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.

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.

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.

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.

Mutation of conserved histidine residues of dengue virus envelope protein impairs viral like particle maturation and secretion.

Dengue virus (DENV) envelope protein plays crucial role in virus entry and maturation of virus during infection. Maturation of DENV occurs in the trans Golgi network at slightly acidic pH which is close to pKa of histidine. When exposed to the acidic environment of the late secretory pathway, dengue virus particles go through a significant conformational change, whereby interactions of structural proteins envelope (E) and prM proteins are reorganised and enable furin protease to cleave prM resulting in mature virus. In order to study the role of histidine of E protein in DENV maturation, we mutated 7 conserved histidine residues of envelope protein and assessed the percent of budding using viral like particle (VLP) system. Histidine mutants; H144A, H244A, H261A and H282A severely disrupted VLP formation without any significant change in expression in cell and its oligomerization ability. Treatment with acidotropic amine reversed the defect for all 4 mutants suggesting that these histidines could be involved in maturation and release. Over expression of capsid protein slightly enhanced VLP release of H244A and H261A. Similarly, furin over expression increased VLP release of these mutants. Co-immunoprecipitation studies revealed that prM and E interaction is lost for H244A, H261A and H282A mutants at acidic pH but not at neutral pH indicating that they could be involved in histidine switch during maturation at acidic pH. Detailed analysis of the mutants could provide novel insights on the interplay of envelop protein during maturation and aid in target for drug development.

Time-resolved RNA-seq analysis to unravel the in vivo competence induction by Streptococcus pneumoniae during pneumonia-derived sepsis.

Competence development in Streptococcus pneumoniae (pneumococcus) is tightly intertwined with virulence. In addition to genes encoding genetic transformation machinery, the competence regulon also regulates the expression of allolytic factors, bacteriocins, and cytotoxins. Pneumococcal competence system has been extensively interrogated in vitro where the short transient competent state upregulates the expression of three distinct phases of "early," "late," and "delayed" genes. Recently, we have demonstrated that the pneumococcal competent state develops naturally in mouse models of pneumonia-derived sepsis. To unravel the underlying adaptive mechanisms driving the development of the competent state, we conducted a time-resolved transcriptomic analysis guided by the spatiotemporal live in vivo imaging system of competence induction during pneumonia-derived sepsis. Mouse lungs infected by the serotype 2 strain D39 expressing a competent state-specific reporter gene (D39-ssbB-luc) were subjected to RNA sequencing guided by monitoring the competence development at 0, 12, 24, and, at the moribund state, >40 hours post-infection (hpi). Transcriptomic analysis revealed that the competence-specific gene expression patterns in vivo were distinct from those under in vitro conditions. There was significant upregulation of early, late, and some delayed phase competence-specific genes as early as 12 hpi, suggesting that the pneumococcal competence regulon is important for adaptation to the lung environment. Additionally, members of the histidine triad (pht) gene family were sharply upregulated at 12 hpi followed by a steep decline throughout the rest of the infection cycle, suggesting that Pht proteins participate in the early adaptation to the lung environment. Further analysis revealed that Pht proteins execute a metal ion-dependent regulatory role in competence induction.IMPORTANCEThe induction of pneumococcal competence for genetic transformation has been extensively studied in vitro but poorly understood during lung infection. We utilized a combination of live imaging and RNA sequencing to monitor the development of a competent state during acute pneumonia. Upregulation of competence-specific genes was observed as early as 12 hour post-infection, suggesting that the pneumococcal competence regulon plays an important role in adapting pneumococcus to the stressful lung environment. Among others, we report novel finding that the pneumococcal histidine triad (pht) family of genes participates in the adaptation to the lung environment and regulates pneumococcal competence induction.

Histidine transport is essential for the growth of Staphylococcus aureus at low pH.

