Cloning, expression, and characterization of human metalloprotease 1: a novel member of the pitrilysin family of metalloendoproteases.

A novel cDNA, designated human metalloendoprotease 1 (hMP1), was identified on the basis of homology to known metalloendoproteases of the pitrilysin family. The full-length MP1 codes for a protein with an open reading frame of 1038 amino acids. The N-terminal region contains the HXXEH(X)76E catalytic domain that is conserved in the members of pitrilysin family, namely insulin-degrading enzyme and NRD convertase. The hMP1 mRNA is expressed in a number of cell lines and tissues as a single species of about 3.4 kb. The expression of hMP1 mRNA is higher in muscle and heart than in brain, pancreas, liver, lung, and placenta. The full-length hMP1 was expressed in the baculovirus system and purified to homogeneity using isoelectrofocusing and ion-exchange chromatography. The enzyme exhibited a neutral pH optimum and high sensitivity to thiol reagents. HMP1 was inactivated by 1,10-phenanthroline, a specific inhibitor of Zn(+2)-dependent metalloproteases. The enzyme was not inhibited by agents that inhibit neutral metalloendoproteases of the thermolysin family such as thimet endo-oligopeptidase, enkephalinase, or angiotensin-converting enzyme. HMP1 cleaved a prodynorphin-derived peptide, leumorphin, N-terminal to Arg in the monobasic processing site, as evidenced by MALDI-TOF mass spectrometry. However, the enzyme did not exhibit strict monobasic cleavage specificity, as peptide substrates with amino acid substitutions around the monobasic site was cleaved efficiently by hMP1. Taken together, these results suggest that hMP1 is a novel member of the metalloendoprotease superfamily with ubiquitous distribution that could play a broad role in general cellular regulation.     

Giant Starship Elements Mobilize Accessory Genes in Fungal Genomes

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a newly discovered group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27 to 393 kb and last shared a common ancestor ca. 400 Ma. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize four additional Starships whose activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager’s activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first dedicated agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.     

Cancer cells adapt FAM134B/BiP mediated ER-phagy to survive hypoxic stress

In the tumor microenvironment, cancer cells experience hypoxia resulting in the accumulation of misfolded/unfolded proteins largely in the endoplasmic reticulum (ER). Consequently, ER proteotoxicity elicits unfolded protein response (UPR) as an adaptive mechanism to resolve ER stress. In addition to canonical UPR, proteotoxicity also stimulates the selective, autophagy-dependent, removal of discrete ER domains loaded with misfolded proteins to further alleviate ER stress. These mechanisms can favor cancer cell growth, metastasis, and long-term survival. Our investigations reveal that during hypoxia-induced ER stress, the ER-phagy receptor FAM134B targets damaged portions of ER into autophagosomes to restore ER homeostasis in cancer cells. Loss of FAM134B in breast cancer cells results in increased ER stress and reduced cell proliferation. Mechanistically, upon sensing hypoxia-induced proteotoxic stress, the ER chaperone BiP forms a complex with FAM134B and promotes ER-phagy. To prove the translational implication of our mechanistic findings, we identified vitexin as a pharmacological agent that disrupts FAM134B-BiP complex, inhibits ER-phagy, and potently suppresses breast cancer progression in vivo.Subject terms: Cell biology, Cancer     

Molecular docking analysis of estrogen receptor binding phytocomponents identified from the ethyl acetate extract of Salicornia herbacea (L)

It is of interest to evaluate the secondary metabolites using high performance thin layer chromatography (HPTLC) finger printing and Gas chromatography-Mass spectroscopy (GC-MS) in S. herbaceaextract. The powdered plant material extracted using different solvents were used for the qualitative analysis of alkaloids, flavonoids, terpenoids and saponins followed by HPTLC finger printing and GC-MS analysis. The components identified in the GC-MS were docked with estrogen receptor (ER) to identify the binding specificity of isolated compounds. The ethyl acetate extract of S. herbaceashowed the presence of high number of secondary metabolites when compared to other solvent system. The qualitative analysis of the plant material also showed the presence of carbohydrates, protein, amino acid, phenol, flavonoids, terpenoids, glycosides, saponins and steroids. The HPTLC finger printing analysis revealed the existence of alkaloid, flavonoid, terpenoid and saponin compounds and GC-MS. GC-MS was performed to identify the phytocomponents constituents in the extract. 8 phytocompounds were identified to analyse binding with ER. The binding affinity score (-6.8 kcal/mol) and interacting ER residues (28) the phyto compound di-n-octyl phthalate showed best docking score with ER α than the standard drugs lasofoxifene, and 4-hydroxytamoxifen. The binding affinity and number of interacting ER residues was -6.9 kcal/mol; 10 and -6.2; 11, respectively. The results identified the presence of ER antagonist in S. herbaceaand warrants further investigation to explore for treating ER regulated diseases.     

