Nitric oxide blocks cellular heme insertion into a broad range of heme proteins

Although the insertion of heme into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process are not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3-h period and then investigated how nitric oxide (NO) released from a chemical donor (NOC-18) might influence heme (protoporphyrin IX) insertion into seven targets that present a range of protein structures, heme ligation states, and functions (three NO synthases, two cytochrome P450's, catalase, and hemoglobin). NO blocked cellular heme insertion into all seven apo-protein targets. The inhibition occurred at relatively low (nM/min) fluxes of NO, was reversible, and did not involve changes in intracellular heme levels, activation of guanylate cyclase, or inhibition of mitochondrial ATP production. These aspects and the range of protein targets suggest that NO can act as a global inhibitor of heme insertion, possibly by inhibiting a common step in the process.     

Thioredoxin and HSP90α modulate ASK1–JNK1/2 signaling in stressed hepatocytes of Mugil cephalus

Induction of antioxidant proteins like thioredoxin (Trx) and heat shock protein 90α (HSP90α) is a crucial step in the cellular response to oxidative stress. Here, we report the impact of environmental stress on Trx and HSP90α expressions in freshly isolated hepatocytes of Mugil cephalus living in either a contaminated (Test; Ennore) or uncontaminated (Control; Kovalam) estuary. Modulation in the activities of signal transduction molecules like apoptosis signal-regulating kinase 1 (ASK1) and c-Jun NH₂-terminal kinase 1/2 (JNK1/2) were also investigated to understand their functional role under natural stressed condition. The expression pattern of the proteins was determined by immunoblotting and the relationship between the proteins was identified by regression analysis. Test fish hepatocytes demonstrated significant upregulation (P < 0.05) in the levels of Trx and HSP90α and insignificant inductions in the expression pattern of ASK1 and JNK1/2 than control fish hepatocytes. These findings provide direct evidence that Trx and HSP90α induction in fish hepatocytes under stress may aid cell survival by negatively regulating ASK1 expression and thereby functionally antagonizing the apoptotic role of JNK1/2 in natural aquatic systems.     

Integrated Seed Proteome and Phosphoproteome Analyses Reveal Interplay of Nutrient Dynamics,Carbon–Nitrogen Partitioning,and Oxidative Signaling in Chickpea

Nutrient dynamics in storage organs is a complex developmental process that requires coordinated interactions of environmental, biochemical, and genetic factors. Although sink organ developmental events have been identified, understanding of translational and post‐translational regulation of reserve synthesis, accumulation, and utilization in legumes is limited. To understand nutrient dynamics during embryonic and cotyledonary photoheterotrophic transition to mature and germinating autotrophic seeds, an integrated proteomics and phosphoproteomics study in six sequential seed developmental stages in chickpea is performed. MS/MS analyses identify 109 unique nutrient‐associated proteins (NAPs) involved in metabolism, storage and biogenesis, and protein turnover. Differences and similarities in 60 nutrient‐associated phosphoproteins (NAPPs) containing 93 phosphosites are compared with NAPs. Data reveal accumulation of carbon–nitrogen metabolic and photosynthetic proteoforms during seed filling. Furthermore, enrichment of storage proteoforms and protease inhibitors is associated with cell expansion and seed maturation. Finally, combined proteoforms network analysis identifies three significant modules, centered around malate dehydrogenase, HSP70, triose phosphate isomerase, and vicilin. Novel clues suggest that ubiquitin–proteasome pathway regulates nutrient reallocation. Second, increased abundance of NAPs/NAPPs related to oxidative and serine/threonine signaling indicates direct interface between redox sensing and signaling during seed development. Taken together, nutrient signals act as metabolic and differentiation determinant governing storage organ reprogramming.     

Chitosan‐triggered immunity to Fusarium in chickpea is associated with changes in the plant extracellular matrix architecture,stomatal closure and remodeling of the plant metabolome and proteome

Pathogen‐/microbe‐associated molecular patterns (PAMPs/MAMPs) initiate complex defense responses by reorganizing the biomolecular dynamics of the host cellular machinery. The extracellular matrix (ECM) acts as a physical scaffold that prevents recognition and entry of phytopathogens, while guard cells perceive and integrate signals metabolically. Although chitosan is a known MAMP implicated in plant defense, the precise mechanism of chitosan‐triggered immunity (CTI) remains unknown. Here, we show how chitosan imparts immunity against fungal disease. Morpho‐histological examination revealed stomatal closure accompanied by reductions in stomatal conductance and transpiration rate as early responses in chitosan‐treated seedlings upon vascular fusariosis. Electron microscopy and Raman spectroscopy showed ECM fortification leading to oligosaccharide signaling, as documented by increased galactose, pectin and associated secondary metabolites. Multiomics approach using quantitative ECM proteomics and metabolomics identified 325 chitosan‐triggered immune‐responsive proteins (CTIRPs), notably novel ECM structural proteins, LYM2 and receptor‐like kinases, and 65 chitosan‐triggered immune‐responsive metabolites (CTIRMs), including sugars, sugar alcohols, fatty alcohols, organic and amino acids. Identified proteins and metabolites are linked to reactive oxygen species (ROS) production, stomatal movement, root nodule development and root architecture coupled with oligosaccharide signaling that leads to Fusarium resistance. The cumulative data demonstrate that ROS, NO and eATP govern CTI, in addition to induction of PR proteins, CAZymes and PAL activities, besides accumulation of phenolic compounds downstream of CTI. The immune‐related correlation network identified functional hubs in the CTI pathway. Altogether, these shifts led to the discovery of chitosan‐responsive networks that cause significant ECM and guard cell remodeling, and translate ECM cues into cell fate decisions during fusariosis.     

