistic proliferation level of liver tissues are also enhanced by exogenous T{beta}4. Hepatocyte proliferation activities negatively correlate with the necrosis extent of the liver tissue. These results suggested firstly exogenous T{beta}4 treatment could enhance liver regeneration and exhibit prosperous potential for application in clinical conditions such as liver transplantation.

EIF2alpha kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2alpha-ATF4 pathway. We report a protocol for producing the minimal kinase domain of full-length human HRI, termed HRIKD-delta-KI, where the unstructured 140 amino acid (aa) kinase insert (KI) within HRI kinase domain (HRIKD) is replaced with a 2aa glycine/serine (GS) linker. X-ray crystal structures were determined of apo-HRIKD-delta-KI and of its complex with ATP at 2.1 & 2.5 Angstrom resolution respectively. Both structures display a canonical bi-lobal kinase fold. However, they remain in a non-productive state with a displaced C-helix, disassembled R-spine, and a disordered activation segment hindering the substrate site. Biophysical assays (fluorescence based thermal shift & Synchrotron Radiation Circular Dichroism) demonstrate HRIKD-delta-KI retains its functional ligand-binding conformation. All together, these findings define structural and ligand-binding features of HRI to support ongoing drug discovery efforts in blood cancer.

Dengue Virus Serotype 2 is a human pathogenic flavivirus that encodes a non-structural protein 3 (DEN2-NS3) containing a helicase domain essential for viral replication. DEN2-NS3 utilizes energy derived from NTP hydrolysis to unwind dsRNA and dsDNA. A galloylated catechin, (-)-epigallocatechin gallate (EGCG), was previously reported to be highly potent against the Zika Virus NS3 helicase, with an IC50 value observed at 295.7 nM. This prompted an investigation to determine if three catechins, namely, (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and EGCG, would act as potent inhibitors of DEN2-NS3. Enzyme-inhibition assays revealed that the helicase catalytic domain, DEN2-NS3(S171-K618), is strongly inhibited by these galloylated catechins. We observed Ki values of 400 {+/-} 86.6 nM for EGCG (mixed-mode inhibition with respect to ATP) and 550 {+/-} 250 nM for ECG (uncompetitive inhibition with respect to ATP). Furthermore, using a computational workflow starting with SiteMap, we provide evidence that a highly druggable pocket exists within the RNA-binding cavity, involving residues ASP290, ARG387, ASP409, MET429, HIS487, ASP541, ARG599, and ASP603. These catechins were each analyzed through 200-ns molecular dynamics (MD) simulations to evaluate the binding stability within the target DEN2-NS3 binding pocket. Computational results revealed that EGCG and ECG maintained high stability, forming shared, highly persistent amino acid contacts (>45% occupancy) with ASP603, ARG599, ASP541, and ARG387. In conclusion, we have demonstrated that EGCG and ECG achieve strong binding and allosteric disruption of the critical RNA-binding channel. We suggest that future structural optimization of these compounds into stable prodrug derivatives could yield promising antiviral therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/733882v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2db363org.highwire.dtl.DTLVardef@5c2fdaorg.highwire.dtl.DTLVardef@49bf8eorg.highwire.dtl.DTLVardef@1bf31f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Endothelial progenitor cells (EPCs) contribute to endothelial repair and neovascularization, and EPC dysfunction is closely associated with oxidative stress-related vascular injury. Forkhead box O3a (FoxO3a) regulates cellular stress responses, whereas miR-34a has been implicated in endothelial dysfunction, senescence, and apoptosis. However, the relationship between FoxO3a and miR-34a-3p in oxidatively injured EPCs remains incompletely defined. Objective: This study investigated the role of FoxO3a in H2O2-induced EPC dysfunction and examined whether miR-34a-3p directly interacts with the FoxO3a 3' untranslated region (3'UTR). Methods: Human umbilical cord blood-derived EPCs were identified by DiI-ac-LDL uptake, FITC-UEA-1 binding, and the expression of EPC-related markers. Oxidative stress was induced by H2O2. Cell viability, apoptosis, and angiogenic capacity were evaluated using CCK-8 assay, Annexin V/7-AAD flow cytometry, and Matrigel tube formation assay, respectively. FoxO3a expression was modulated using adenoviral overexpression or knockdown vectors, and miR-34a was modulated using mimics or antagomir. FoxO3a and miR-34a expression levels were detected by Western blot and qPCR. A dual-luciferase reporter assay was used to verify the interaction between hsa-miR-34a-3p and the FoxO3a 3'UTR. Results: H2O2 reduced EPC viability, increased apoptosis, and impaired tube formation in a concentration-dependent manner. H2O2 increased FoxO3a protein abundance and miR-34a expression, whereas FoxO3a mRNA did not change markedly. FoxO3a overexpression aggravated, whereas FoxO3a knockdown partially alleviated, H2O2-induced EPC dysfunction. Similarly, miR-34a mimics further suppressed EPC viability and tube formation, while miR-34a antagomir exerted a protective effect. Dual-luciferase reporter analysis showed that hsa-miR-34a-3p significantly reduced the activity of the wild-type FoxO3a 3'UTR reporter, while mutation of the predicted binding site abolished this suppression. Conclusion: FoxO3a and miR-34a participate in oxidative stress-induced EPC dysfunction. The dual-luciferase data demonstrate that hsa-miR-34a-3p directly targets the FoxO3a 3'UTR, suggesting the presence of miR-34a-3p-mediated post-transcriptional feedback within the FoxO3a-related stress-response network in EPCs.

