The extracellular β-glucosidase BGL2 has two variants with different molecular sizes and hydrolytic activities in the stipe or pilei of Coprinopsis cinerea

Two variants of extracellular β-glucosidase (BGL2) were purified from the stipe and pilei of Coprinopsis cinerea. In the stipe, BGL2 was a monomeric protein with an apparent molecular mass of approximately 220 kDa, representing a mature full-length peptide of BGL2. However, in the pilei, the apparent molecular mass of BGL2 was only approximately 120 kDa, consisting of the 60 kDa N-terminal fragment and 55 kDa C-terminal fragment. The hydrolytic activities of BGL2 purified from the pilei were higher than those of BGL2 purified from the stipe. No mRNA splice variants of bgl2 were detected. Therefore, the different variants of BGL2 in the stipe and pilei were not formed by differential RNA splicing. Furthermore, in vitro experiments showed that full-length BGL2 could be cleaved by endogenous proteases from pilei or commercial trypsin at a similar site to form an oligomeric protein consisting of the N-terminal fragment and C-terminal fragment similar to BGL2 from pilei. The hydrolytic activity of BGL2 increased after cleavage by those proteases in vitro. We conclude that the 120 kDa variant of BGL2 in the pilei of C. cinerea is formed by posttranslational proteolytic cleavage. Posttranslational proteolytic cleavage is an efficient way to regulate the activity of BGL2 to adapt to the needs of different physiological functions in the elongation stipe and expansion pilei of C. cinerea.

Cleavage state of γENaC in mouse and rat kidney

Extracellular proteases can activate the epithelial Na channel (ENaC) by cleavage of the g subunit. Here we investigate the cleavage state of the channel in the kidneys of mice and rats on a low-salt diet. We identified the cleaved species of channels expressed in FRT cells by co-expressing the apical-membrane bound protease CAP1 (prostasin). To compare the peptides produced in the heterologous system with those in the mouse kidney we treated both lysates with PNGaseF to remove N-linked glycosylation. The apparent molecular mass of the smallest C-terminal fragment of gENaC (52 kDa) was indistinguishable from that of the CAP1-induced species in FRT cells. Similar cleaved peptides were observed in total and cell surface fractions of rat kidney. This suggests that most of the subunits at the surface have been processed by extracellular proteases. This was confirmed using non-reducing gels, in which the N- and C-terminal fragments of gENaC are linked by a disulfide bond. Under these conditions the major cleaved form in rat kidney had an apparent molecular mass of 56 kDa, ~4 kDa lower than that of the full-length form, consistent with excision of a short peptide by two proteolytic events. We conclude that the most abundant gENaC species in the apical membrane of rat and mouse kidney on a low-Na diet is the twice-cleaved, presumably activated form.

Ubiquitination of renal ENaC subunits in vivo

Ubiquitination of the epithelial Na+ channel (ENaC) in epithelial cells may influence trafficking and hormonal regulation of the channels. We assessed ENaC ubiquitination (ub-ENaC) in mouse and rat kidneys using affinity beads to capture ubiquitinated proteins from tissue homogenates and Western blot analysis with anti-ENaC antibodies. Ub-αENaC was observed primarily as a series of proteins of apparent molecular mass of 40–70 kDa, consistent with the addition of variable numbers of ubiquitin molecules primarily to the NH2-terminal cleaved fragment (~30 kDa) of the subunit. No significant Ub-βENaC was detected, indicating that ubiquitination of this subunit is minimal. For γENaC, the protein eluted from the affinity beads had the same apparent molecular mass as the cleaved COOH-terminal fragment of the subunit (~65 kDa). This suggests that the ubiquitinated NH2 terminus remains attached to the COOH-terminal moiety during isolation through disulfide bonds. Consistent with this, under nonreducing conditions, eluates contained material with increased molecular mass (90–150 kDa). In mice with a Liddle syndrome mutation (β566X) deleting a putative binding site for the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2, the amount of ub-γENaC was reduced as expected. To assess aldosterone dependence of ubiquitination, we fed rats either control or low-Na+ diets for 7 days before kidney harvest. Na+ depletion increased the amounts of ub-αENaC and ub-γENaC by three- to fivefold, probably reflecting increased amounts of fully cleaved ENaC. We conclude that ubiquitination occurs after complete proteolytic processing of the subunits, contributing to retrieval and/or disposal of channels expressed at the cell surface. Diminished ubiquitination does not appear to be a major factor in aldosterone-dependent ENaC upregulation.

Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

CTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions. One such modifications linked to cancer is poly(ADP-ribosyl)ation (PARylation). The highly PARylated CTCF form has an apparent molecular mass of 180 kDa (referred to as CTCF180), which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130 kDa (referred to as CTCF130). The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites). The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present.