Staphylococcus aureus is an opportunistic pathogen capable of causing many different human diseases. During colonization and infection, S. aureus will encounter a range of hostile environments, including acidic conditions such as those found on the skin and within macrophages. However, little is known about the mechanisms that S. aureus uses to detect and respond to low pH. Here, we employed a transposon sequencing approach to determine on a genome-wide level the genes required or detrimental for growth at low pH. We identified 31 genes that were essential for the growth of S. aureus at pH 4.5 and confirmed the importance of many of them through follow up experiments using mutant strains inactivated for individual genes. Most of the genes identified code for proteins with functions in cell wall assembly and maintenance. These data suggest that the cell wall has a more important role than previously appreciated in promoting bacterial survival when under acid stress. We also identified several novel processes previously not linked to the acid stress response in S. aureus. These include aerobic respiration and histidine transport, the latter by showing that one of the most important genes, SAUSA300_0846, codes for a previously uncharacterized histidine transporter. We further show that under acid stress, the expression of the histidine transporter gene is increased in WT S. aureus. In a S. aureus SAUSA300_0846 mutant strain expression of the histidine biosynthesis genes is induced under acid stress conditions allowing the bacteria to maintain cytosolic histidine levels. This strain is, however, unable to maintain its cytosolic pH to the same extent as a WT strain, revealing an important function specifically for histidine transport in the acid stress response of S. aureus.

Rapid diagnostic tests for malaria diagnosis in Cameroon: impact of histidine rich protein 2/3 deletions and lactate dehydrogenase gene polymorphism.

Malaria rapid diagnostic tests (mRDT) play a vital role in malaria control in endemic areas. In this study, histidine-rich protein (hrp) and lactate dehydrogenase (ldh) genes were genotyped in Plasmodium falciparum (Pf) and Plasmodium ovale (Po) spp. isolates. Deletions in P. falciparum hrp2/3 (pfhrp2/3) proteins and single nucleotide polymorphisms (SNPs) were analyzed. Twenty-four samples were analyzed for pfhrp2/3 gene deletions and 25 for SNPs in ldh gene (18 Pf and 7 Po spp.). Deletions in pfhrp2/3 genes were observed in 1.9% malaria positive isolates. The pfldh gene sequences showed one SNP at codon 272 (D272N) in 22.2% of samples while in Po spp., sequences were 100% similar to P. ovale curtisi but when compared to P. ovale wallikeri reference sequence, SNPs at positions 143 (P143S), 168 (K168N), 204 (V204I) were found. Findings suggest low prevalence in pfhrp2/3 genes and highlight the circulation of P. ovale curtisi in the studies areas.

Lack of Evidence for the Role of the p.(Ser96Ala) Polymorphism in Histidine-Rich Calcium Binding Protein as a Secondary Hit in Cardiomyopathies.

Inherited forms of arrhythmogenic and dilated cardiomyopathy (ACM and DCM) are characterized by variable disease expression and age-related penetrance. Calcium (Ca2+) is crucially important for proper cardiac function, and dysregulation of Ca2+ homeostasis seems to underly cardiomyopathy etiology. A polymorphism, c.286T>G p.(Ser96Ala), in the gene encoding the histidine-rich Ca2+ binding (HRC) protein, relevant for sarcoplasmic reticulum Ca2+ cycling, has previously been associated with a marked increased risk of life-threatening arrhythmias among idiopathic DCM patients. Following this finding, we investigated whether p.(Ser96Ala) affects major cardiac disease manifestations in carriers of the phospholamban (PLN) c.40_42delAGA; p.(Arg14del) pathogenic variant (cohort 1); patients diagnosed with, or predisposed to, ACM (cohort 2); and DCM patients (cohort 3). We found that the allele frequency of the p.(Ser96Ala) polymorphism was similar across the general European-American population (control cohort, 40.3-42.2%) and the different cardiomyopathy cohorts (cohorts 1-3, 40.9-43.9%). Furthermore, the p.(Ser96Ala) polymorphism was not associated with life-threatening arrhythmias or heart failure-related events across various patient cohorts. We therefore conclude that there is a lack of evidence supporting the important role of the HRC p.(Ser96Ala) polymorphism as a modifier in cardiomyopathy, refuting previous findings. Further research is required to identify bona fide genomic predictors for the stratification of cardiomyopathy patients and their risk for life-threatening outcomes.