Glyceraldehyde (GA) contributes to the development of various diseases, such as diabetes and Alzheimer's disease via protein glycation and the formation of advanced glycation end products (AGEs); however, effective strategies for neutralizing GA are limited. Carnosine (Car), an imidazole dipeptide (IDP) that is abundant in meat, suppresses protein glycation by scavenging reactive aldehydes. There are only a few reports on the antiglycation activity of Car against GA. For other IDPs, such as anserine, balenine (Bal), and homocarnosine, there are almost no reports on their antiglycation activity. In this study, we demonstrated the antiglycation activity of four types of IDPs and 2-oxocarnosine (2-oxo-Car), an oxidized form of Car, against GA-induced intracellular protein glycation and neuronal cytotoxicity. Car and Bal exhibited significantly higher reactivity with GA compared with other IDPs and 2-oxo-Car. An in silico analysis suggested that the difference in reactivity is dependent upon intramolecular hydrogen bond formation and the conformation of each IDP. Although there were differences in reactivity with GA, LC-MS analysis revealed that all of the IDPs and 2-oxo-Car reacted with two molecules of GA to form adducts containing pyridinium rings. Car and Bal exhibited high reactivity with GA and markedly suppressed GA-induced cytotoxicity in SH-SY5Y cells. Western blot and qPCR analyses revealed that IDPs suppressed GA-induced protein glycation and the upregulation of endoplasmic reticulum and oxidative stress response genes. Our results indicate that IDPs represent a novel preventive approach to AGE-related diseases and provide a foundation for the development of strategies to treat GA-related neurotoxicity.