Tissue kallikrein activation of the epithelial Na channel

Epithelial Na Channels (ENaC) are responsible for the apical entry of Na+ in a number of different epithelia including the renal connecting tubule and cortical collecting duct. Proteolytic cleavage of γ-ENaC by serine proteases, including trypsin, furin, elastase, and prostasin, has been shown to increase channel activity. Here, we investigate the ability of another serine protease, tissue kallikrein, to regulate ENaC. We show that excretion of tissue kallikrein, which is secreted into the lumen of the connecting tubule, is stimulated following 5 days of a high-K+ or low-Na+ diet in rats. Urinary proteins reconstituted in a low-Na buffer activated amiloride-sensitive currents ( INa) in ENaC-expressing oocytes, suggesting an endogenous urinary protease can activate ENaC. We next tested whether tissue kallikrein can directly cleave and activate ENaC. When rat ENaC-expressing oocytes were exposed to purified tissue kallikrein from rat urine (RTK), ENaC currents increased threefold in both the presence and absence of a soybean trypsin inhibitor (SBTI). RTK and trypsin both decreased the apparent molecular mass of cleaved cell-surface γ-ENaC, while immunodepleted RTK produced no shift in apparent molecular mass, demonstrating the specificity of the tissue kallikrein. A decreased effect of RTK on Xenopus ENaC, which has variations in the putative prostasin cleavage sites in γ-ENaC, suggests these sites are important in RTK activation of ENaC. Mutating the prostasin site in mouse γ-ENaC (γRKRK186QQQQ) abolished ENaC activation and cleavage by RTK while wild-type mouse ENaC was activated and cleaved similar to that of the rat. We conclude that tissue kallikrein can be a physiologically relevant regulator of ENaC activity.

Rutin synthase in fava d’anta: purification and influence of stressors

The flavonoid rutin is synthesized in plants from quercetin, via a process in which isoquercitrin is an intermediary metabolite. In this work, the activities of isoquercitrin synthase and rutin synthase, and the quercetin, isoquercitrin and rutin contents of fava d’anta plants stressed for water (drought and flooding) and salt (NaCl) were studied. In general, stress increased the contents of the three compounds and both enzyme activities. Semi-purified rutin synthase and isoquercitrin synthase showed Km values of 1.816 and 2.10 µM, respectively, with optimum reaction pHs of 5 and 7, respectively, and an optimum reaction temperature of 35°C. Rutin synthase was purified from leaf buds and showed an apparent molecular mass of 39 KDa by SDS-PAGE. Mass spectrometry analysis of the purified protein did not reveal any similarity to the few known sequenced glycosyltransferases.Key words: Dimorphandra mollis, faveiro, flavonoid, isoquercitrin, quercetin.

The small domain of cytochromeffrom the psychrophileChlamydomonas raudensisUWO 241 modulates the apparent molecular mass and decreases the accumulation of cytochromefin the mesophileChlamydomonas reinhardtii

Cytochrome f from the psychrophile Chlamydomonas raudensis UWO 241 has a lower thermostability of its c-type heme and an apparent molecular mass that is 7 kDa lower than that of the model mesophilic green alga Chlamydomonas reinhardtii. We combined chloroplast transformation, site-directed mutagensis, and the creation of chimeric fusion constructs to assess the contribution of specific domains and (or) amino acids residues to the structure, stability, and accumulation of cytochrome f, as well as its function in photosynthetic intersystem electron transport. We demonstrate that differences in the amino acid sequence of the small domain and specific charged amino acids in the large domain of cytochrome f alter the physical properties of this protein but do not affect either the thermostability of the c-type heme, the apparent half-life of cytochrome f in the presence of the chloroplastic protein synthesis inhibitor chloramphenicol, or the capacity for photosynthetic intersystem electron transport, measured as e–/P700. However, pulse-labeling with [14C]acetate, combined with immunoblotting, indicated that the negative autoregulation of cytochrome f accumulation observed in mesophilic C. reinhardtii transformed with chimeric constructs from the psychrophile was likely the result of the defective association of the chimeric forms of cytochrome f with the other subunits of the cytochrome b6/f complex native to the C. reinhardtii wild type. These results are discussed in terms of the unique fatty acid composition of the thylakoid membranes of C. raudensis UWO 241 adapted to cold environments.

Modification of a Mammalian Cell Protein in the Presence of [32P-Adenylate]NAD: Evidence for ADP Ribosylation Activity Associated with Helicobacter pylori

ABSTRACT Culture filtrates from Helicobacter pylori promote the transfer of the radiolabel from [32P-adenylate]NAD to one or more heat-labile factors within extracts prepared from several mammalian cell lines, with the predominate radiolabeled species exhibiting an apparent molecular mass of greater than 130 kDa. Our results suggest that several H. pylori strains release a factor that ADP-ribosylates a mammalian target protein.

Methylcrotonyl-CoA and geranyl-CoA carboxylases are involved in leucine/isovalerate utilization (Liu) and acyclic terpene utilization (Atu), and are encoded by liuB/liuD and atuC/atuF, in Pseudomonas aeruginosa

Pseudomonas aeruginosa is able to grow on acyclic monoterpenes (citronellol, citronellate, geraniol and geranylate), and on other methyl-branched compounds such as leucine or isovalerate. The catabolic pathway of citronellol (Atu, acyclic terpene utilization) enters that of leucine/isovalerate (Liu, leucine and isovalerate utilization) at the level of methylcrotonyl-CoA. Key enzymes of the combined pathways are geranyl-CoA carboxylase (GCase) and methylcrotonyl-CoA carboxylase (MCase). In this study, isovalerate-grown cells specifically expressed MCase (apparent molecular mass of the biotin-containing subunit, 74 kDa) only, and the GCase biotin-containing subunit (71 kDa) was not detected. Citronellol- or citronellate-grown cells produced both carboxylases. Biotin-dependent proteins were purified from crude extracts by avidin-affinity chromatography, and assigned to the corresponding coding genes by trypsin fingerprint analysis. The two subunits of MCase corresponded to liuB/liuD (PA2014/PA2012) of the P. aeruginosa genome database, and atuC/atuF (PA2888/PA2891) encoded GCase subunits. This finding is contrary to that reported by others. The identified genes are part of two separate gene clusters [liuRABCDE (PA2011–PA2016) and atuABCDEFGH (PA2886–PA2893)] that are thought to encode most of the genes of the Atu and Liu pathways.