Hb H disease associated with compound heterozygosity for --SEA deletion and a novel alpha globin chain variant (HBA2:c.175C>A) on the distal histidine in a Chinese family.

OBJECTIVES: In clinical practice, the majority of α-thalassaemia cases arise from deletions of the α-globin genes. However, a subset of cases is attributed to rare haemoglobin variants, which can manifest with borderline or normal screening results, potentially leading to missed diagnoses in clinical practice. METHODS: Blood samples were collected from family members and underwent haematological, DNA and RNA analysis. RESULTS: The five-month-old proband presented a haematological phenotype consistent with Hb H disease. The mother's haematology profile was consistent with an α-thalassaemia carrier, while the father exhibited a borderline reduction in MCV and MCH. MALDI-TOF identified an abnormal α-chain in the proband. DNA analysis revealed a novel α-globin variant (HBA2:c.175C>A, α58His>Asn, Hb DG-Nancheng) affecting the distal histidine in the family. The father and the mother had α-genotype of --SEA/αα and αDG-Nanchengα/αα, respectively; while the proband inherited both mutant alleles (--SEA/αDG-Nanchengα). Sequencing of cDNA from HBA2 gene identified an equal ratio of normal and mutant alleles. CONCLUSION: This rare case highlighted the importance of identifying rare haemoglobin variant during prenatal screening. The clinical and genetic data provides useful information on the pathogenicity of this variant and further insight into the role of distal histidine residue of α-globin.

Functional expression, purification, biochemical and biophysical characterizations, and molecular dynamics simulation of a histidine acid phosphatase from Saccharomyces cerevisiae.

A histidine acid phosphatase (HAP) (PhySc) with 99.50% protein sequence similarity with PHO5 from Saccharomyces cerevisiae was expressed functionally with the molecular mass of ∼110 kDa through co-expression along with the set of molecular chaperones dnaK, dnaJ, GroESL. The purified HAP illustrated the optimum activity of 28.75 ± 0.39 U/mg at pH 5.5 and 40 ˚C. The Km and Kcat values towards calcium phytate were 0.608 ± 0.09 mM and 650.89 ± 3.6 s- 1. The half-lives (T1/2) at 55 and 60 ˚C were 2.75 min and 55 s, respectively. The circular dichroism (CD) demonstrated that PhySc includes 30.5, 28.1, 21.3, and 20.1% of random coils, α-Helix, ß-Turns, and ß-Sheet, respectively. The Tm recorded by CD for PhySc was 56.5 ± 0.34˚C. The molecular docking illustrated that His59 and Asp322 act as catalytic residues in the PhySc. MD simulation showed that PhySc at 40 ˚C has higher structural stability over those of the temperatures 60 and 80 ˚C that support the thermodynamic in vitro investigations. Secondary structure content results obtained from MD simulation indicated that PhySc consists of 34.03, 33.09, 17.5, 12.31, and 3.05% of coil, helix, turn, sheet, and helix310, respectively, which is almost consistent with the experimental results.

Functions and Metabolism of Amino Acids in the Hair and Skin of Dogs and Cats.