Acetyl-coenzyme A (CoA) is a central metabolic intermediate that links carbon and energy metabolism across all domains of life. The conversion of acetate and acetyl-CoA is carried out by three enzyme pathways: acetate kinase/phosphotransacetylase, ADP-forming acetyl-CoA synthetase, and AMP-forming acetyl-CoA synthetase (Acs). Acs enzymes serve critical physiological roles across diverse organisms generally by catalyzing a reversible two-step reaction forming acetyl-CoA and AMP from acetate and ATP. Isolated from the wastewater reclamation facility in Norman, Oklahoma, Syntrophus aciditrophicus strain SB (Sa) relies on an AMP-forming acetyl-CoA synthetase (SaAcs1) that favors synthesizing acetate and ATP from acetyl-CoA and AMP, in contrast to all previously characterized Acs enzymes. The origin of this preference and the structural determinants of both the thioester-forming step and catalytic directionality remain poorly understood. Here, we report a 2.2 [A] crystal structure of full-length SaAcs1 in the adenylation conformation with acetyl-AMP bound in the active site. Structural comparison to the extensively characterized Acs enzymes from Salmonella enterica (SeAcs) and Cryptococcus neoformans (CnAcs) revealed a displaced CoA-binding loop in SaAcs1. Enzymatic assays confirmed that SaAcs1 preferentially catalyzes the ATP-forming reaction. Site-directed mutagenesis demonstrated that reversion of two residues, G196 and T197, at the beginning of the CoA-binding loop to the consensus sequence repositions the loop and shifts catalytic preference toward the AMP-forming direction. Together, these results establish the CoA-binding loop and G196 and T197 as the primary structural determinants of directional preference in SaAcs1.

Background: Uncontrolled bleeding complicates trauma, surgery and many medical conditions. While currently available procoagulant therapies (e.g., plasma-derived factors, recombinant proteins, antifibrinolytics) have crucial limitations. Methods: N389 (CQQTVTLLPAADLDDFSC) was synthesized by Fmoc solid-phase chemistry, characterized by HPLC and LC-MS, then tested in normal human pooled plasma in microplate mechanical clot-formation assays using incubated and immediate addition formats. Kinetic parameters (plasma recalcification, PRT; maximum absorbance, Amax) were obtained from absorbance curves fit to four-parameter logistic models. Mixing studies with modified (i.e., aged, adsorbed) plasma probed factor dependence. Results: In plasma coagulation assays activated with 25 mM CaCl2, baseline clotting showed a PRT of 23.74 +/- 0.27 min and Amax of 0.1813 +/- 0.0043 (n = 3), whereas N389 significantly reduced PRT to 8.442 +/- 6.0395 min without incubation (p = 0.0012), further decreased PRT after incubation (p < 0.0001), increased Amax to 0.2523, and retained comparable activity across normal, adsorbed, and aged plasma, in contrast to S1255 which showed a faster but incubation-labile effect with PRT 2.353 +/- 1.3685 min (p = 0.0007) and marked attenuation in factor-depleted and aged plasma. Mixing studies showed N389 activity persisted across normal, aged and adsorbed plasma, consistent with a mechanism that does not require intact plasma coagulation factor profiles (specifically factors II, V, VIII, VII, IX, X). Discussion: Collectively with prior evidence on anionic surfaces, Ca2+-binding Gla domains, and peptide-modulated fibrin polymerization, these results support a model in which N389 functions as a stable, charge-based scaffold that coordinates divalent cations and/or directly nucleates fibrin(ogen), while highlighting limitations of bulk clotting assays and the need for targeted thrombin generation, binding, aggregation, and contact-activation studies. Conclusions: The aspartate-rich peptide N389 is a sustained, factor-independent procoagulant at least in vitro. N389 thus merits further mechanistic and translational evaluation as a synthetic hemostatic agent.

Emerin (EMD), an inner nuclear membrane protein essential for nuclear architecture integrity, gene expression, cellular signaling, and chromatin stability, interacts with the LINC complex and participates in cytoskeleton-nucleoskeleton communication by binding to nuclear actin filaments. EMD is implicated in migration, invasion, and metastasis in some tumors, but its role in glioblastoma (GBM) remains unclear. This study evaluated the effects of EMD knockdown and overexpression in GBM cell lines following genotoxic treatment with cisplatin. In both wild-type p53 (U87-MG) and mutant p53 (U138-MG) GBM cells, EMD expression is high, and cisplatin treatment did not affect these protein levels. EMD knockdown in U87-MG cells significantly increased cisplatin IC50, viability, and proliferation. Conversely, stable overexpression of EMD in U87-MG cells led to reduced cisplatin IC50, viability, proliferation, and migration. EMD knockdown or overexpression did not affect any U138-MG phenotypes, with or without cisplatin treatment. Modulation of EMD levels causes morphological changes in stress fiber cytoskeleton, whereas overexpression of EMD in U87-MG cells promotes an increase and a decrease in nuclear and cytoplasmic actin levels, respectively. These biological responses of U87-MG cells overexpressing EMD were coincidentally associated with alterations in the levels of pH2AX(Ser139), p-p53(Ser15), p53, and p21Kip1 proteins after cisplatin exposure. In sum, modulation of EMD levels affects the viability, migration, and proliferation of wild-type p53 GBM cells treated with cisplatin, suggesting unknown roles in the DNA damage response and repair. This work highlights EMD as a potential regulator of GBM chemoresistance and a target for therapeutic intervention.