Entrapment of lipase into K-carrageenan beads and its use in hydrolysis of olive oil in biphasic system

The porcine pancrease lipase was immobilized by entrapment in the beads of K-carrageenan and cured by treatment with polyethyleneimine (PEI) in the phosphate buffer. The retention of hydrolytic activity of lipase and compressive strength of the beads were examined. The activity of free and immobilized lipase was assessed by using olive oil as the substrate. The immobilized enzyme exhibited a little shift towards acidic pH for its optimal activity and retained 50% of its activity after 5 cycles. When the enzyme concentration was kept constant and substrate concentration was varied the K_m and V_max were observed to be 0.18 × 10⁻² and 0.10, and 0.10 × 10⁻² and 0.09 respectively, for free and for entrapped enzymes. When the substrate concentration was kept constant and enzyme concentration was varied, the values of K_m and V_max were observed to be 0.19 × 10⁻⁷ and 0.41, and 0.18 × 10⁻⁷ and 0.41 for free and entrapped enzymes. Though this indicates that there is no conformational change during immobilization, it also shows that the reaction velocity depends on the concentration. Immobilized enzyme showed improved thermal and storage stability. Hydrolysis of olive oil in organic–aqueous two-phase system using fixed bed reactor was carried out and conditions were optimized. The enzyme in reactor retained 30% of its initial activity after 480 min (12 cycles).     

A Novel Cell Fixation Method that Greatly Enhances Protein Identification in Microproteomic Studies Using Laser Capture Microdissection and Mass Spectrometry

Microproteomic studies have improved our knowledge of cell biology. Yet, with mass spectrometry (MS) analysis, accuracy can be lost for protein identification and quantification when using heterogeneous samples. Laser capture microdissection (LCM) allows for the enrichment of specific subsets of cells to study their proteome; however, sample fixation is necessary. Unfortunately, fixation hampers MS results due to protein cross‐linking. The aim of this study was to identify both a fixation protocol and an extraction method that returns the best yield of proteins for downstream MS analysis, while preserving cellular structures. We compared glutaraldehyde (GLU), a common fixative to preserve cells, to dithiobispropionimidate (DTBP), a cleavable cross‐linker. Our DTBP fixation/extraction protocol greatly increased the protein recovery. In fact, while 1000 GLU fixed cells returned only 159 unique protein hits, from 1464 unique peptides of 1994 unique collected spectra, 1000 DTBP fixed cells resulted in 567 unique collected protein hits, from 7542 unique peptides, of 10,401 unique collected spectra. That is, a 3.57‐fold increase in protein hits, 5.15‐fold increase in unique peptides, and a 5.22‐fold increase in unique collected spectra. Overall, the novel protocol introduced here allows for a very efficient protein recovery and good sample quality for MS after sample collection using LCM.     

Clinical application of nerve growth factor on human corneal ulcer

Nerve growth factor (NGF) is a neurotrophic and immunomodulatory factor contributing of the control of cutaneous morphogenesis, wound healing and inflammatory responses. Following the evidence that topical administration of NGF leads to healing of human corneal ulcers, we investigate the therapeutic action of NGF on immune and/or autoimmune cutaneous ulcers. We found that 1-10 micrograms of highly purified murine NGF dissolved in 50 microliters of physiological solution and topically applied to skin ulcer leads, after 4-12 weeks of daily treatment (depending on the size and depth of the lesion) to complete healing of the ulcer. Thus, NGF was able to promote complete repair in human skin and corneal ulcers which were poorly or non-responsive to conventional topical and systemic treatments. No side effects were observed and a follow up after 4 months showed no signs of relapse. These findings indicate that failure of cutaneous tissues to produce sufficient amounts of NGF might represent a prominent mechanism implicated in the clinical manifestation of ocular ulcers.     

Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients

First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.