The hair and skin of domestic cats or dogs account for 2% and 12-24% of their body weight, respectively, depending on breed and age. These connective tissues contain protein as the major constituent and provide the first line of defense against external pathogens and toxins. Maintenance of the skin and hair in smooth and elastic states requires special nutritional support, particularly an adequate provision of amino acids (AAs). Keratin (rich in cysteine, serine and glycine) is the major protein both in the epidermis of the skin and in the hair. Filaggrin [rich in some AAs (e.g., serine, glutamate, glutamine, glycine, arginine, and histidine)] is another physiologically important protein in the epidermis of the skin. Collagen and elastin (rich in glycine and proline plus 4-hydroxyproline) are the predominant proteins in the dermis and hypodermis of the skin. Taurine and 4-hydroxyproline are abundant free AAs in the skin of dogs and cats, and 4-hydroxyproline is also an abundant free AA in their hair. The epidermis of the skin synthesizes melanin (the pigment in the skin and hair) from tyrosine and produces trans-urocanate from histidine. Qualitative requirements for proteinogenic AAs are similar between cats and dogs but not identical. Both animal species require the same AAs to nourish the hair and skin but the amounts differ. Other factors (e.g., breeds, coat color, and age) may affect the requirements of cats or dogs for nutrients. The development of a healthy coat, especially a black coat, as well as healthy skin critically depends on AAs [particularly arginine, glycine, histidine, proline, 4-hydroxyproline, and serine, sulfur AAs (methionine, cysteine, and taurine), phenylalanine, and tyrosine] and creatine. Although there are a myriad of studies on AA nutrition in cats and dogs, there is still much to learn about how each AA affects the growth, development and maintenance of the hair and skin. Animal-sourced foodstuffs (e.g., feather meal and poultry by-product meal) are excellent sources of the AAs that are crucial to maintain the normal structure and health of the skin and hair in dogs and cats.

A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

Comparison of the Backbone Dynamics of Dehaloperoxidase-Hemoglobin Isoenzymes.

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.

Widespread pfhrp2/3 deletions and HRP2-based false-negative results in southern Ethiopia.

BACKGROUND: Rapid diagnostic tests (RDTs) play a significant role in expanding case management in peripheral healthcare systems. Histidine-rich protein-2 (HRP2) antigen detection RDTs are predominantly used to diagnose Plasmodium falciparum infection. However, the evolution and spread of P. falciparum parasite strains with deleted hrp2/3 genes, causing false-negative results, have been reported. This study assessed the diagnostic performance of HRP2-detecting RDTs for P. falciparum cases and the prevalence of pfhrp2/3 deletions among symptomatic patients seeking malaria diagnosis at selected health facilities in southern Ethiopia. METHODS: A multi-health facilities-based cross-sectional study was conducted on self-presenting febrile patients seeking treatment in southern Ethiopia from July to September 2022. A purposive sampling strategy was used to enroll patients with microscopically confirmed P. falciparum infections. A capillary blood sample was obtained to prepare a blood film for microscopy and a RDT using the SD Bioline™ Malaria Pf/Pv Test. Dried blood spot samples were collected for further molecular analysis. DNA was extracted using gene aid kits and amplification was performed using nested PCR assay. Exon 2 of hrp2 and hrp3, which are the main protein-coding regions, was used to confirm its deletion. The diagnostic performance of RDT was evaluated using PCR as the gold standard test for P. falciparum infections. RESULTS: Of 279 P. falciparum PCR-confirmed samples, 249 (89.2%) had successful msp-2 amplification, which was then genotyped for hrp2/3 gene deletions. The study revealed that pfhrp2/3 deletions were common in all health centres, and it was estimated that 144 patients (57.8%) across all health facilities had pfhrp2/3 deletions, leading to false-negative PfHRP2 RDT results. Deletions spanning exon 2 of hrp2, exon 2 of hrp3, and double deletions (hrp2/3) accounted for 68 (27.3%), 76 (30.5%), and 33 (13.2%) of cases, respectively. The study findings revealed the prevalence of P. falciparum parasites lacking a single pfhrp2-/3-gene and that both genes varied across the study sites. This study also showed that the sensitivity of the SD Bioline PfHRP2-RDT test was 76.5% when PCR was used as the reference test. CONCLUSION: This study confirmed the existence of widespread pfhrp2/3- gene deletions, and their magnitude exceeded the WHO-recommended threshold (> 5%). False-negative RDT results resulting from deletions in Pfhrp2/3- affect a country's attempts at malaria control and elimination. Therefore, the adoption of non-HRP2-based RDTs as an alternative measure is required to avoid the consequences associated with the continued use of HRP-2-based RDTs, in the study area in particular and in Ethiopia in general.

Pleiotropic Nanostructures Built from l-Histidine Show Biologically Relevant Multicatalytic Activities.