Ovate Family Proteins (OFPs) are a class of plant-specific, negative nuclear transcriptional regulators characterized by conserved C-terminal OVATE domain. This study on comparative functional characterization of two head-to-head arranged OFPs - AtOFP2 (Ovate-OFP with full ovate domain) and AtOFP17 (Ovate-Like OFP with partial ovate domain) provides critical insight into how structural variations in ovate domain leads to functional divergence. Detailed phenotypic analysis of 28 physical and physiological traits of loss- and gain-of-function mutants revealed that both genes act as broad, pleotropic repressors of plant growth and development. Removal of repression in knock-down mutants of both genes exhibited reduced duration of seed dormancy, faster rate of germination and growth, bigger plants and significantly higher seed yield. In contrast, constitutive over-expression showed a generalized repressive nature of both genes, with nuanced differences for fine tuning of specific traits. For example, both genes showed antagonistic behaviours on root hair architecture. AtOFP2 act as a strong repressor of root hair development whereas AtOFP17 is a stronger repressor of hypocotyl and root cell architecture. AtOFP17 owing to partial ovate domain exerts a mild level of repression throughout life span as indicated by smaller plants and lesser yield in knock-down AtOFP17 mutants. On the contrary, AtOFP2 exerted a much stronger repressor effect in which > 90% over-expression mutants died at the juvenile stage ; the survival of remaining 10% is probably owing to activation of dosage-dependent feedback loop mechanism as indicated by normal growth of mature plants, and is also evident by transcriptome data. Transcriptome analysis of roots of 7-day old seedling of knock-down and over-expression mutants of AtOFP2 showed downregulation of OFP2 in over-expressed mutants. However, severely stunted phenotype indicated presence of stable OFP2 protein to exert effects. Analysis of DEGs in OFP2 mutants revealed that it acts as an important regulator working at intersection of hormonal signalling affecting critical genes required for auxin, cytokinin, GA, BR and ABA functioning. Perturbations across hormonal signalling pathways affects cell wall remodelling factors such as EXPANSINS, Xyloglucan hydrolases (XTHs) and cellulose synthases (CSLs) causing overall stunted growth; and epidermal patterning genes such as WER, GL1, EGL3, TTG1 leading to severely reduced root length and root hairs. Significantly, functional analysis of this master regulator highlighted a significant economic potential. Knockdown of both these genes relieves their natural repression on reproductive traits, leading to longer siliques, bigger and heavier seeds, and substantially increased overall seed yield, positioning AtOFP2 and AtOFP17 as highly valuable targets for agricultural crop improvement.