The transcriptional factor Clr-5 is involved in cellulose degradation through regulation of amino acid metabolism in Neurospora crassa.

BACKGROUND: Filamentous fungi are efficient degraders of plant biomass and the primary producers of commercial cellulolytic enzymes. While the transcriptional regulation mechanisms of cellulases have been continuously explored in lignocellulolytic fungi, the induction of cellulase production remains a complex multifactorial system, with several aspects still largely elusive. RESULTS: In this study, we identified a Zn2Cys6 transcription factor, designated as Clr-5, which regulates the expression of cellulase genes by influencing amino acid metabolism in Neurospora crassa during growth on cellulose. The deletion of clr-5 caused a significant decrease in secreted protein and cellulolytic enzyme activity of N. crassa, which was partially alleviated by supplementing with yeast extract. Transcriptomic profiling revealed downregulation of not only the genes encoding main cellulases but also those related to nitrogen metabolism after disruption of Clr-5 under Avicel condition. Clr-5 played a crucial role in the utilization of multiple amino acids, especially leucine and histidine. When using leucine or histidine as the sole nitrogen source, the Δclr-5 mutant showed significant growth defects on both glucose and Avicel media. Comparative transcriptomic analysis revealed that the transcript levels of most genes encoding carbohydrate-active enzymes and those involved in the catabolism and uptake of histidine, branched-chain amino acids, and aromatic amino acids, were remarkably reduced in strain Δclr-5, compared with the wild-type N. crassa when grown in Avicel medium with leucine or histidine as the sole nitrogen source. These findings underscore the important role of amino acid metabolism in the regulation of cellulase production in N. crassa. Furthermore, the function of Clr-5 in regulating cellulose degradation is conserved among ascomycete fungi. CONCLUSIONS: These findings regarding the novel transcription factor Clr-5 enhance our comprehension of the regulatory connections between amino acid metabolism and cellulase production, offering fresh prospects for the development of fungal cell factories dedicated to cellulolytic enzyme production in bio-refineries.

Assessing the histidine-rich protein 2/3 gene deletion in Plasmodium falciparum isolates from Burkina Faso.

BACKGROUND: Dual hrp2/hrp3 genes deletions in P. falciparum isolates are increasingly reported in malaria-endemic countries and can produce false negative RDT results leading to inadequate case management. Data on the frequency of hrp2/hrp3 deleted parasites are rarely available and it has become necessary to investigate the issue in Burkina Faso. METHODS: Plasmodium falciparum-positive dried blood spots were collected during a cross-sectional household survey of the malaria asymptomatic children from Orodara, Gaoua, and Banfora. Amplicons from the target regions (exon 2 of hrp2 and hrp3 genes) were generated using multiplexed nested PCR and sequenced according to Illumina's MiSeq protocol. RESULTS: A total of 251 microscopically positive parasite isolates were sequenced to detect hrp2 and hrp3 gene deletions. The proportion of RDTs negative cases among microscopy positive slides was 12.7% (32/251). The highest prevalence of negative RDTs was found in Orodara 14.3% (5/35), followed by Gaoua 13.1%(24/183), and Banfora 9.1% (3/33). The study found that 95.6% of the parasite isolates were wild type hrp2/ hrp3 while 4.4% (11/251) had a single hrp2 deletion. Of the 11 hrp2 deletion samples, 2 samples were RDT negative (mean parasitaemia was 83 parasites/ µL) while 9 samples were RDT positive with a mean parasitaemia of 520 parasites /µL (CI95%: 192-1239). The highest frequency of hrp2 deletion 4/35 (11.4%) was found in Orodara, while it was similar in the other two sites (< 3.5%). No single deletion of the hrp3 or dual deletion hrp2/3 gene was detected in this study. CONCLUSION: These results demonstrate that P. falciparum isolates lacking hrp2 genes are present in 4.4% of samples obtained from the asymptomatic children population in three sites in Burkina Faso. These parasites are circulating and causing malaria, but they are also still detectable by HRP2-based RTDs due to the presence of the intact pfhrp3 gene.