The essential amino acid histidine plays a central role in the manifestation of several metabolic processes, including protein synthesis, enzyme-catalysis, and key biomolecular interactions. However, excess accumulation of histidine causes histidinemia, which shows brain-related medical complications, and the molecular mechanism of such histidine-linked complications is largely unknown. Here, we show that histidine undergoes a self-assembly process, leading to the formation of amyloid-like cytotoxic and catalytically active nanofibers. The kinetics of histidine self-assembly was favored in the presence of Mg(II) and Co(II) ions. Molecular dynamics data showed that preferential noncovalent interactions dominated by H-bonds between histidine molecules facilitate the formation of histidine nanofibers. The histidine nanofibers induced amyloid cross-seeding reactions in several proteins and peptides including pathogenic Aß1-42 and brain extract components. Further, the histidine nanofibers exhibited oxidase activity and enhanced the oxidation of neurotransmitters. Cell-based studies confirmed the cellular internalization of histidine nanofibers in SH-SY5Y cells and subsequent cytotoxic effects through necrosis and apoptosis-mediated cell death. Since several complications including behavioral abnormality, developmental delay, and neurological disabilities are directly linked to abnormal accumulation of histidine, our findings provide a foundational understanding of the mechanism of histidine-related complications. Further, the ability of histidine nanofibers to catalyze amyloid seeding and oxidation reactions is equally important for both biological and materials science research.

Dual-functional porphyrinic zirconium-based metal-organic framework for the fluorescent sensing of histidine enantiomers and Hg2.

A novel fluorescence sensor based on a porphyrinic zirconium-based metal-organic framework, L-cysteine-modified PCN-222 (L-Cys/PCN-222), was developed to selectively recognize histidine enantiomers and sensitively detect Hg2+. The dual-functional sensor was successfully prepared via the solvent-assisted ligand incorporation method and characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), 1H nuclear magnetic resonance (1H NMR) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD), X-ray photoelectron spectroscopy (XPS), and nitrogen adsorption-desorption analyses. L-Cys/PCN-222 not only showed a higher quenching response for L-histidine than that for D-histidine with a fast fluorescent response rate of <40 s but also exhibited low detection limits for L- and D-histidine (2.48 µmol L-1 and 3.85 µmol L-1, respectively). Moreover, L-Cys/PCN-222 was employed as a fluorescent and visual sensor for the highly sensitive detection of Hg2+ in the linear range of 10-500 µmol L-1, and the detection limit was calculated to be 2.79 µmol L-1 in surface water. The specific and selective recognition of chiral compounds and metal ions by our probe make it suitable for real field applications.

Histidine tag modified magnetic beads for analysis of arsenic binding proteins.

BACKGROUND: Many proteins with thiol groups can bind with trivalent arsenic which are termed as arsenic binding proteins, thus change their physiological functions. Therefore, it is vital to analyze the arsenic binding proteins in cells. The Pull-Down strategy based on biotinylated phenylarsenic acid (Bio-PAO(III)) probes is an effective way for analysis of arsenic binding proteins. In this strategy, streptavidin magnetic beads (SA-MBs) was applied to capture the arsenic binding proteins conjugating with Bio-PAO(III) probe. However, strong interaction between SA and biotin makes the elution of arsenic binding proteins not easy. RESULTS: We developed a novel affinity separation strategy to address the challenge of eluting arsenic binding proteins, a key issue with the existing Bio-PAO(III) Pull-Down method. By employing magnetic beads modified with Nα-Bis(carboxymethyl)-l-lysine (NTA-Lys), polyhistidine-tag (His6-Tag), and SA (MB-NTA(Ni)-His6-SA), we established a more efficient purification process. This innovative approach enables selective capture of arsenic binding proteins in HepG2 cells labeled by Bio-PAO(III) probes, facilitating gentle digestion by trypsin for precise identification through capillary high performance liquid chromatography (Cap HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). What is more, the magnetic beads can be regenerated by using imidazole as the eluent, and the obtained MB-NTA(Ni) can be reloaded with His6-SA for next use. Our method successfully identified 41 arsenic binding proteins, including those involved in cytoskeletal structure, heat shock response, transcriptional regulation, DNA damage repair, redox state regulation, mitochondrial dehydrogenase function, and protein synthesis and structure. SIGNIFICANCE: This work contributes to a more comprehensive understanding of the toxic mechanisms of arsenic, potentially providing valuable insights for the prevention or treatment of arsenic-related diseases.