Structure-based drug discovery is known to apply computational methods in a tiered hierarchy, with each layer narrowing the candidate set and refining the binding picture before committing to the next, more expensive step. We present a four-tiered computational benchmarking study evaluating five engines against a panel of 36 compounds targeting B-secretase 1 (BACE1), a validated Alzheimer's disease target with extensive co-crystal ground truth. This study evaluates Flexible Docking and Boltz2 Cofolding as the primary tier, followed by Ensemble Docking, and then Protein-Ligand MD with MM/PBSA and MM/GBSA post-processing. This is then concluded with Relative Binding Free Energy Perturbation (RevFEP) as the terminal refinement layer. Each method was benchmarked against the experimental binding free energies derived from the co-crystal structures spanning -7.85 to -11.35 kcal/mol. Our findings revealed that Flexible Docking reproduced the co-crystal binding mode for 35 of 36 ligands (97.2% within 2.0 A RMSD) but did not rank potency at this resolution. Boltz2 CoFolding provided an orthogonal structural cross-check with a receptor backbone RMSD of 0.293 A against the experimental co-crystal structure. Ensemble Docking identified the optimal receptor conformation for downstream FEP setup. MD with MM/GBSA decomposition identified van der Waals complementarity as the primary potency driver (Pearson r = +0.855, R2 = 0.732 on a 10-compound subset). RevFEP delivered the highest affinity correlation of any method (Pearson r = +0.662, R2 = 0.438, Spearman p = +0.624, mean absolute error 1.02 kcal/mol across all 36 ligands), resolving potency differences within a narrow 3.5 kcal/mol congeneric window that no other engine could discriminate. We characterize what each engine contributes independently and where RevFEP delivers signals no other engine achieves.

Hydropersulfides have gained attention in cell biology as excellent nucleophiles and membrane-protective radical scavengers. They form perthiyl radicals, which terminate radical chain reactions through self-recombination, leading to the formation of polysulfides. It is currently unknown how polysulfides are subsequently reduced again in non-enzymatic or enzymatic metabolic pathways. Here we used stopped-flow kinetic measurements in combination with mass spectrometry to show that the model class I glutaredoxin from the malaria parasite Plasmodium falciparum (PfGrx) rapidly reduces the polysulfides glutathione trisulfide (GS3G) and glutathione tetrasulfide (GS4G), yielding the glutathionylated enzyme and the corresponding glutathione hydropersulfide GSSH and hydrotrisulfide GS3H. The second-order rate constants of these enzymatic reductions [&ge;]107 M-1s-1 are even slightly higher than for glutathione disulfide (GSSG). In contrast, PfGrx was inactive or only moderately active using cystine or cysteine trisulfide as oxidants. GSSH and GS3H are further reduced by PfGrx with second-order rate constants on the order of 106-107 M-1s-1, yielding the glutathionylated enzyme as well as hydrogen sulfide (H2S) and hydrogen disulfide (H2S2), respectively. Thus, glutaredoxins specifically recognize the glutathione moiety of glutathione (hydro)polysulfides and glutathione hydropersulfide. Due to the rapid reduction of glutathionylated glutaredoxins by reduced glutathione (GSH), glutathione (hy-dro)per/polysulfides are efficiently converted to GSSG and H2S or the corresponding hydrogen polysulfides. As a consequence, the steady-state concentration of glutathione (hydro)per/polysulfides should be tightly controlled in subcellular compartments containing active glutaredoxins and high GSH concentrations.

Abstract Background: Acute Lymphoblastic Leukemia (ALL) is a highly heterogeneous pediatric malignancy. Despite high survival rates, relapse and the involvement of central nervous system (CNS) remains a significant clinical challenge. Traditional clinical parameters often lack the precision required for early detection and risk stratification. This study utilizes high-throughput proteomics and machine learning to identify molecular signatures in cerebrospinal fluid (CSF) that characterize disease effect and treatment response. Methods: 82 CSF samples from 41 pediatric ALL patients at diagnosis (VD) and remission (VR) were analyzed. Proteomic profiling of 276 proteins was performed using Olink Proximity Extension Assay. Differentially abundant proteins were identified (q-value< 0.05, |Log_2FC| > 0.5) using the Wilcoxon rank-sum test. Three machine-learning algorithms - Random Forest, LASSO, and SVM-RFE - were integrated to select the differentially abundant proteins in VR and VD and between CNS involvement levels. To validate the data Pan-Cancer Atlas analysis was done using two different platforms. Results: In the remission phase, we observed significant alterations in the expression of key proteins compared to diagnosis, with ADGRG1 and KYNU showing a marked increase, while CCL17, CD5, CD27, CXCL9, CXCL11, FASLG, GZMA, and TNFRSF9 were significantly downregulated. Furthermore, our analysis identified distinct protein signatures associated with CNS involvement: CCL4, CTSC, CXCL10, CXCL9, and MMP7 were differentially abundant at the VD stage, whereas CAIX, CASP-8, HAGH, CXCL9, MMP7, MCP-2, and VWC2 at the VR stage. Conclusion: Integrating Olink proteomics with machine learning identified molecular signatures in ALL that have the potential to be further developed to a biomarker panel for monitoring treatment response and guiding personalized therapeutic strategies shifting the focus toward the Precision One Health approaches.