Inoculum cell count influences separation efficiency and variance in Ames plate incorporation and Ames RAMOS test.

The Ames test is one of the most applied tools in mutagenicity testing of chemicals ever since its introduction by Ames et al. in the 1970s. Its principle is based on histidine auxotrophic bacteria that regain prototrophy through reverse mutations. In the presence of a mutagen, more reverse mutations occur that become visible as increased bacterial growth on medium without histidine. Many miniaturized formats of the Ames test have emerged to enable the testing of environmental water samples, increase experimental throughput, and lower the required amounts of test substances. However, most of these formats still rely on endpoint determinations. In contrast, the recently introduced Ames RAMOS test determines mutagenicity through online monitoring of the oxygen transfer rate. In this study, the oxygen transfer rate of Salmonella typhimurium TA100 during the Ames plate incorporation test was monitored and compared to the Ames RAMOS test to prove its validity further. Furthermore, the Ames RAMOS test in 96-well scale is newly introduced. For both the Ames plate incorporation and the Ames RAMOS test, the influence of the inoculum cell count on the negative control was highlighted: A lower inoculum cell count led to a higher coefficient of variation. However, a lower inoculum cell count also led to a higher separation efficiency in the Ames RAMOS test and, thus, to better detection of a mutagenic substance at lower concentrations.

Histidine N1-position-specific methyltransferase CARNMT1 targets C3H zinc finger proteins and modulates RNA metabolism.

Histidine (His) residues are methylated in various proteins, but their roles and regulation mechanisms remain unknown. Here, we show that carnosine N-methyltransferase 1 (CARNMT1), a known His methyltransferase of dipeptide carnosine (ßAla-His), is a major His N1-position-specific methyltransferase. We found that 52 His sites in 20 proteins underwent CARNMT1-mediated methylation. The consensus methylation site for CARNMT1 was identified as Cx(F/Y)xH, a C3H zinc finger (C3H ZF) motif. CARNMT1-deficient and catalytically inactive mutant mice showed embryonic lethality. Among the CARNMT1 target C3H ZF proteins, RNA degradation mediated by Roquin and tristetraprolin (TTP) was affected by CARNMT1 and its enzymatic activity. Furthermore, the recognition of the 3' splice site of the CARNMT1 target C3H ZF protein U2AF1 was perturbed, and pre-mRNA alternative splicing (AS) was affected by CARNMT1 deficiency. These findings indicate that CARNMT1-mediated protein His methylation, which is essential for embryogenesis, plays roles in diverse aspects of RNA metabolism by targeting C3H ZF-type RNA-binding proteins and modulating their functions, including pre-mRNA AS and mRNA degradation regulation.

A seven-transmembrane methyltransferase catalysing N-terminal histidine methylation of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in industrial biotechnology. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identify the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663. Targeted proteomics confirm that NHMT was solely responsible for His1 methylation of LPMOs. NHMT is predicted to encode a unique seven-transmembrane segment anchoring a soluble methyltransferase domain. Co-localization studies show endoplasmic reticulum residence of NHMT and co-expression in the industrial production yeast Komagataella phaffii with LPMOs results in His1 methylation of the LPMOs. This demonstrates the biotechnological potential of recombinant production of proteins and peptides harbouring this specific post-translational modification.

Causal associations of histidine and 12 site-specific cancers: a bidirectional Mendelian randomization study.

