The Integration of Proteogenomics and Ribosome Profiling Circumvents Key Limitations to Increase the Coverage and Confidence of Novel Microproteins.

There has been a dramatic increase in the identification of non-conical translation and a significant expansion of the protein-coding genome and proteome. Among the strategies used to identify novel small ORFs (smORFs), Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple sites in the genome are computationally removed since they cannot unambiguously be assigned to a specific genomic location, or to a specific transcript in the case of multiple isoforms. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of ambiguous and multi-mapping alignments, such that smORFs that reside in these regions cannot be identified by Ribo-Seq. Here, we show that the inclusion of proteogenomics to create a Ribosome Profiling and Proteogenomics Pipeline (RP3) bypasses this limitation to identify a group of microprotein-encoding smORFs that are missed by current Ribo-Seq pipelines. Moreover, we show that the microproteins identified by RP3 have different sequence compositions from the ones identified by Ribo-Seq-only pipelines, which can affect proteomics identification. In aggregate, the development of RP3 maximizes the detection and confidence of protein-encoding smORFs and microproteins.

Profiling mouse brown and white adipocytes to identify metabolically relevant small ORFs and functional microproteins.

Microproteins (MPs) are a potentially rich source of uncharacterized metabolic regulators. Here, we use ribosome profiling (Ribo-seq) to curate 3,877 unannotated MP-encoding small ORFs (smORFs) in primary brown, white, and beige mouse adipocytes. Of these, we validated 85 MPs by proteomics, including 33 circulating MPs in mouse plasma. Analyses of MP-encoding mRNAs under different physiological conditions (high-fat diet) revealed that numerous MPs are regulated in adipose tissue in vivo and are co-expressed with established metabolic genes. Furthermore, Ribo-seq provided evidence for the translation of Gm8773, which encodes a secreted MP that is homologous to human and chicken FAM237B. Gm8773 is highly expressed in the arcuate nucleus of the hypothalamus, and intracerebroventricular administration of recombinant mFAM237B showed orexigenic activity in obese mice. Together, these data highlight the value of this adipocyte MP database in identifying MPs with roles in fundamental metabolic and physiological processes such as feeding.

One enzyme, many faces: urease is also canatoxin.

Ureases catalyze the hydrolysis of urea into carbamate and ammonia. Well-conserved proteins, most plant ureases are hexamers of a single chain subunit, like the most abundant isoform of the jack bean (Canavalia ensiformis) urease (JBU). Canatoxin (CNTX) was originally isolated from these seeds as a neurotoxic protein, and later characterized as an isoform of JBU with lower molecular mass and enzyme activity. Inactive CNTX oligomers form upon storage and stabilization of CNTX was achieved by treatment with low concentration of formaldehyde, avoiding its oligomerization. Here, nano-LC-MS/MS-based peptide analysis of CNTX revealed 804 amino acids identical to those of JBU's sequence (840 amino acids). De novo sequencing of CNTX revealed 15 different peptides containing substitution of amino acid residues, denoting CNTX as a product of a paralog gene of JBU. The MS/MS analysis of formaldehyde-treated CNTX showed that amino acid residues located at the trimer-trimer interface of JBU's hexamer were modified. The data confirmed that CNTX is an isoform of JBU and elucidated that stabilization by formaldehyde treatment occurs by modification of amino acids at the protein's surface that prevents the formation of the hexamer and of higher molecular mass inactive aggregates. HIGHLIGHTSCanatoxin (CNTX) is an isoform of jack bean urease (JBU, hexamer of 90 kDa chains)MS/MS sequencing of CNTX showed 804 amino acids identical in JBU (840 residues)Formaldehyde treatment of CNTX stabilizes its toxicity and avoids oligomerizationModified amino acid residues in CNTX are at the trimer-trimer interface of JBUCommunicated by Ramaswamy H. Sarma.

Bacterial Enoyl-Reductases: The Ever-Growing List of Fabs, Their Mechanisms and Inhibition.