The tick Ixodes ricinus is the main vector of human and animal pathogens in Europe. Despite its importance in epidemiology and medicine, our understanding of physiological mechanisms controlling blood feeding, osmoregulation, or development are still limited. Here, we identify novel neuropeptide invertebrate parathyroid hormone-like peptide (iPTH) and its two receptors - PTHR1 and PTHR2 in I. ricinus. Functional aequorin-based assay confirmed specific activation of both receptors by iPTH. Using RT-qPCR we detected the PTHR1 transcript in the synganglion, while increased expression levels of PTHR2 were found in the salivary glands, hindgut and female gonads. RNA-mediated knockdown of iPTH receptors in nymphs resulted in delayed blood feeding, and a high incidence of defects in adult ecdysis. Consistent with observed phenotypes, iPTH is expressed in multiple neurons of the synganglion which project arborizing axons to the salivary glands, rectal sack and skeletal muscles. iPTH was colocalized with orcokinin-immunoreactivity (OK-IR) in all neurons that innervate these peripheral tissues. iPTH is further colocalized with tachykinin (TK) in Pd1DL1 neurons, suggesting coordinated action with other neuropeptides. Our findings indicate that iPTH signaling is required for normal feeding, development and successful ecdysis.

Aldehyde dehydrogenases (ALDH) are enzymes that catalyze the NAD(P)+-dependent oxidation of aldehydes into carboxylic acids, playing roles in detoxification, biosynthesis, and regulatory functions. Dysfunction of ALDH is associated with serious conditions such as alcohol intolerance, cancer, cardiovascular problems, and neurological disorders. In humans, ALDH1A1 and ALDH1A3 isoforms act as retinaldehyde dehydrogenases and are overexpressed in various cancers, where high levels are associated with increased tumor malignancy, cancer stem cell traits, and therapeutic resistance. ALDH1A3 is recognized as a promising target for anticancer therapies, with several inhibitors, mainly reversible, developed to specifically target it or the enzyme family. Since ALDH enzymes can also display esterase activity, we used this property to develop an in vitro assay specifically targeting the esterase function of ALDH1A3. A highly conserved active-site cysteine in ALDH1A3 is located at the bottom of two converging channels, which define the substrate- and cofactor-binding pockets. To target this catalytic cysteine, we screened a library of 3,200 cysteine-focused covalent fragments. This led to the identification of Z3405279217 (Z34), an acrylamide-based covalent compound that inhibits both ALDH1A1 and ALDH1A3 at sub-micromolar levels. Biochemical and biophysical tests confirmed that Z34 acts as a time-dependent, covalent, and irreversible binder to the active-site cysteine. In this work, we determined the Cryo-EM structure of the ALDH1A3-Z34 complex at 2.26 [A] resolution, confirming the covalent attachment to the catalytic cysteine of Z34. Notably, two mutually exclusive covalent binding modes were observed: one occupying the substrate-binding pocket and the other the cofactor-binding region. Z34 displayed unexpected binding modes within the active site and holds promise as a lead compound for future drug development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/738401v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@982d1forg.highwire.dtl.DTLVardef@ba86f2org.highwire.dtl.DTLVardef@1f19f2borg.highwire.dtl.DTLVardef@8e807_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nearly all bacteria are surrounded by a mesh-like macromolecule called peptidoglycan that gives them their shape and helps them resist turgor pressure. To grow and maintain their peptidoglycan, bacteria produce a wide range of enzymes, including the relatively understudied ,[x1D05]-transpeptidase (LDT) family. LDTs can catalyse several different reactions and vary widely in copy number: some bacteria have none, whilst others have more than twenty. To better understand why some bacteria have so many LDTs, we examined 18 putative ones from Rhizobium johnstonii, a nitrogen-fixing, symbiotic bacterium. Heterologous expression revealed several highly active enzymes, one of which, LdtRj8, we further characterized in detail. In vitro assays showed that LdtRj8 was capable of ,[x1D05]-transpeptidation, carboxypeptidation, substitution, and endopeptidation, but that its preferred activity differed at different pHs. LdtRj8 particularly excelled at ,[x1D05]-substitution, utilizing all of the tested [x1D05]-amino acids, and, surprisingly, most of the -amino acids as well. LdtRj8's pH-modulated activity could help R. johnstonii respond to acidic conditions encountered throughout the rhizobium-legume symbiosis, and its -amino acid substitution activity, which we show to be a more general property of LDTs, may regulate ,[x1D05]-transpeptidation and explain the existence of isomeric muropeptides often reported in the literature.