Small angle X-ray scattering analysis of thermophilic cytochrome P450 CYP119 and the effects of the N-terminal histidine tag.

Combining size exclusion chromatography-small angle X-ray scattering (SEC-SAXS) and molecular dynamics (MD) analysis is a promising approach to investigate protein behavior in solution, particularly for understanding conformational changes due to substrate binding in cytochrome P450s (CYPs). This study investigates conformational changes in CYP119, a thermophilic CYP from Sulfolobus acidocaldarius that exhibits structural flexibility similar to mammalian CYPs. Although the crystal structure of ligand-free (open state) and ligand-bound (closed state) forms of CYP119 is known, the overall structure of the enzyme in solution has not been explored until now. It was found that theoretical scattering profiles from the crystal structures of CYP119 did not align with the SAXS data, but conformers from MD simulations, particularly starting from the open state (46 % of all frames), agreed well. Interestingly, a small percentage of closed-state conformers also fit the data (9 %), suggesting ligand-free CYP119 samples ligand-bound conformations. Ab initio SAXS models for N-His tagged CYP119 revealed a tail-like unfolded structure impacting protein flexibility, which was confirmed by in silico modeling. SEC-SAXS analysis of N-His CYP119 indicated pentameric structures in addition to monomers in solution, affecting the stability and activity of the enzyme. This study adds insights into the conformational dynamics of CYP119 in solution.

D-histidine combated biofilm formation and enhanced the effect of amikacin against Pseudomonas aeruginosa in vitro.

Pseudomonas aeruginosa is an opportunistic gram-negative pathogenic microorganism that poses a significant challenge in clinical treatment. Antibiotics exhibit limited efficacy against mature biofilm, culminating in an increase in the number of antibiotic-resistant strains. Therefore, novel strategies are essential to enhance the effectiveness of antibiotics against Pseudomonas aeruginosa biofilms. D-histidine has been previously identified as a prospective anti-biofilm agent. However, limited attention has been directed towards its impact on Pseudomonas aeruginosa. Therefore, this study was undertaken to explore the effect of D-histidine on Pseudomonas aeruginosa in vitro. Our results demonstrated that D-histidine downregulated the mRNA expression of virulence and quorum sensing (QS)-associated genes in Pseudomonas aeruginosa PAO1 without affecting bacterial growth. Swarming and swimming motility tests revealed that D-histidine significantly reduced the motility and pathogenicity of PAO1. Moreover, crystal violet staining and confocal laser scanning microscopy demonstrated that D-histidine inhibited biofilm formation and triggered the disassembly of mature biofilms. Notably, D-histidine increased the susceptibility of PAO1 to amikacin compared to that in the amikacin-alone group. These findings underscore the efficacy of D-histidine in combating Pseudomonas aeruginosa by reducing biofilm formation and increasing biofilm disassembly. Moreover, the combination of amikacin and D-histidine induced a synergistic effect against Pseudomonas aeruginosa biofilms, suggesting the potential utility of D-histidine as a preventive strategy against biofilm-associated infections caused by Pseudomonas aeruginosa.

Improving the Performance of Selective Solid-State Nanopore Sensing Using a Polyhistidine-Tagged Monovalent Streptavidin.

