Leishmania donovani 6-phosphogluconolactonase: Crucial for growth and host infection?

The hexose monophosphate shunt is a crucial pathway in a variety of microorganisms owing to its vital metabolic products and intermediates such as NADPH, ribose 5-phosphate etc. The enzyme 6-phosphogluconolactonase catalyses the second step of this pathway, converting 6-phosphogluconolactone to 6-phosphogluconic acid. This enzyme has been known to have a significant involvement in growth, pathogenesis and sensitivity to oxidative stress in bacterial and protozoal pathogens. However, the functional role of kinetoplastid Leishmania donovani 6-phospohogluconolactonase (Ld6PGL) remains unexplored. L. donovani is the second largest parasitic killer and causative organism of life threatening visceral leishmaniasis. To understand its possible functional role in the parasite, the alleles of Ld6PGL were sequentially knocked-out followed by gene complementation. The Ld6PGL mutant cell lines showed decrease in transcriptional and translational expression as well as in the enzyme activity. In case of Ld6PGL null mutants, approximately 2-fold reduction was observed in growth. The null mutants also showed ∼38% decrease in infectivity, which recovered to ∼15% on complementation. Scanning electron microscopy showed a marked decrease in flagellar length in the knockout parasites. When treated with the standard drug miltefosine, the mutant strains had no significant change in the drug sensitivity. However, the Ld6PGL mutants were more susceptible to oxidative stress. Our findings suggest that 6PGL is required for parasite growth and infection, but it is not essential.

Pseudokinases repurpose flexibility signatures associated with the protein kinase fold for noncatalytic roles.

The bilobal protein kinase-like fold in pseudokinases lack one or more catalytic residues, conserved in canonical protein kinases, and are considered enzymatically deficient. Tertiary structures of pseudokinases reveal that their loops topologically equivalent to activation segments of kinases adopt contracted configurations, which is typically extended in active conformation of kinases. Herein, anisotropic network model based normal mode analysis (NMA) was conducted on 51 active conformation structures of protein kinases and 26 crystal structures of pseudokinases. Our observations indicate that although backbone fluctuation profiles are similar for individual kinase-pseudokinase families, low intensity mean square fluctuations in pseudo-activation segment and other sub-structures impart rigidity to pseudokinases. Analyses of collective motions from functional modes reveal that pseudokinases, compared to active kinases, undergo distinct conformational transitions using the same structural fold. All-atom NMA of protein kinase-pseudokinase pairs from each family, sharing high amino acid sequence identities, yielded distinct community clusters, partitioned by residues exhibiting highly correlated fluctuations. It appears that atomic fluctuations from equivalent activation segments guide community membership and network topologies for respective kinase and pseudokinase. Our findings indicate that such adaptations in backbone and side-chain fluctuations render pseudokinases competent for catalysis-independent roles.

Genome-wide and structural analyses of pseudokinases encoded in the genome of Arabidopsis thaliana provide functional insights.

Protein Kinase-Like Non-Kinases (PKLNKs), commonly known as "pseudokinases", are homologous to eukaryotic Ser/Thr/Tyr protein kinases (PKs) but lack the crucial aspartate residue in the catalytic loop, indispensable for phosphotransferase activity. Therefore, they are predicted to be "catalytically inactive" enzyme homologs. Analysis of protein-kinase like sequences from Arabidopsis thaliana led to the identification of more than 120 pseudokinases lacking catalytic aspartate, majority of which are closely related to the plant-specific receptor-like kinase family. These pseudokinases engage in different biological processes, enabled by their diverse domain architectures and specific subcellular localizations. Structural comparison of pseudokinases with active and inactive conformations of canonical PKs, belonging to both plant and animal origin, revealed unique structural differences. The currently available crystal structures of pseudokinases show that the loop topologically equivalent to activation segment of PKs adopts a distinct-folded conformation, packing against the pseudoenzyme core, in contrast to the extended and inhibitory geometries observed for active and inactive states, respectively, of catalytic PKs. Salt-bridge between ATP-binding Lys and DFG-Asp as well as hydrophobic interactions between the conserved nonpolar residue C-terminal to the equivalent DFG motif and nonpolar residues in C-helix mediate such a conformation in pseudokinases. This results in enhanced solvent accessibility of the pseudocatalytic loop in pseudokinases that can possibly serve as an interacting surface while associating with other proteins. Specifically, our analysis identified several residues that may be involved in pseudokinase regulation and hints at the repurposing of pseudocatalytic residues to achieve mechanistic control over noncatalytic functions of pseudoenzymes.