Prion diseases are neurodegenerative disorders associated with the structural conversion of the cellular prion protein (PrPc) into its misfolded infectious isoform (PrPSc). Despite substantial efforts, no disease-modifying therapy or cure is currently available. Here, we present an integrated computational-experimental pipeline for the rational design of cyclic peptides targeting PrPc to inhibit its pathogenic conversion. Starting from crystal structures of antibody-bound mouse PrPc, we develop a rational design strategy combined with iterative molecular dynamics simulations and sequence optimization to generate peptides with enhanced binding and structural impact. Three candidates were selected for experimental validation. Our results show that PH1 (49YGPDPSDSYT58, antibody numbering) that binds stably to the &alpha2-&alpha3 interface most effectively reduced PrPSc levels in GT1-7 cells, essentially by inducing allosteric rearrangements that reinforce the intramolecular helical bundle. PL1 (89GQSNTKPYT97) and PL2 (89RQSNTWPYT97) binding the &beta1-&alpha1/&alpha3 junction exerted more modest effects due to the potential competition of the flexible tail to bind at this site. These results establish a mechanistic link between peptide-induced stabilization of PrPc and inhibition of prion propagation and provide a generalizable framework for designing conformational stabilizers of aggregation-prone proteins.

Background: Diabetic kidney disease (DKD) remains a leading cause of end-stage renal disease worldwide, characterized by progressive structural and metabolic alterations secondary to chronic hyperglycemia. While numerous type 1 and type 2 rodent models have been developed to study the pathophysiology of DKD, no single model perfectly recapitulates the full clinical spectrum of human disease. The selection of an optimal model depends deeply on the specific research objective, as phenotypic expression and histopathological severity vary significantly across different strains and induction methods. The present study provides a comparative analysis of the renal histological of three widely utilized murine models: the chemically induced streptozotocin (STZ) model and the genetic Akita (type 1) and db/db (type 2) models. Methods: Male STZ-induced (28 weeks post-induction), heterozygous Akita (28 weeks old), and db/db mice at two different age intervals (18-21 and 16-24 weeks old) were assessed. Renal injury was quantified using four light-microscopic parameters: glomerulomegaly, mesangial hypercellularity, tubular vacuolization and arteriolar hyalinosis. Due to observed discrepancies between metabolic and structural findings in the db/db strain, transmission electron microscopy (TEM) was employed for subcellular characterization. Results: All models exhibited significant hyperglycemia and albuminuria. At the light-microscopic level, STZ and Akita mice demonstrated consistent and pronounced renal lesions. In contrast, db/db mice despite increasing albuminuria and obesity, light microscopy revealed heterogeneous and inconsistent histopathological changes. However, TEM analysis of db/db mice kidneys successfully captured early ultrastructural injury, including irregular glomerular basement membrane (GBM) thickening and focal podocyte foot process effacement, which were undetectable by light microscopy. Conclusions: Our findings indicate that the Akita and STZ-induced models exhibit prominent structural alterations detectable by conventional light microscopy, whereas the db/db model requires ultrastructural evaluation by TEM to reliably confirm renal injury. This study underscores the limitation of routine histology in certain type 2 diabetes models and highlights the complementary value of TEM for accurate histopathological characterization. Collectively, the alternative histopathological markers identified herein offer sensitive and readily accessible indices for monitoring early-to-moderate DKD progression, providing a more robust framework for preclinical model selection and therapeutic evaluation in future studies.