Enoyl-ACP reductases (ENRs) are enzymes that catalyze the last step of the elongation cycle during fatty acid synthesis. In recent years, new bacterial ENR types were discovered, some of them with structures and mechanisms that differ from the canonical bacterial FabI enzymes. Here, we briefly review the diversity of structural and catalytic properties of the canonical FabI and the new FabK, FabV, FabL, and novel ENRs identified in a soil metagenome study. We also highlight recent efforts to use the newly discovered Fabs as targets for drug development and consider the complex evolutionary history of this diverse set of bacterial ENRs.

Evaluation of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS) as a Vulnerable Target in Mycobacterium tuberculosis.

Tuberculosis (TB) remains one of the leading causes of death due to a single pathogen. The emergence and proliferation of multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) represent compelling reasons to invest in the pursuit of new anti-TB agents. The shikimate pathway, responsible for chorismate biosynthesis, which is a precursor of important aromatic compounds, is required for Mycobacterium tuberculosis growth. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (MtbDAHPS) catalyzes the first step in the shikimate pathway and it is an attractive target for anti-tubercular agents. Here, we used a CRISPRi system to evaluate the DAHPS as a vulnerable target in M. tuberculosis. The silencing of aroG significantly reduces the M. tuberculosis growth in both rich medium and, especially, in infected murine macrophages. The supplementation with amino acids was only able to partially rescue the growth of bacilli, whereas the Aro supplement (aromix) was enough to sustain the bacterial growth at lower rates. This study shows that MtbDAHPS protein is vulnerable and, therefore, an attractive target to develop new anti-TB agents. In addition, the study contributes to a better understanding of the biosynthesis of aromatic compounds and the bacillus physiology. IMPORTANCE Determining the vulnerability of a potential target allows us to assess whether its partial inhibition will impact bacterial growth. Here, we evaluated the vulnerability of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) from M. tuberculosis by silencing the DAHPS-coding aroG gene in different contexts. These results could lead to the development of novel and potent anti-tubercular agents in the near future.

Short-chain fatty acid acetate triggers antiviral response mediated by RIG-I in cells from infants with respiratory syncytial virus bronchiolitis.

BACKGROUND: Gut microbiota-derived short-chain fatty-acid (SFCA) acetate protects mice against RSV A2 strain infection by increasing interferon-ß production and expression of interferon-stimulated genes (ISGs). However, the role of SFCA in RSV infection using strains isolated from patients is unknown. METHODS: We first used RSV clinical strains isolated from infants hospitalized with RSV bronchiolitis to investigate the effects of in vitro SCFA-acetate treatment of human pulmonary epithelial cells. We next examined whether SCFA-acetate treatment is beneficial in a mouse model of RSV infection using clinical isolates. We sought to investigate the relationship of gut microbiota and fecal acetate with disease severity among infants hospitalized with RSV bronchiolitis, and whether treating their respiratory epithelial cells with SCFA-acetate ex-vivo impacts viral load and ISG expression. We further treated epithelial cells from SARS-CoV-2 infected patients with SCFA-acetate. FINDINGS: In vitro pre-treatment of A549 cells with SCFA-acetate reduced RSV infection with clinical isolates and increased the expression of RIG-I and ISG15. Animals treated with SCFA-acetate intranasally recovered significantly faster, with reduction in the RSV clinical isolates viral load, and increased lung expression of IFNB1 and the RIG-I. Experiments in RIG-I knockout A549 cells demonstrated that the protection relies on RIG-I presence. Gut microbial profile was associated with bronchiolitis severity and with acetate in stool. Increased SCFA-acetate levels were associated with increasing oxygen saturation at admission, and shorter duration of fever. Ex-vivo treatment of patients' respiratory cells with SCFA-acetate reduced RSV load and increased expression of ISGs OAS1 and ISG15, and virus recognition receptors MAVS and RIG-I, but not IFNB1. These SCFA-acetate effects were not found on cells from SARS-CoV-2 infected patients. INTERPRETATION: SCFA-acetate reduces the severity of RSV infection and RSV viral load through modulation of RIG-I expression. FUNDING: FAPERGS (FAPERGS/MS/CNPq/SESRS no. 03/2017 - PPSUS 17/2551-0001380-8 and COVID-19 20/2551-0000258-6); CNPq 312504/2017-9; CAPES) - Finance Code 001.

Stem-loop formation drives RNA folding in mechanical unzipping experiments.

Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force-distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping-rezipping process, we introduce a barrier energy landscape of the stem-loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem-loops that kinetically trap the long-lived intermediates observed in the FDC. Stem-loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.

Effects of tafenoquine against active, dormant and resistant Mycobacterium tuberculosis.

Antimalarial drugs have been suggested as promising scaffolds with anti-tubercular activities. In this work, we demonstrated, for the first time, the effectiveness of tafenoquine against mycobacteria. Firstly, tafenoquine inhibited the growth of Mycobacterium smegmatis and Mycobacterium tuberculosis with lower MICs values as compared to other antimalarial drugs, such as mefloquine, chloroquine, and primaquine. Importantly, tafenoquine was active against three multi-drug resistant strains of M. tuberculosis with MIC values similar to pan-sensitive strains, suggesting that tafenoquine is capable of evading the major mechanisms of resistance found in drug-resistant clinical isolates of M. tuberculosis. Importantly, tafenoquine displayed a synergistic effect when combined with mefloquine. In addition, tafenoquine displayed an improved activity compared to the groups treated with both isoniazid and rifampicin in the six-week nutrient starved M. tuberculosis cultures. This finding suggests that further investigations of tafenoquine against dormant mycobacteria are worth pursuing. Moreover, different concentrations of tafenoquine ranging from 1.25 to 80 µM displayed different effects against M. tuberculosis, from moderate (reduction of a 1.8 log CFU/mL) to potent bactericidal (reduction of a 4.2 log CFU/mL) activities. Tafenoquine may represent a hit for further drug optimization and for future clinical development as a new anti-mycobacterial agent, especially in cases of resistant and/or dormant forms of tuberculosis.

Handling the Hurdles on the Way to Anti-tuberculosis Drug Development.

The global epidemic of tuberculosis (TB) imposes a sustained epidemiologic vigilance and investments in research by governments. Mycobacterium tuberculosis, the main causative agent of TB in human beings, is a very successful pathogen, being the main cause of death in the population among infectious agents. In 2018, ~10 million individuals were contaminated with this bacillus and became ill with TB, and about 1.2 million succumbed to the disease. Most of the success of the M. tuberculosis to linger in the population comes from its ability to persist in an asymptomatic latent state into the host and, in fact, the majority of the individuals are unaware of being contaminated. Even though TB is a treatable disease and is curable in most cases, the treatment is lengthy and laborious. In addition, the rise of resistance to first-line anti-TB drugs elicits a response from TB research groups to discover new chemical entities, preferably with novel mechanisms of action. The pathway to find a new TB drug, however, is arduous and has many barriers that are difficult to overcome. Fortunately, several approaches are available today to be pursued by scientists interested in anti-TB drug development, which goes from massively testing chemical compounds against mycobacteria, to discovering new molecular targets by genetic manipulation. This review presents some difficulties found along the TB drug development process and illustrates different approaches that might be used to try to identify new molecules or targets that are able to impair M. tuberculosis survival.

Mycobacterium tuberculosis Shikimate Pathway Enzymes as Targets for the Rational Design of Anti-Tuberculosis Drugs.

Roughly a third of the world's population is estimated to have latent Mycobacterium tuberculosis infection, being at risk of developing active tuberculosis (TB) during their lifetime. Given the inefficacy of prophylactic measures and the increase of drug-resistant M. tuberculosis strains, there is a clear and urgent need for the development of new and more efficient chemotherapeutic agents, with selective toxicity, to be implemented on patient treatment. The component enzymes of the shikimate pathway, which is essential in mycobacteria and absent in humans, stand as attractive and potential targets for the development of new drugs to treat TB. This review gives an update on published work on the enzymes of the shikimate pathway and some insight on what can be potentially explored towards selective drug development.

Nonclinical evaluation of IQG-607, an anti-tuberculosis candidate with potential use in combination drug therapy.