Gatekeeper-Activation Loop Cross-Talk Determines Distinct Autoactivation States of Symbiosis Receptor Kinase.

Plant receptor-like kinases (RLKs) have a Tyr in the "gatekeeper" position adjacent to the hinge region. The gatekeeper is phosphorylated in several RLKs, including symbiosis receptor kinase (SYMRK), but the significance of this remains unknown. Gatekeeper substitution did not inactivate Arachis hypogaea SYMRK but affected autophosphorylation at selected sites. Herein, we show that nonphosphorylatable gatekeepers (Y670F and Y670A) restrict SYMRK to be a Ser/Thr kinase with a basal level of phosphorylation (∼5 P/polypeptide, termed state I) whereas phosphorylatable gatekeepers (Y670 and Y670T) allowed SYMRK to be dual specific (Ser/Thr/Tyr) with a maximal level of phosphorylation (∼10 P/polypeptide, termed state II). State II SYMRKs were phosphorylated on gatekeeper residues, and the phosphocode in their activation segment was distinct from state I. The kcat/ Km for substrate phosphorylation was ∼10-fold higher for state II, though for autophosphorylation, it was comparable with those of state I SYMRKs. To identify other determinants of state I features, we mutagenized all nine sites where phosphorylation was affected by nonphosphorylatable gatekeepers (Y670F and Y670A). Only two such mutants, S754A and S757A, located on the activation loop failed to phosphorylate gatekeeper Tyr and restricted SYMRK in state I. Double mutants like Y670F/S754A retained the features of state I, but Y670F/S757A was significantly inactivated, indicating a nonphosphorylatable gatekeeper can bypass phosphorylation of S754 but not S757 in the activation segment. We propose a working model for the hierarchical phosphorylation of SYMRK on gatekeeper and activation segments for its pS757-mediated activation as a Ser/Thr kinase in selfie mode (autophosphorylation) to a pS754/pY670-mediated activation as a Ser/Thr/Tyr kinase that functions in dual mode (both autophosphorylation and substrate phosphorylation).

p53 gain-of-function mutations increase Cdc7-dependent replication initiation.

Cancer-associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7-dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7-dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53-driven cancer phenotypes in vitro and in vivo Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7-dependent replication initiation is a hallmark of p53 gain-of-function mutations.

Chemo-profiling of anthocyanins and fatty acids present in pomegranate aril and seed grown in Indian condition and its bioaccessibility study.

The goal of the present study was to investigate the bioactive molecules (anthocyanins and fatty acids) present in the aril of pomegranate. Major anthocyanins present in the aril of pomegranate were identified by HRMS as delphinidin 3,5-diglucoside, cyanidin 3,5-diglucoside, pelargonidin 3,5-diglucoside, cyanidin 3-glucoside and delphinidin 3-glucoside. In-vitro study revealed that bioaccessibility of anthocyanin in duodenal condition was varied between 7.3 and 9.7%. Encapsulation enhances the bioaccessibility of both the phenolics to some extent in gastric as well as duodenal condition. Seed oil contains significant amount of unsaturated fatty acids especially ω-5 fatty acids. Geometrical isomers of ω-5 fatty acids were also identified by GC-MS. The spray dried anthocyanin formulation has potential for food application.

Gatekeeper Tyrosine Phosphorylation of SYMRK Is Essential for Synchronizing the Epidermal and Cortical Responses in Root Nodule Symbiosis.

Symbiosis receptor kinase (SYMRK) is indispensable for activation of root nodule symbiosis (RNS) at both epidermal and cortical levels and is functionally conserved in legumes. Previously, we reported SYMRK to be phosphorylated on "gatekeeper" Tyr both in vitro as well as in planta. Since gatekeeper phosphorylation was not necessary for activity, the significance remained elusive. Herein, we show that substituting gatekeeper with nonphosphorylatable residues like Phe or Ala significantly affected autophosphorylation on selected targets on activation segment/αEF and ß3-αC loop of SYMRK. In addition, the same gatekeeper mutants failed to restore proper symbiotic features in a symrk null mutant where rhizobial invasion of the epidermis and nodule organogenesis was unaffected but rhizobia remain restricted to the epidermis in infection threads migrating parallel to the longitudinal axis of the root, resulting in extensive infection patches at the nodule apex. Thus, gatekeeper phosphorylation is critical for synchronizing epidermal/cortical responses in RNS.