An increasing number of studies indicate that cancer patients' histidine (HIS) circulating levels have changed. However, the causality between HIS and cancer is still not well established. Thus, to ascertain the causal link between HIS and cancers, we performed a bidirectional Mendelian randomization (MR) analysis. Summary-level data are derived from publicly available genome-wide association studies (GWAS). The causal effects were mainly estimated using the inverse-variance weighted method (IVW). The weighted-median (WM) method and MR-Egger regression were conducted as sensitivity analyses. In the forward-MR, we found malignant neoplasm of respiratory system and intrathoracic organs (OR: 1.020; 95% CI: 1.006-1.035; pIVW = 0.007) genetically associated with circulating HIS. And there was no significant genetic correlation between HIS and another 11 site-specific cancers using IVW method. In the reversed-MR, we did not observe the causal relationship between HIS and 12 site-specific cancers. Our findings help clarify that HIS, as a biomarker for malignant neoplasms of respiratory system and intrathoracic organs, is causal rather than a secondary biomarker of the cancerous progression. The mechanism between histidine and cancer progression deserves further investigation.

An arginine-to-histidine mutation in flavanone-3-hydroxylase results in pink strawberry fruits.

Fruit color is a very important external commodity factor for consumers. Compared to the most typical red octoploid strawberry (Fragaria × ananassa), the pink strawberry often sells for a more expensive price and has a higher economic benefit due to its outstanding color. However, few studies have examined the molecular basis of pink-colored strawberry fruit. Through an EMS mutagenesis of woodland strawberry (Fragaria vesca), we identified a mutant with pink fruits and green petioles. Bulked-segregant analysis sequencing analysis and gene function verification confirmed that the responsible mutation resides in a gene encoding flavanone-3-hydroxylase (F3H) in the anthocyanin synthesis pathway. This nonsynonymous mutation results in an arginine-to-histidine change at position 130 of F3H. Molecular docking experiments showed that the arginine-to-histidine mutation results in a reduction of intermolecular force-hydrogen bonding between the F3H protein and its substrates. Enzymatic experiments showed a greatly reduced ability of the mutated F3H protein to catalyze the conversion of the substrates and hence a blockage of the anthocyanin synthesis pathway. The discovery of a key residue in the F3H gene controlling anthocyanin synthesis provides a clear target of modification for the molecular breeding of strawberry varieties with pink-colored fruits, which may be of great commercial value.

Mimicking the last step of gene elongation: hints from the bacterial hisF gene.

Gene elongation consists in an in-tandem duplication of a gene and divergence and fusion of the two copies, resulting in a gene constituted by two divergent paralogous modules. Many present-day proteins show internal repeats of amino acid sequences, generated by gene elongation events; however, gene elongation is still a poorly studied evolutionary molecular mechanism. The most documented case is that of the histidine biosynthetic genes hisA and hisF, which derive from the gene elongation of an ancestral gene half the size of the extant ones. The aim of this work was to experimentally simulate the possible last step of the gene elongation event occurred during hisF gene evolution under selective pressure conditions. Azospirillum brasilense hisF gene, carrying a single nucleotide mutation that generates a stop codon between the two halves of the gene, was used to transform the histidine-auxotrophic Escherichia coli strain FB182 (hisF892). The transformed strain was subjected to selective pressure (i.e., low concentration/absence of histidine in the growth medium) and the obtained mutants were characterized. The restoration of prototrophy was strongly dependent on the time of incubation and on the strength of the selective pressure. The mutations involved the introduced stop codon with a single base substitution and none of the mutants restored the wild-type codon. Possible correlations between the different mutations and i) E. coli codon usage, ii) three-dimensional structures of the mutated HisF proteins, and iii) growth ability of the mutants were investigated. On the contrary, when the experiment was repeated by mutating a more conserved codon, only a synonymous substitution was obtained. Thus, experiments performed in this study allowed to mimic a possible gene elongation event occurred during the evolution of hisF gene, evidencing the ability of bacterial cells to modify their genome in short times under selective conditions.

Molecular Engineering of E. coli Bacterioferritin: A Versatile Nanodimensional Protein Cage.