New effective compounds to treat tuberculosis are urgently needed. IQG-607 is an orally active anti-tuberculosis drug candidate, with promising preliminary safety profile and anti-mycobacterial activity in both in vitro and in vivo models of tuberculosis infection. Here, we evaluated the mutagenic and genotoxic effects of IQG-607, and its interactions with CYP450 isoforms. Moreover, we describe for the first time a combination study of IQG-607 in Mycobacterium tuberculosis-infected mice. Importantly, IQG-607 had additive effects when combined with the first-line anti-tuberculosis drugs rifampin and pyrazinamide in mice. IQG-607 presented weak to moderate inhibitory potential against CYP450 isoforms 3A4, 1A2, 2C9, 2C19, 2D6, and 2E1. The Salmonella mutagenicity test revealed that IQG-607 induced base pair substitution mutations in the strains TA100 and TA1535. However, in the presence of human metabolic S9 fraction, no mutagenic effect was detected in any strain. Additionally, IQG-607 did not increase micronucleus frequencies in mice, at any dose tested, 25, 100, or 250 mg/kg. The favorable activity in combination with first-line drugs and mild to moderate toxic events described in this study suggest that IQG-607 represents a candidate for clinical development.

Is IQG-607 a Potential Metallodrug or Metallopro-Drug With a Defined Molecular Target in Mycobacterium tuberculosis?

The emergence of strains of Mycobacterium tuberculosis resistant to isoniazid (INH) has underscored the need for the development of new anti-tuberculosis agents. INH is activated by the mycobacterial katG-encoded catalase-peroxidase, forming an acylpyridine fragment that is covalently attached to the C4 of NADH. This isonicotinyl-NAD adduct inhibits the activity of 2-trans-enoyl-ACP(CoA) reductase (InhA), which plays a role in mycolic acid biosynthesis. A metal-based INH analog, Na3[FeII(CN)5(INH)]·4H2O, IQG-607, was designed to have an electronic redistribution on INH moiety that would lead to an intramolecular electron transfer to bypass KatG activation. HPLC and EPR studies showed that the INH moiety can be oxidized by superoxide or peroxide yielding similar metabolites and isonicotinoyl radical only when associated to IQG-607, thereby supporting redox-mediated drug activation as a possible mechanism of action. However, IQG-607 was shown to inhibit the in vitro activity of both wild-type and INH-resistant mutant InhA enzymes in the absence of KatG activation. IQG-607 given by the oral route to M. tuberculosis-infected mice reduced lung lesions. Experiments using early and late controls of infection revealed a bactericidal activity for IQG-607. HPLC and voltammetric methods were developed to quantify IQG-607. Pharmacokinetic studies showed short half-life, high clearance, moderate volume of distribution, and low oral bioavailability, which was not altered by feeding. Safety and toxic effects of IQG-607 after acute and 90-day repeated oral administrations in both rats and minipigs showed occurrence of mild to moderate toxic events. Eight multidrug-resistant strains (MDR-TB) were resistant to IQG-607, suggesting an association between katG mutation and increasing MIC values. Whole genome sequencing of three spontaneous IQG-607-resistant strains harbored katG gene mutations. MIC measurements and macrophage infection experiments with a laboratorial strain showed that katG mutation is sufficient to confer resistance to IQG-607 and that the macrophage intracellular environment cannot trigger the self-activation mechanism. Reduced activity of IQG-607 against an M. tuberculosis strain overexpressing S94A InhA mutant protein suggested both the need for KatG activation and InhA as its target. Further efforts are suggested to be pursued toward attempting to translate IQG-607 into a chemotherapeutic agent to treat tuberculosis.

Synthesis and mechanistic investigation of iron(II) complexes of isoniazid and derivatives as a redox-mediated activation strategy for anti-tuberculosis therapy.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) represents a major threat to global health. Isoniazid (INH) is a prodrug used in the first-line treatment of tuberculosis. It undergoes oxidation by a catalase-peroxidase KatG, leading to generation of an isonicotinoyl radical that reacts with NAD(H) forming the INH-NADH adduct as the active metabolite. A redox-mediated activation of isoniazid using an iron metal complex was previously proposed as a strategy to overcome isoniazid resistance due to KatG mutations. Here, we have prepared a series of iron metal complexes with isoniazid and analogues, containing alkyl substituents at the hydrazide moiety, and also with pyrazinamide derivatives. These complexes were activated by H2O2 and studied by ESR and LC-MS. For the first time, the formation of the oxidized INH-NAD adduct from the pentacyano(isoniazid)ferrate(II) complex was detected by LC-MS, supporting a redox-mediated activation, for which a mechanistic proposition is reported. ESR data showed all alkylated hydrazides, in contrast to non-substituted hydrazides, only generated alkyl-based radicals. The structural modifications did not improve minimal inhibitory concentration (MIC) against MTB in comparison to isoniazid iron complex, providing support to isonicotinoyl radical formation as a requirement for activity. Nonetheless, the pyrazinoic acid hydrazide iron complex showed redox-mediated activation using H2O2 with generation of a pyrazinoyl radical intermediate and production of pyrazinoic acid, which is in fact the active metabolite of pyrazinamide prodrug. Thereby, this strategy can also unveil new opportunities for activation of this type of drug.