Pyridoxal kinase gene deletion leads to impaired growth, deranged redox metabolism and cell cycle arrest in Leishmania donovani.

Pyridoxal kinase (PdxK) is a vitamin B6 salvage pathway enzyme which produces pyridoxal phosphate. We have investigated the impact of PdxK deletion in Leishmania donovani on parasite survivability, infectivity and cellular metabolism. LdPdxK mutants were generated by gene replacement strategy. All mutants showed significant reduction in growth in comparison to wild type. For PdxK mediated biochemical perturbations, only heterozygous mutants and complementation mutants were used as the growth of null mutants were compromised. Heterozygous mutant showed reduction invitro infectivity and higher cytosolic and mitochondrial ROS levels. Glutathione levels decreased significantly in heterozygous mutant indicating its involvement in cellular oxidative metabolism. Pyridoxal kinase gene deletion resulted in reduced ATP levels in parasites and arrest at G0/G1 phase of cell cycle. All these perturbations were rescued by PdxK gene complementation. This is the first report to confirm that LdPdxK plays an indispensable role in cell survival, pathogenicity, redox metabolism and cell cycle progression of L. donovani parasites. These results provide substantial evidence supporting PdxK as a therapeutic target for the development of specific antileishmanial drug candidates.

Clotrimazole causes membrane depolarization and induces sub G0 cell cycle arrest in Leishmania donovani.

Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC50) of 35.75 ± 1.06 µM, 12.75 ± 0.35 µM and 73 ± 1.41 µM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G0 phase suggesting apoptotic mode of cell death.

Mechanistic and structural insights into vitamin B2 metabolizing enzyme riboflavin kinase from Leishmania donovani.

Leishmania donovani relies on specific vitamins and cofactors crucial for its survival and pathogenesis. Tailoring therapies to disrupt these pathways offers a promising strategy for the treatment of Visceral Leishmaniasis. Current treatment regimens are limited due to drug resistance and high costs. The dependency of Leishmania parasites on Vitamin B2 and its metabolic products is not known. In this study, we have biochemically and biophysically characterized a Vitamin B2 metabolism enzyme, riboflavin kinase from L. donovani (LdRFK) which converts riboflavin (vitamin B2) into flavin mononucleotide (FMN). Sequence comparison with human counterpart reflects 31.58 % identity only, thus opening up the possibility of exploring it as drug target. The rfk gene was cloned, expressed and the recombinant protein was purified. Kinetic parameters of LdRFK were evaluated with riboflavin and ATP as substrates which showed differential binding affinity when compared with the human RFK enzyme. Thermal and denaturant stability of the enzyme was evaluated. The rfk gene was overexpressed in the parasites and its role in growth and cell cycle was evaluated. In the absence of crystal structure, homology modelling and molecular dynamic simulation studies were performed to predict LdRFK structure. The data shows differences in substrate binding between human and parasite enzyme. This raises the possibility of exploring LdRFK for specific designing of antileishmanial molecules. Gene disruption studies can further validate its candidature as antileishmanial target.

Visceral leishmaniasis in the COVID-19 pandemic era.

Visceral l eishmaniasis (VL), also known as kala-azar, had once been targeted for elimination in 2020, which now has been shifted to 2030. The year 2020 was also the year in which the world was gripped by the coronavirus disease 2019 (COVID-19) pandemic. This review sheds light on the impact of COVID-19 on VL elimination programmes and the increasing incidences of COVID-19/VL cases. Lockdowns were imposed worldwide that led to the suspension of surveys, active case finding and mass drug administration, which are important activities to manage neglected tropical diseases. Healthcare machinery was redirected to control the pandemic and acute resource shortages were seen. Budget cuts from funding agencies and donors also came as a severe blow. Priority changes for manufacturers of drugs and diagnostic kits have also exacerbated the situation. Cases where patients were co-infected with VL and COVID-19 were reported across various settings and in people of various age groups, posing unprecedented challenges in diagnosis and treatment. Concerted efforts from all stakeholders are required to understand and deal with the impact that this pandemic has had on VL.

Insights into Leishmania donovani potassium channel family and their biological functions.