Solid-state (SS-) nanopore sensing has gained tremendous attention in recent years, but it has been constrained by its intrinsic lack of selectivity. To address this, we previously established a novel SS-nanopore assay that produces translocation signals only when a target biotinylated nucleic acid fragment binds to monovalent streptavidin (MS), a protein variant with a single high-affinity biotin-binding domain. While this approach has enabled selective quantification of diverse nucleic acid biomarkers, sensitivity enhancements are needed to improve the detection of low-abundance translational targets. Because the translocation dynamics that determine assay efficacy are largely governed by constituent charge characteristics, we here incorporate a polyhistidine-tagged MS (hMS) to alter the component detectability. We investigate the effects of buffer pH, salt concentration, and SS-nanopore diameter on the performance with the alternate reagent, achieve significant improvements in measurement sensitivity and selectivity, and expand the range of device dimensions viable for the assay. We used this improvement to detect as little as 1 nM miRNA spiked into human plasma. Overall, our findings improve the potential for broader applications of SS-nanopores in the quantitative analyses of molecular biomarkers.

Metabolomic response to acute resistance exercise in healthy older adults by 1H-NMR.

BACKGROUND: The favorable health-promoting adaptations to exercise result from cumulative responses to individual bouts of physical activity. Older adults often exhibit anabolic resistance; a phenomenon whereby the anabolic responses to exercise and nutrition are attenuated in skeletal muscle. The mechanisms contributing to age-related anabolic resistance are emerging, but our understanding of how chronological age influences responsiveness to exercise is incomplete. The objective was to determine the effects of healthy aging on peripheral blood metabolomic response to a single bout of resistance exercise and whether any metabolites in circulation are predictive of anabolic response in skeletal muscle. METHODS: Thirty young (20-35 years) and 49 older (65-85 years) men and women were studied in a cross-sectional manner. Participants completed a single bout of resistance exercise consisting of eight sets of 10 repetitions of unilateral knee extension at 70% of one-repetition maximum. Blood samples were collected before exercise, immediately post exercise, and 30-, 90-, and 180-minutes into recovery. Proton nuclear magnetic resonance spectroscopy was used to profile circulating metabolites at all timepoints. Serial muscle biopsies were collected for measuring muscle protein synthesis rates. RESULTS: Our analysis revealed that one bout of resistance exercise elicits significant changes in 26 of 33 measured plasma metabolites, reflecting alterations in several biological processes. Furthermore, 12 metabolites demonstrated significant interactions between exercise and age, including organic acids, amino acids, ketones, and keto-acids, which exhibited distinct responses to exercise in young and older adults. Pre-exercise histidine and sarcosine were negatively associated with muscle protein synthesis, as was the pre/post-exercise fold change in plasma histidine. CONCLUSIONS: This study demonstrates that while many exercise-responsive metabolites change similarly in young and older adults, several demonstrate age-dependent changes even in the absence of evidence of sarcopenia or frailty. TRIAL REGISTRATION: Clinical trial registry: ClinicalTrials.gov NCT03350906.

pH Dependence of HSF1 trimerization is shaped by intramolecular interactions.

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.

SP-CHAP, an endolysin with enhanced activity against biofilm pneumococci and nasopharyngeal colonization.

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn, designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections. IMPORTANCE: Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae-targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.

The impact of orphan histidine kinases and phosphotransfer proteins on the regulation of clostridial sporulation initiation.

Sporulation is an important feature of the clostridial life cycle, facilitating survival of these bacteria in harsh environments, contributing to disease transmission for pathogenic species, and sharing common early steps that are also involved in regulating industrially important solvent production by some non-pathogenic species. Initial genomics studies suggested that Clostridia lack the classical phosphorelay that phosphorylates Spo0A and initiates sporulation in Bacillus, leading to the hypothesis that sporulation in Clostridia universally begins when Spo0A is phosphorylated by orphan histidine kinases (OHKs). However, components of the classical Bacillus phosphorelay were recently identified in some Clostridia. Similar Bacillus phosphorelay components have not yet been found in the pathogenic Clostridia or the solventogenic Clostridia of industrial importance. For some of those Clostridia lacking a classical phosphorelay, the involvement of OHKs in sporulation initiation has received support from genetic studies demonstrating the involvement of several apparent OHKs in their sporulation. In addition, several clostridial OHKs directly phosphorylate Spo0A in vitro. Interestingly, there is considerable protein domain diversity among the sporulation-associated OHKs in Clostridia. Further adding to the emergent complexity of sporulation initiation in Clostridia, several candidate OHK phosphotransfer proteins that were OHK candidates were shown to function as phosphatases that reduce sporulation in some Clostridia. The mounting evidence indicates that no single pathway explains sporulation initiation in all Clostridia and supports the need for further study to fully understand the unexpected and biologically fascinating mechanistic diversity of this important process among these medically and industrially important bacteria.