Revisiting Activation of and Mechanism of Resistance to Compound IQG-607 in Mycobacterium tuberculosis.

IQG-607 is a metal complex previously reported as a promising anti-tuberculosis (TB) drug against isoniazid (INH)-resistant strains of Mycobacterium tuberculosis Unexpectedly, we found that INH-resistant clinical isolates were resistant to IQG-607. Spontaneous mutants resistant to IQG-607 were subjected to whole-genome sequencing, and all sequenced colonies carried alterations in the katG gene. The katG(S315T) mutation was sufficient to confer resistance to IQG-607 in both MIC assays and inside macrophages. Moreover, overexpression of the InhA(S94A) protein caused IQG-607's resistance.

Preclinical pharmacokinetic profiling of IQG-607, a potential oral metallodrug to treat tuberculosis.

IQG-607 is an analog of isoniazid with anti-tuberculosis activity. This work describes the development and validation of an HPLC method to quantify pentacyano(isoniazid)ferrate(II) compound (IQG-607) and the pharmacokinetic studies of this compound in mice. The method showed linearity in the 0.5-50µg/mL concentration range (r=0.9992). Intra- and inter-day precision was <5%, and the recovery ranged from 92.07 to 107.68%. IQG-607 was stable in plasma for at least 30days at -80°C and, after plasma processing, for 4h in the auto-sampler maintained on ice (recovery >85%). The applicability of the method for pharmacokinetic studies was determined after intravenous (i.v.) and oral (fasted and fed conditions) administration to mice. IQG-607 levels in plasma were quantified at time points for up to 2.5h. A short half-life (t1/2) (1.14h), a high clearance (CL) (3.89L/h/kg), a moderate volume of distribution at steady state (Vdss) of 1.22L/kg, were observed after i.v. (50mg/kg) administration. Similar results were obtained for oral administration (250mg/kg) under fasted and fed conditions. The oral bioavailability (F), approximately 4%, was not altered by feeding. Plasma protein binding was 88.87±0.9%. The results described here provide novel insights into a pivotal criterion to warrant further efforts to be pursued towards attempts to translate this chemical compound into a chemotherapeutic agent to treat TB.

Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only.

The cellular milieu is a complex and crowded aqueous solution. Macromolecular crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects, if any, of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (K m , k cat , and k cat /K m ) of Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase (InhA). Negligible effects on InhA activity were observed for ficoll 70, ficoll 400 and dextran 70. A complex effect was observed for PEG 6000. Glucose and sucrose showed, respectively, no effect on InhA activity and decreased k cat /K m for NADH and k cat for 2-trans-dodecenoyl-CoA. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution. The effects of the crowding agents on the energy (E a and E η ), enthalpy (∆H # ), entropy (∆S # ), and Gibbs free energy (∆G # ) of activation were determined. The ∆G # values for all crowding agents were similar to buffer, suggesting that excluded volume effects did not facilitate stable activated ES # complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that "soft" interactions play a role in crowding effects. The results on InhA do not unequivocally meet the criteria for crowding effect due to exclude volume only.

Pulse Proteolysis and Precipitation for Target Identification.

In recent years, phenotypic screening has assumed a leading role in drug discovery efforts. However, development of new drugs from bioactive compounds obtained in screening campaigns requires identification of the cellular targets responsible for their biological activities. A new energetics-based method for target identification is presented: pulse proteolysis and precipitation for target identification (PePTID). In this method, proteins incubated with or without a ligand and submitted to a brief proteolytic pulse are directly analyzed and compared using a label-free semiquantitative mass spectrometry strategy, dispensing the SDS-PAGE readout and greatly improving the throughput. As a proof-of-concept, we applied the PePTID method to identify ATP-binding proteins in Mycobacterium smegmatis, a model system for Mycobacterium tuberculosis, the etiological agent of tuberculosis.