Leishmania donovani is the causative organism for visceral leishmaniasis. Although this parasite was discovered over a century ago, nothing is known about role of potassium channels in L. donovani. Potassium channels are known for their crucial roles in cellular functions in other organisms. Recently the presence of a calcium-activated potassium channel in L. donovani was reported which prompted us to look for other proteins which could be potassium channels and to investigate their possible physiological roles. Twenty sequences were identified in L. donovani genome and subjected to estimation of physio-chemical properties, motif analysis, localization prediction and transmembrane domain analysis. Structural predictions were also done. The channels were majorly α-helical and predominantly localized in cell membrane and lysosomes. The signature selectivity filter of potassium channel was present in all the sequences. In addition to the conventional potassium channel activity, they were associated with gene ontology terms for mitotic cell cycle, cell death, modulation by virus of host process, cell motility etc. The entire study indicates the presence of potassium channel families in L. donovani which may have involvement in several cellular pathways. Further investigations on these putative potassium channels are needed to elucidate their roles in Leishmania. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03692-y.

Poly(3-hydroxybuyrate) production from industrial hemp waste pretreated with a chemical-free hydrothermal process.

In this study, a mild two-stage hydrothermal pretreatment was employed to optimally valorize industrial hemp (Cannabis sativa sp.) fibrous waste into sugars for Poly(3-hydroxybuyrate) (PHB) production using recombinant Escherichia coli LSBJ. Biomass was pretreated using hot water at 160, 180, and 200 °C for 5 and 10 min (15% solids), followed by disk refining. The sugar yields during enzymatic hydrolysis were found to improve with increasing temperature and the yields for hot water-disk refining pretreatment (HWDM) were higher compared to only hot water pretreatment at all conditions. The maximum glucose (56 g/L) and cellulose conversion (92%) were achieved for HWDM at 200 °C for 10 min. The hydrolysate obtained was fermented at a sugar concentration of 20 g/L. The PHB inclusion and concentration of 48% and 1.8 g/L, respectively, were similar to those from pure sugars. A pH-controlled fermentation resulted in a near bi-fold increase in PHB yield (3.46 g/L).

Molecular explorations of the Leishmania donovani 6-phosphogluconolactonase enzyme, a key player in the pentose phosphate pathway.

The enzymes of the pentose phosphate pathway are vital to survival in kinetoplastids. The second step of the pentose phosphate pathway involves hydrolytic cleavage of 6-phosphogluconolactone to 6-phosphogluconic acid by 6- phosphogluconolactonase (6PGL). In the present study, Leishmania donovani 6PGL (Ld6PGL) was cloned and overexpressed in bacterial expression system. Comparative sequence analysis revealed the conserved sequence motifs, functionally and structurally important residues in 6PGL family. In silico amino acid substitution study and interacting partners of 6PGL were predicted. The Ld6PGL enzyme was found to be active in the assay and in the parasites. Specificity was confirmed by Western blot analysis. The ∼30 kDa protein was found to be a dimer in MALDI, glutaraldehyde crosslinking and size exclusion chromatography studies. Kinetic analysis and structural stability studies of Ld6PGL were performed with denaturants and at varied temperature. Computational 3D Structural modelling of Ld6PGL elucidates that it has a similar α/ß hydrolase fold structural topology as in other members of 6PGL family. The three loops are found in extended form when the structure is compared with the human 6PGL (Hs6PGL). Further, enzyme substrate binding mode and its mechanism were investigated using the molecular docking and molecular simulation studies. Interesting dynamics action of substrate 6-phosphogluconolactone was observed into active site during MD simulation. Interesting differences were observed between host and parasite enzyme which pointed towards its potential to be explored as an antileishmanial drug target. This study forms the basis for further analysis of the role of Ld6PGL in combating oxidative stress in Leishmania.

A liquid chromatographic method for determination of acetamiprid and buprofezin residues and their dissipation kinetics in paddy matrices and soil.