NME3 is a gatekeeper for DRP1-dependent mitophagy in hypoxia.

NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.

A Redox Mediator-Free Highly Selective and Sensitive Electrochemical Aptasensor for Patulin Mycotoxin Detection in Apple Juice Using Ni-NiO Pseudocapacitive Nanomaterials.

Pseudocapacitive nanomaterials have recently gained significant attention in electrochemical biosensors due to their rapid response, long cycle life, high surface area, biomolecule compatibility, and superior energy storage capabilities. In our study, we introduce the potential of using Ni-NiO nanofilm's pseudocapacitive traits as transducer signals in electrochemical aptasensors. Capitalizing on the innate affinity between histidine and nickel, we immobilized histidine-tagged streptavidin (HTS) onto Ni-NiO-modified electrodes. Additionally, we employed a biolayer interferometry-based SELEX to generate biotinylated patulin aptamers. These aptamers, when placed on Ni-NiO-HTS surfaces, make a suitable biosensing platform for rapid patulin mycotoxin detection in apple juice using electrochemical amperometry in microseconds. The novelty lies in optimizing pseudocapacitive nanomaterials structurally and electrochemically, offering the potential for redox mediator-free electrochemical aptasensors. Proof-of-concept is conducted by applying this surface for the ultrasensitive detection of a model analyte, patulin mycotoxin. The aptamer-functionalized bioelectrode showed an excellent linear response (10-106 fg/mL) and an impressive detection limit (1.65 fg/mL, +3σ of blank signal). Furthermore, reproducibility tests yielded a low relative standard deviation of 0.51%, indicating the good performance of the developed biosensor. Real sample analysis in freshly prepared apple juice revealed no significant difference (P < 0.05) in current intensity between spiked and real samples. The sensor interface maintained excellent stability for up to 2 weeks (signal retention 96.45%). The excellent selectivity, stability, and sensitivity of the electrochemical aptasensor exemplify the potential for using nickel-based pseudocapacitive nanomaterials for a wide variety of electrochemical sensing applications.

Histidine re-sensitizes pediatric acute lymphoblastic leukemia to 6-mercaptopurine through tetrahydrofolate consumption and SIRT5-mediated desuccinylation.

Despite progressive improvements in the survival rate of pediatric B-cell lineage acute lymphoblastic leukemia (B-ALL), chemoresistance-induced disease progression and recurrence still occur with poor prognosis, thus highlighting the urgent need to eradicate drug resistance in B-ALL. The 6-mercaptopurine (6-MP) is the backbone of ALL combination chemotherapy, and resistance to it is crucially related to relapse. The present study couples chemoresistance in pediatric B-ALL with histidine metabolism deficiency. Evidence was provided that histidine supplementation significantly shifts the 6-MP dose-response in 6-MP-resistant B-ALL. It is revealed that increased tetrahydrofolate consumption via histidine catabolism partially explains the re-sensitization ability of histidine. More importantly, this work provides fresh insights into that desuccinylation mediated by SIRT5 is an indispensable and synergistic requirement for histidine combination therapy against 6-MP resistance, which is undisclosed previously and demonstrates a rational strategy to ameliorate chemoresistance and protect pediatric patients with B-ALL from disease progression or relapse.

A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme.

Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.