EST-SSRs reveal genetic distinction between lac and grain yielding genotypes of pigeonpea

Journal of Plant Biochemistry and Biotechnology - Pigeonpea, an important legume crop is a good host plant for lac cultivation in North East India. In the present study, sixty-three polymorphic EST...     

Comparative analysis of metabolites in contrasting chickpea cultivars

Journal of Plant Biochemistry and Biotechnology - Chickpea (Cicer arietinum L.) is a good source of nutrients for animals and human consumption. In the present study, we analyzed the anthocyanin...     

Pathophysiological relationship between hypoxia associated oxidative stress,Epithelial-mesenchymal transition,stemness acquisition and alteration of Shh/ Gli-1 axis during oral sub-mucous fibrosis and oral squamous cell carcinoma

Oral sub-mucous fibrosis (OSF) is a pathophysiological state of oral cavity or oropharynx having a high chance of conversion to oral squamous cell carcinoma (OSCC). It involves fibrotic transformation of sub-epithelial matrix along with epithelial abnormalities. The present work aims to unveil the mechanistic domain regarding OSF to OSCC conversion exploring the scenario of hypoxia associated oxidative stress, epithelial-mesenchymal transition (EMT), metastasis and stemness acquisition. The study involves histopathological analysis of the diseased condition along with the exploration of oxidative stress status, assessment of mitochondrial condition, immunohistochemical analysis of HIF-1α, E-cadherin, vimentin, ERK, ALDH-1, CD133, Shh, Gli-1 and survivin expressions in the oral epithelial region together with the quantitative approach towards collagen deposition in the sub-epithelial matrix. Oxidative stress was found to be associated with type-II EMT in case of OSF attributing the development of sub-epithelial fibrosis and type-III EMT in case of OSCC favoring malignancy associated metastasis. Moreover, the acquisition of stemness during OSCC can also be correlated with EMT. Alteration of Shh and Gli-1 expression pattern revealed the mechanistic association of hypoxia with the phenotypic plasticity and disease manifestation in case of OSF as well as OSCC. Shh/ Gli-1 signaling can also be correlated with survivin mediated cytoprotective phenomenon under oxidative stress. Overall, the study established the correlative network of hypoxia associated oxidative stress, EMT and manifestation of oral pre-cancerous and cancerous condition in a holistic approach that may throw rays of hope in the therapeutic domain of the concerned diseases.     

A375 melanoma cells are sensitized to cisplatin-induced toxicity by a synthetic nitro-flavone derivative 2-(4-Nitrophenyl)-4H-chromen-4-one through inhibition of PARP1

Background

Cisplatin has been extensively used in therapeutics for its broad-spectrum anticancer activity and frequently used for the treatment of solid tumors. However, it presents several side-effects and several cancers develop resistance. Combination therapy of cisplatin with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors has been effective in increasing its efficacy at lower doses.

Methods and results

In this work, we have shown that the nitro-flavone derivative, 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO), can improve the sensitivity of cancer cells to cisplatin through inhibition of PARP1. The effect of 4NCO on cisplatin toxicity was studied through combination therapy in both exponential and density inhibited A375 melanoma cells. Combination index (CI) was determined from isobologram analysis. The mechanism of cell killing was assessed by lactate dehydrogenase (LDH) assay. Temporal nicotinamide adenine dinucleotide (NAD⁺) assay was done to show the inhibition of PARP1. We also performed in silico molecular modeling studies to know the binding mode of 4NCO to a modeled PARP1-DNA complex containing cisplatin-crosslinked adduct. The results from both in silico and in cellulo studies confirmed that PARP1 inhibition by 4NCO was most effective in sensitizing A375 melanoma cells to cisplatin. Isobologram analysis revealed that 4NCO reduced cell viability both in exponential and density inhibited A375 cells synergistically. The combination led to cell death through apoptosis.

Conclusion

The synthetic nitro-flavone derivative 4NCO effectively inhibited the important nuclear DNA repair enzyme PARP1 and therefore, could complement the DNA-damaging anticancer drug cisplatin in A375 cells and thus, could act as a potential adjuvant to cisplatin in melanoma therapy.

     

STATISTICAL APPROACH FOR THE ENHANCED PRODUCTION OF COLD-ACTIVE β-GALACTOSIDASE FROM Thalassospira frigidphilosprofundus: A NOVEL MARINE PSYCHROPHILE FROM DEEP WATERS OF BAY OF BENGAL

In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active β-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for β-galactosidase production. Under optimal conditions, the production of β-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. β-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active β-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active β-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.