The present study was conducted to investigate the residue status of two insecticides (acetamiprid and buprofezin) and their dissipation kinetics in three matrices viz. paddy grain, straw, and soil. The extraction procedure for residues of these two insecticides was executed using acetonitrile solvent. The analytical method was validated, which showed good linearity with the limit of quantification (LOQ) value of 0.01 and 0.02 mg kg-1 for acetamiprid and buprofezin, respectively. The recovery range was 79.67-98.33 % concerning all the matrices in both the insecticides. Acetamiprid (20% SP) and Buprofezin (25% SC) were applied separately in the paddy field in two doses: single dose (recommended dose) and double dose along with untreated control throughout the experiment. Residue analysis of these two insecticides in paddy (grain and straw) and soil was accomplished employing high-performance liquid chromatography (HPLC) with ultraviolet (UV) detector and confirmed by ultra-performance liquid chromatography (UPLC) coupled with mass spectrometry (UPLC-MS/MS). The dissipation data showed that acetamiprid exhibited higher dissipation in comparison with buprofezin. However, their persistence was found slightly higher in soil. The dissipation dynamics in the rice and soil were discussed with biological half-lives of both the insecticides. Consumer risk assessment study was also made considering its fate to the consumers.

A Core-Linker-Polyamine (CLP) Strategy Enables Rapid Discovery of Antileishmanial Aminoalkylquinolinecarboxamides That Target Oxidative Stress Mechanism.

A Core-Linker-Polyamine (CLP) strategy has been exploited to develop new antileishmanial agents. It involves the linker-based assembly of alkyl-polyamine side chain as a potential pharmacophore motif with a privileged heterocyclic motif, 4-arylquinoline. A series of aminoalkyl 4-arylquinoline-2-carboxamides and their analogs were synthesized and tested against L. donovani promastigotes. Among all synthesized derivatives, 10 compounds showed significant antipromastigote activities with more efficacy (IC50 : 4.75-8 µM) than an antileishmanial oral drug Miltefosine (IC50 : 8.9±1.55 µM). Most active aminoalkyl-quinoline-carboxamides 9 a and 9 b, displayed negligible cytotoxicity towards human monocytic (THP-1) macrophages. The compounds show antileishmanial activity by generating mitochondrial superoxide radicals. However, they did not show interference with trypanothione reductase, a redox enzyme of Leishmania. Significant change in the morphology of the L. donovani promastigote by the compounds was observed. The Structure-activity relationship analysis suggest the pharmacophoric importance of alkylpolyamine and carboxamide motifs. In silico evaluation indicated that the investigated active molecules 9 a and 9 b possess important drug-likeness, physicochemical and pharmacokinetic-relevant properties.

Recent trends and developments on integrated biochemical conversion process for valorization of dairy waste to value added bioproducts: A review.

In this review article, discuss the many ways utilized by the dairy sector to treat pollutants, emphasizing their influence on the quality and efficiency with which contamination is removed. It focuses on biotechnology possibilities for valorizing dairy waste in particular. The findings revealed that dairy waste may be treated using physicochemical, biological, and biotechnological techniques. Notably, this article highlighted the possibility of dairy waste being used as a feedstock not only for the generation of biogas, bioethanol, biohydrogen, microbial fuel cells, lactic acid, and fumaric acid via microbial technology but also for the production of biooil and biochar by pyrolysis. In addition, this article critically evaluates the many treatment techniques available for recovering energy and materials from dairy waste, their combinations, and implementation prospects. Valorization of dairy waste streams presents an opportunity to extend the dairy industry's presence in the fermented functional beverage sector.

Identification of a novel calcium activated potassium channel from Leishmania donovani and in silico predictions of its antigenic features.

Visceral Leishmaniasis is a major neglected tropical disease with increasing incidences of drug resistance. This has led to the search for a suitable drug target for chemotherapeutic intervention. Potassium channels are a family of membrane proteins which play a vital role in homeostasis and any perturbation in them alters cell survival which makes them an attractive target. To characterize a calcium-activated potassium channel from Leishmania donovani (LdKCa), a putative ion-channel like protein which showed sequence similarity with other Trypanosoma cruzi putative potassium channels was selected. It was cloned and expressed with a histidine tag. MALDI confirmed that it is a potassium channel. Homology model of LdKCa was generated by I-TASSER. It is a transmembrane protein localized in the plasma membrane as predicted by DeepLoc tool. In silico analyses of the protein showed that it is a small conductance calcium activated potassium channel. Point mutation in conserved signature domain 'TXGYGD' affects the protein function as predicted by heat map analysis. The LdKCa model predicted amino acids S207, T208 and M236 as ligand-binding sites. The sequence HSLRGRSARVIQLAWRLRKARKVGPHAPSLKQKVYTLVLSWLLT was the highest scoring B-cell epitope. The highest scoring T-cell epitope was RLYSVIVYL. This study may provide new insights into antigenicity features of leishmanial calcium-activated potassium channels.