Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism

Proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity clamp for eukaryotic DNA polymerases and a binding platform for many DNA replication and repair proteins. The enzymatic activities of PCNA loading and unloading have been studied extensively in vitro. However, the subcellular locations of PCNA loaders, replication complex C (RFC) and CTF18-RFC-like-complex (RLC), and PCNA unloader ATAD5-RLC remain elusive, and the role of their subunits RFC2-5 is unknown. Here we used protein fractionation to determine the subcellular localization of RFC and RLCs and affinity purification to find molecular requirements for the newly defined location. All RFC/RLC proteins were detected in the nuclease-resistant pellet fraction. RFC1 and ATAD5 were not detected in the non-ionic detergent-soluble and nuclease-susceptible chromatin fractions, independent of cell cycle or exogenous DNA damage. We found that small RFC proteins contribute to maintaining protein levels of the RFC/RLCs. RFC1, ATAD5, and RFC4 co-immunoprecipitated with lamina-associated polypeptide 2 (LAP2) α which regulates intranuclear lamin A/C. LAP2α knockout consistently reduced detection of RFC/RLCs in the pellet fraction, while marginally affecting total protein levels. Our findings strongly suggest that PCNA-mediated DNA transaction occurs through regulatory machinery associated with nuclear structures, such as the nuclear matrix.

Salt-Mediated Organic Solvent Precipitation for Enhanced Recovery of Peptides Generated by Pepsin Digestion

Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow—including the digestion step—can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.

Abstract MP36: Human Urinary Exosomes Contain Functional ACE2

The Ang II convertase and SARS-COV-2 co-receptor ACE2 is highly expressed on proximal tubules within the kidney. ACE2 is also present in urine and reportedly correlates with various renal pathologies that may reflect enhanced shedding of the peptidase through activation of ADAMs. Indeed, 95 kDa ACE2 is typically detected in urine consistent with a shorter, soluble form of the peptidase; however, the full-length, membrane-bound form of ACE2 (120 kDa) is also evident in urine which is difficult to reconcile with ACE2 shedding. To account for these isoforms, we evaluated ACE2 expression in exosomes isolated from human urine. Morning collections from males [50 to 64 years of age, non-smokers] were immediately processed for exosome isolation by cibacron blue binding of albumin followed by 0.2 μmicron filtration to remove microvesicles and apoptotic bodies, Amicon 100 kDa concentration, and ultracentrifugation (UC) to pellet exosomes. Analysis of the UC pellet fraction revealed the exosomal markers ALIX, CD63 and HSP70, as well as the proximal tubule peptidases neprilysin (NEP) and ACE2. Exosomal ACE2 content was 45 ± 11 ng/mL (mean ± SEM; N=5) by ELISA and exosomal activity hydrolyzed Ang II to Ang-(1-7) that was abolished by the ACE2 inhibitor MLN4760. Fluorescent nanotracking analysis (f-NTA) with Alexa Fluor antibodies and CellMask Deep Red membrane stain (CMDR) demonstrate a similar density of ACE2+ and NEP+ exosomes that were ~50% of total urinary exosomes (*P<0.05 vs. CD63+, N=3) while particle sizes were comparable and in the expected range of exosomes (100-150 nm). We conclude that human urinary exosomes express functional ACE2 which may originate from proximal tubule release.

Results of geological and geophysical research on the Subotska structure of Ingulskiy megablock of the Ukrainian shield

The article presents the results of diamond prospecting studies in the Subotska structure of the Inhulskyi megablock of Ukrainian Shield. For the results, it is indicated that the Subotska structure is mimicked by crater rocks and in some cases by manifestations of the vent facies with signs of kimberlitic-lamproitic magmatism. The typical local features of manifestations of explosive structures from maar volcanism in Subotska area are determined. The article presents the results of petrographic and mineralogical study of the core material from exploratory wells on the Subotska structure, the results of study of material composition of the clay fraction, X-ray diffraction analysis of the pellet fraction. Data of the X-ray structural analysis of the pellet fraction of samples taken from the core material from exploratory wells on the Subotska structure indicates the obvious mechanical sum, the head folder of such is calciferous montmorilonite, and also saponite, nontronite, hydromica and kaolinit. The availability of the nontronite and saponite is confirmed by the results of electronic-microscopic reports. Also the article presents the results of studying the secondary lithochemical halos of Cr, Ni, Mg, Co, Ti, V, Fe, covering the geochemical spectrum inherent in alkaline-ultrabasic rocks and their weathering crust. These halos are combined with negative gravitational anomalies associated with the explosive structures in the Subotska area. The structural control of the great part of the detected geochemical anomalies, geochemical halos are determined. According to the degree of manifestation of the complex of criteria five potential diamond-prospective structures are discovered on the Subotska area. There were developed recommendations for further research on the Subotska area.

