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133 tissue samples spanning 11 cancer types and matching non‐cancerous specimens are profiled from 467 published and unpublished PGC‐LC‐MS/MS N‐glycome datasets collected over a decade.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.     

Colonization of nasal cavities by Staphylococcus epidermidis mitigates SARS-CoV-2 nucleocapsid phosphoprotein-induced interleukin (IL)-6 in the lung

Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can trigger excessive interleukin (IL)-6 signalling, leading to a myriad of biological effects including a cytokine storm that contributes to multiple organ failure in severe coronavirus disease 2019 (COVID-19). Using a mouse model, we demonstrated that nasal inoculation of nucleocapsid phosphoprotein (NPP) of SARS-CoV-2 increased IL-6 content in bronchoalveolar lavage fluid (BALF). Nasal administration of liquid coco-caprylate/caprate (LCC) onto Staphylococcus epidermidis (S. epidermidis)-colonized mice significantly attenuated NPP-induced IL-6. Furthermore, S. epidermidis-mediated LCC fermentation to generate electricity and butyric acid that promoted bacterial colonization and activated free fatty acid receptor 2 (Ffar2) respectively. Inhibition of Ffar2 impeded the effect of S. epidermidis plus LCC on the reduction of NPP-induced IL-6. Collectively, these results suggest that nasal S. epidermidis is part of the first line of defence in ameliorating a cytokine storm induced by airway infection of SARS-CoV-2.     

Calcineurin activation is only one calcium-dependent step in cytotoxic T lymphocyte granule exocytosis

We have tested the idea that calcineurin, a calcium-dependent phosphatase that is critical for activating cytokine gene expression in helper T cells, plays a role in lytic granule exocytosis in cytotoxic T lymphocytes (CTLs). We used TALL-104 human leukemic CTLs as a model. Our results confirm an earlier report (Dutz, J. P., Fruman, D. A., Burakoff, S. J., and Bierer, B. E. (1993) J. Immunol. 150, 2591-2598) that immunosuppressive drugs inhibit exocytosis in CTLs stimulated either via the T cell receptor (TCR) or via TCR-independent soluble agents. Of the two recently reported alternate targets of immunosuppressive drugs (Matsuda, S., Shibasaki, F., Takehana, K., Mori, H., Nishida, E., and Koyasu, S. (2000) EMBO Rep. 1, 428-434 and Matsuda, S., and Koyasu, S. (2000) Immunopharmacology 47, 119-125), JNK is not required for lytic granule exocytosis, but we were not able to exclude a role for P38. Exocytosis could be inhibited by expressing GFP fused to a C-terminal fragment of CAIN (cabin 1), but not by expressing VIVIT-GFP. Finally, expressing either full-length or truncated constitutively active mutant calcineurin A enhanced lytic granule exocytosis. However, the mutant calcineurin was unable to support exocytosis when cells were stimulated in the absence of Ca2+ influx. Taken together, our results support the idea that activation of calcineurin is required for lytic granule exocytosis but suggest that it is not the sole Ca2+-dependent step.     

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.     

Enlightening energy parasitism by analysis of an ATP/ADP transporter from chlamydiae

Energy parasitism by ATP/ADP transport proteins is an essential, common feature of intracellular bacteria such as chlamydiae and rickettsiae, which are major pathogens of humans. Although several ATP/ADP transport proteins have so far been characterized, some fundamental questions regarding their function remained unaddressed. In this study, we focused on the detailed biochemical analysis of a representative ATP/ADP transporter (PamNTT1), from the amoeba symbiont Protochlamydia amoebophila (UWE25) to further clarify the principle of energy exploitation. We succeeded in the purification of the first bacterial nucleotide transporter (NTT) and its functional reconstitution into artificial lipid vesicles. Reconstituted PamNTT1 revealed high import velocities for ATP and an unexpected and previously unobserved stimulating effect of the luminal ADP on nucleotide import affinities. Latter preference of the nucleotide hetero-exchange is independent of the membrane potential, and therefore, PamNTT1 not only structurally but also functionally differs from the well-characterized mitochondrial ADP/ATP carriers. Reconstituted PamNTT1 exhibits a bidirectional orientation in lipid vesicles, but interestingly, only carriers inserted with the N-terminus directed to the proteoliposomal interior are functional. The data presented here comprehensively explain the functional basis of how the intracellular P. amoebophila manages to exploit the energy pool of its host cell effectively by using the nucleotide transporter PamNTT1. This membrane protein mediates a preferred import of ATP, which is additionally stimulated by a high internal (bacterial) ADP/ATP ratio, and the orientation-dependent functionality of the transporter ensures that it is not working in a mode that is detrimental to P. amoebophila. Heterologous expression and purification of high amounts of PamNTT1 provides the basis for its crystallization and detailed structure/function analyses. Furthermore, functional reconstitution of this essential chlamydial protein paves the way for high-throughput uptake studies in order to screen for specific inhibitors potentially suitable as anti-chlamydial drugs.     

Fosfomycin Permeation through the Outer Membrane Porin OmpF

Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.     

Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice

Heat shock proteins (HSPs) are highly conserved among all organisms from prokaryotes to eukaryotes. In mice, the HSP genes Hsp70.1 and Hsp70.3 are induced by both endogenous and exogenous stressors, such as heat and toxicants. In order to determine whether such proteins specifically influence genomic instability, mice deficient for Hsp70.1 and Hsp70.3 (Hsp70.1/3(-/-) mice) were generated by gene targeting. Mouse embryonic fibroblasts (MEFs) prepared from Hsp70.1/3(-/-) mice did not synthesize Hsp70.1 or Hsp70.3 after heat-induced stress. While the Hsp70.1/3(-/-) mutant mice were fertile, their cells displayed genomic instability that was enhanced by heat treatment. Cells from Hsp70.1/3(-/-) mice also display a higher frequency of chromosome end-to-end associations than do control Hsp70.1/3(+/+) cells. To determine whether observed genomic instability was related to defective chromosome repair, Hsp70.1/3(-/-) and Hsp70.1/3(+/+) fibroblasts were treated with ionizing radiation (IR) alone or heat and IR. Exposure to IR led to more residual chromosome aberrations, radioresistant DNA synthesis (a hallmark of genomic instability), increased cell killing, and enhanced IR-induced oncogenic transformation in Hsp70.1/3(-/-) cells. Heat treatment prior to IR exposure enhanced cell killing, S-phase-specific chromosome damage, and the frequency of transformants in Hsp70.1/3(-/-) cells in comparison to Hsp70.1/3(+/+) cells. Both in vivo and in vitro studies demonstrate for the first time that Hsp70.1 and Hsp70.3 have an essential role in maintaining genomic stability under stress conditions.     

Partial purification of chlorophyll degrading enzymes from cavendish banana (Musa Cavendishi)

Cavendish banana (Musa Cavendishi, subgroup AAA) remains green upon ripening at tropical temperature (25-30 degrees C), due to incomplete degradation of chlorophyll (Chl). Earlier, evidence for the existence of two distinct degradative pathways--chlorophyllase and chlorophyll oxidase pathways in these bananas was provided. Here, an attempt has been made to understand further the mechanism of inhibition of Chl degradation at different stages of ripening and detecting various enzyme activities by partial purification. Soluble and Triton-solubilized protein fractions obtained from peel acetone powder from green-unripe, green-ripe and yellow-ripe bananas efficiently degraded Chl a. About 2-fold increase in Chl hydrolyzing/oxidizing and magnesium-dechelatase activities was observed in ripe, as compared to green-unripe bananas. The electrophoretic pattern of the soluble and detergent-solubilized proteins from the three stages of ripening revealed that the latter fraction contained only three slow moving proteins, which were found to be glycoproteins, as revealed in PAS staining. The soluble enzyme fraction contained all other bands along with the above three bands, as observed in the Native-PAGE of DEAE-Sepharose purified fractions. Only soluble fraction from 'green-ripe' bananas, catalyzed formation of an unknown intermediate (retention time 8.6 min), which was formed by the action of Triton-solubilized enzyme fractions, obtained from 'green-unripe' and 'yellow-ripe' bananas. The enzyme responsible for the formation of this intermediate might be involved in the stay-green character and could be a component of Chl oxidase pathway. Partial purification of soluble protein fraction by DEAE-Sepharose showed the presence of chlorophyllase, magnesium-dechelatase, pheophorbide a oxygenase, red fluorescent catabolite reductase and Chl oxidase. Native PAGE of pooled fractions showed separation of proteins in different bands. Pooled fractions IV and VI showed the presence of a single major band, resulting in almost a homogenous preparation in a single step. Fraction IV catalyzed dechelation of Mg by Mg-dechelatase, while fraction VI catalyzed the formation of 132-OH-Chl a by chlorophyll oxidase. Chlorophyll oxidase activity was stimulated by linolenic acid, indicating involvement of lipoxygenase in oxidative Chl degradation, thereby resulting in the formation of 13(2)-OH-Chl a as product. The results show the presence of various enzymes of chlorophyllase and chlorophyll oxidase pathways in soluble enzyme fraction.     

Computational method for discovery of estrogen responsive genes

Estrogen has a profound impact on human physiology and affects numerous genes. The classical estrogen reaction is mediated by its receptors (ERs), which bind to the estrogen response elements (EREs) in target gene's promoter region. Due to tedious and expensive experiments, a limited number of human genes are functionally well characterized. It is still unclear how many and which human genes respond to estrogen treatment. We propose a simple, economic, yet effective computational method to predict a subclass of estrogen responsive genes. Our method relies on the similarity of ERE frames across different promoters in the human genome. Matching ERE frames of a test set of 60 known estrogen responsive genes to the collection of over 18,000 human promoters, we obtained 604 candidate genes. Evaluating our result by comparison with the published microarray data and literature, we found that more than half (53.6%, 324/604) of predicted candidate genes are responsive to estrogen. We believe this method can significantly reduce the number of testing potential estrogen target genes and provide functional clues for annotating part of genes that lack functional information.