Lanthanides function as enzyme cofactors in bacteria, where they are widely distributed in pyrroloquinoline quinone-dependent 8-bladed beta-propeller dehydrogenases. No lanthanide-dependent enzymes, however, have been described outside prokaryotes. Here, we combined structural bioinformatics, phylogenetics, AlphaFold3 co-folding, coordination-sphere comparison, and quantum-mechanical cluster modeling to search for and rank putative lanthanide-coordinating 8-bladed beta-propeller enzymes in Eukarya. We identified candidate lanthanide-coordinating proteins in a diverse range of eukaryotes, predominantly plants and fungi, including species of clear industrial and agricultural relevance. A high-confidence subset matched validated bacterial Ln-binders based on both geometric similarity to canonical Ln-binding sites and on predicted Ln3+ versus Ca2+ selectivity. Our findings indicate that lanthanide biology likely extends beyond bacteria, with implications for plant, fungal, and broader eukaryotic metabolism, and warrant targeted biochemical investigation.

Background: Stress-induced childhood-onset neurodegeneration with variable ataxia and seizures (CONDSIAS) is a rare autosomal recessive disorder caused by biallelic variants in ADPRHL2, which encodes ADP-ribosylhydrolase 3 (ARH3), a key enzyme involved in poly (ADP-ribose) (PAR) metabolism. Although Minocycline has been reported to attenuate PAR-mediated neurotoxicity primarily through modulation of PARP-dependent pathways, whether it may also interact with ARH3 or influence the structural behavior of pathogenic ARH3 variants remains unknown. This study was designed to explore this possibility by integrating clinical observation with computational structural analyses. Methods: Comprehensive clinical evaluation, targeted Sanger sequencing, and in silico pathogenicity analyses were performed. Protein modeling, molecular docking, and 100-ns molecular dynamics simulations were conducted to evaluate the predicted structural consequences of the p.Thr79Pro variant and to explore potential interactions between ARH3 and Minocycline. Results: A homozygous ADPRHL2 variant (NM_017825.3:c.235A>C; p.Thr79Pro) was identified in a child with CONDSIAS. Computational analyses predicted reduced structural stability and increased conformational flexibility of the mutant ARH3 protein relative to the wild-type structure. MM-GBSA calculations estimated differences in binding free energies between the wild-type (-34.51 kcal/mol) and mutant (-39.76 kcal/mol) ARH3-Minocycline complexes, suggesting subtle differences in their predicted energetic profiles. Clinically, neurological progression appeared stable, with improved motor function observed during approximately one year of follow-up and no notable treatment-related adverse effects. Conclusions: By integrating clinical observations with computational structural analyses, this study provides preliminary computational support for the hypothesis that Minocycline may influence ARH3 conformational behavior in addition to its proposed effects on PARP-dependent pathways. Although these findings do not demonstrate direct molecular binding or therapeutic efficacy, they provide a biologically plausible framework for future biochemical, cellular, and functional investigations. Keywords: CONDSIAS; ADPRHL2; ARH3; Minocycline; molecular docking; molecular dynamics simulation; structural bioinformatics; translational medicine