Structure-based virtual screening as a tool for the identification of novel inhibitors against Mycobacterium tuberculosis 3-dehydroquinate dehydratase.

3-Dehydroquinate dehydratase (DHQase), the third enzyme of the shikimate pathway, catalyzes the reversible reaction of 3-dehydroquinate into 3-dehydroshikimate. The aim of the present study was to identify new drug-like molecules as inhibitors for Mycobacterium tuberculosis DHQase employing structure-based pharmacophore modeling technique using an in house database consisting of about 2500 small molecules. Further the pharmacophore models were validated using enrichment calculations, and finally three models were employed for high-throughput virtual screening and docking to identify novel small molecules as DHQase inhibitors. Five compounds were identified, out of which, one molecule (Lead 1) showed 58% inhibition at 50µ M concentration in the Mtb DHQase assay. Chemical derivatives of the Lead 1 when tested evolved top two hits with IC50s of 17.1 and 31.5 µM as well as MIC values of 25 and 6.25 µg/mL respectively and no cytotoxicity up to 100 µM concentration.

Production of recombinant human annexin V by fed-batch cultivation.

BACKGROUND: Annexin V, a 35.8 kDa intracellular protein, is a Ca⁺²-dependent phospholipid binding protein with high affinity to phosphatidylserine (PS), which is a well-known hallmark of apoptosis. Annexin V is a sensitive probe for PS exposure upon the cell membrane, and used for detection of apoptotic cells both in vivo and in vitro. Large-scale production of recombinant human annexin V is worth optimization, because of its wide use in nuclear medicine, radiolabeled with (99m)Tc, for the evaluation of cancer chemotherapy treatments, and its use in identification of apoptotic cells in histologic studies. Here we describe the high-yield production of a tag-free version of human annexin V recombinant protein by linear fed-batch cultivation in a bioreactor. RESULTS: We cloned the human ANXA5 coding sequence into the pET-30a (+) expression vector and expressed rhANXA5 in batch and fed-batch cultures. Using E. coli BL21 (DE3) in a semi-defined medium at 37°C, pH 7 in fed-batch cultures, we obtained a 45-fold increase in biomass production, respective to shaker cultivations. We developed a single-step protocol for rhANXA5 purification using a strong anion-exchange column (MonoQ HR16/10). Using these procedures, we obtained 28.5 mg of homogeneous, nontagged and biologically functional human annexin V recombinant protein from 3 g wet weight of bacterial cells from bioreactor cultures. The identity and molecular mass of rhANXA5 was confirmed by mass spectrometry. Moreover, the purified rhANXA5 protein was functionally evaluated in a FITC-annexin V binding experiment and the results demonstrated that rhANXA5 detected apoptotic cells similarly to a commercial kit. CONCLUSIONS: We describe a new fed-batch method to produce recombinant human annexin V in large scale, which may expand the commercial utilities for rhANXAV to applications such as in vivo imaging studies.

Targeting the histidine pathway in Mycobacterium tuberculosis.

Worldwide, tuberculosis is the leading cause of morbidity and mortality due to a single bacterial pathogen, Mycobacterium tuberculosis (Mtb). The increasing prevalence of this disease, the emergence of multi-, extensively, and totally drug-resistant strains, complicated by co-infection with the human immunodeficiency virus, and the length of tuberculosis chemotherapy have led to an urgent and continued need for the development of new and more effective antitubercular drugs. Within this context, the L-histidine biosynthetic pathway, which converts 5-phosphoribosyl 1-pyrophosphate to L-histidine in ten enzymatic steps, has been reported as a promising target of antimicrobial agents. This pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants but is absent in mammals, making these enzymes highly attractive targets for the drug design of new antimycobacterial compounds with selective toxicity. Moreover, the biosynthesis of L-histidine has been described as essential for Mtb growth in vitro. Accordingly, a comprehensive overview of Mycobacterium tuberculosis histidine pathway enzymes as attractive targets for the development of new antimycobacterial agents is provided, mainly summarizing the previously reported inhibition data for Mtb or orthologous proteins.