Currently, intense interest is focused on the discovery and application of new multisubunit cage proteins and spherical virus capsids to the fields of bionanotechnology, drug delivery, and diagnostic imaging as their internal cavities can serve as hosts for fluorophores or bioactive molecular cargo. Bacterioferritin is unusual in the ferritin protein superfamily of iron-storage cage proteins in that it contains twelve heme cofactors and is homomeric. The goal of the present study is to expand the capabilities of ferritins by developing new approaches to molecular cargo encapsulation employing bacterioferritin. Two strategies were explored to control the encapsulation of a diverse range of molecular guests compared to random entrapment, a predominant strategy employed in this area. The first was the inclusion of histidine-tag peptide fusion sequences within the internal cavity of bacterioferritin. This approach allowed for the successful and controlled encapsulation of a fluorescent dye, a protein (fluorescently labeled streptavidin), or a 5 nm gold nanoparticle. The second strategy, termed the heme-dependent cassette strategy, involved the substitution of the native heme with heme analogs attached to (i) fluorescent dyes or (ii) nickel-nitrilotriacetate (NTA) groups (which allowed for controllable encapsulation of a histidine-tagged green fluorescent protein). An in silico docking approach identified several small molecules able to replace the heme and capable of controlling the quaternary structure of the protein. A transglutaminase-based chemoenzymatic approach to surface modification of this cage protein was also accomplished, allowing for future nanoparticle targeting. This research presents novel strategies to control a diverse set of molecular encapsulations and adds a further level of sophistication to internal protein cavity engineering.

Biochemical and mechanistic analysis of the cleavage of branched DNA by human ANKLE1.

ANKLE1 is a nuclease that provides a final opportunity to process unresolved junctions in DNA that would otherwise create chromosomal linkages blocking cell division. It is a GIY-YIG nuclease. We have expressed an active domain of human ANKLE1 containing the GIY-YIG nuclease domain in bacteria, that is monomeric in solution and when bound to a DNA Y-junction, and unilaterally cleaves a cruciform junction. Using an AlphaFold model of the enzyme we identify the key active residues, and show that mutation of each leads to impairment of activity. There are two components in the catalytic mechanism. Cleavage rate is pH dependent, corresponding to a pKa of 6.9, suggesting an involvement of the conserved histidine in proton transfer. The reaction rate depends on the nature of the divalent cation, likely bound by glutamate and asparagine side chains, and is log-linear with the metal ion pKa. We propose that the reaction is subject to general acid-base catalysis, using a combination of tyrosine and histidine acting as general base and water directly coordinated to the metal ion as general acid. The reaction is temperature dependent; activation energy Ea = 37 kcal mol-1, suggesting that cleavage is coupled to opening of DNA in the transition state.

SLC38A10 Deficiency in Mice Affects Plasma Levels of Threonine and Histidine in Males but Not in Females: A Preliminary Characterization Study of SLC38A10-/- Mice.

Solute carriers belong to the biggest group of transporters in the human genome, but more knowledge is needed to fully understand their function and possible role as therapeutic targets. SLC38A10, a poorly characterized solute carrier, is preliminary characterized here. By using a knockout mouse model, we studied the biological effects of SLC38A10 deficiency in vivo. We performed a transcriptomic analysis of the whole brain and found seven differentially expressed genes in SLC38A10-deficient mice (Gm48159, Nr4a1, Tuba1c, Lrrc56, mt-Tp, Hbb-bt and Snord116/9). By measuring amino acids in plasma, we found lower levels of threonine and histidine in knockout males, whereas no amino acid levels were affected in females, suggesting that SLC38A10-/- might affect sexes differently. Using RT-qPCR, we investigated the effect of SLC38A10 deficiency on mRNA expression of other SLC38 members, Mtor and Rps6kb1 in the brain, liver, lung, muscle, and kidney, but no differences were found. Relative telomere length measurement was also taken, as a marker for cellular age, but no differences were found between the genotypes. We conclude that SLC38A10 might be important for keeping amino acid homeostasis in plasma, at least in males, but no major effects were seen on transcriptomic expression or telomere length in the whole brain.