Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.     

Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau

It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolytic activity and eventual degradation of recombinant protein.     

P. falciparum RH5‐Basigin interaction induces changes in the cytoskeleton of the host RBC

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte‐binding protein homologues (RHs) and erythrocyte‐binding like proteins are two families involved in the invasion leading to merozoite‐red blood cell (RBC) junction formation. Ca²⁺ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca²⁺ signaling, which triggers the erythrocyte‐binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5‐basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5‐Basigin interaction on its own triggers a Ca²⁺ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca²⁺ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.     

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics‐centric study investigates a possible link between protein paucimannosylation, an under‐studied class of human N‐glycosylation [Man_1‐3GlcNAc₂Fuc_0‐1], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non‐cancerous specimens are profiled from 467 published and unpublished PGC‐LC‐MS/MS N‐glycome datasets collected over a decade. PMGs, particularly Man_2‐3GlcNAc₂Fuc₁, are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0–50.2%). Analyses of paired (tumor/non‐tumor) and stage‐stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N‐acetyl‐β‐hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.     

Elasto-regenerative properties of polyphenols

Abdominal aortic aneurysms (AAA) are progressive dilatations of infra-renal aorta causing structural weakening rendering the aorta prone to rupture. AAA can be potentially stabilized by inhibiting inflammatory enzymes such as matrix metalloproteinases (MMP); however, active regression of AAA is not possible without new elastic fiber regeneration. Here we report the elastogenic benefit of direct delivery of polyphenols such as pentagalloyl glucose (PGG), epigallocatechin gallate (EGCG), and catechin, to smooth muscle cells obtained either from healthy or from aneurysmal rat aorta. Addition of 10 μg/ml PGG and ECGC induce elastin synthesis, organization, and crosslinking while catechin does not. Our results indicate that polyphenols bind to monomeric tropoelastin and enhance coacervation, aid in crosslinking of elastin by increasing lysyl oxidase (LOX) synthesis, and by blocking MMP-2 activity. Thus, polyphenol treatments leads to increased mature elastin fibers synthesis without increasing the production of intracellular tropoelastin.     

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.