Semi-purification procedures of prions from a prion-infected brain using sucrose has no influence on the nonenzymatic glycation of the disease-associated prion isoform

Previous studies have shown that the Nε-carboxymethyl group is linked to not only one or more N-terminal Lys residues but also to one or more Lys residues of the protease-resistant core region of the pathogenic prion isoform (PrPSc) in prion-infected brains. Using an anti-advanced glycation end product (AGE) antibody, we detected nonenzymatically glycated PrPSc (AGE-PrPSc) in prion-infected brains following concentration by a series of ultracentrifugation steps with a sucrose cushion. In the present study, the levels of in vitro nonenzymatic glycation of PrPSc using sucrose were investigated to determine whether sucrose cushion can artificially and nonenzymatically induce in vitro glycation during ultracentrifugation. The first insoluble pellet fraction following the first ultracentrifugation (PU1st) collected from 263K scrapie-infected brains was incubated with sucrose, glucose or colloidal silica coated with polyvinylpyrrolidone (percoll). None of the compounds in vitro resulted in AGE-PrPSc. Nonetheless, glucose and percoll produced AGEs in vitro from other proteins within PU1st of the infected brains. This reaction could lead to the AGE-modified polymer(s) of nonenzymatic glycation-prone protein(s). This study showed that PrPSc is not nonenzymatically glycated in vitro with sucrose, glucose or percoll and that AGE-modified PrPSc can be isolated and enriched from prion-infected brains.

K+-p-nitrophenylphosphatase activity in rat brain and liver

K+-p-nitrophenylphosphatase (K+pNPPase) is the enzyme, which is considered to be involved in K+-dependent hydrolysis of the phosphoenzyme in the reaction cycle of Na+, K+-ATPase. The aim of our present study was to characterize some features of K+pNPPase in homogenates of the rat brain and liver. We determined p-nitrophenylphosphatase (pNPPase) activity in the presence of various ion combinations (Mg2++K+, Mg2+, K+). We found a higher total pNPPase activity in the brain (0.8±0.079 nkat/mg protein) than in the liver (0.08±0.01 nkat/mg protein). Contrary to the liver, the main part of the total brain activity was K+-dependent. The activity of K+pNPPase was significantly higher in cerebral cortex homogenates (0.86±0.073 nkat/mg protein) in comparison to those of the whole brain (0.57±0.075 nkat/mg protein). The specific K+pNPPase activity was two times higher in the isolated pellet fraction (0.911±0.07 nkat/mg protein), rich in synaptosomes, compared to the whole brain homogenate (0.57±0.075 nkat/mg protein). Our results demonstrate the high activity of K+pNPPase in the brain tissue and its distribution mainly into the pellet fraction, what might indicate a possible role of K+pNPPase in specific structures of the brain, e.g. in synaptosomes.

Hexose synthesis by cell wall invertase activity and its effects on the roasting behaviour of macadamia kernel

After roast darkening (ARD) is a defect concealed in the raw macadamia kernel, only becoming evident upon roasting. Kernels susceptible to some forms of ARD reportedly have a higher glucose and fructose concentration. By developing a procedure to simulate ARD and through the inclusion of effector molecules we have demonstrated that the enzyme invertase is key to this form of ARD. Biochemical analysis of raw mature kernel has shown high invertase activity. Separating the extract into pellet and soluble fractions showed that the high invertase activity occurred in the pellet fraction containing the cell wall isoform and that the soluble fraction had little activity. A broad peak in crude cell wall invertase activity occurred between pH 3.75 and 5.0. Enzyme kinetics of the cell wall invertase from crude extracts assayed at pH 4 indicated a high level of activity (Vmax = 4.11 ± 0.55 mg glucose produced/g fresh weight tissue.h), a high affinity for sucrose (Km = 2.02 ± 0.96 mm), and inhibition by MgCl2 (K i = 71.2 ± 12.5 mm). We propose that the initial step in the processes leading to ARD in macadamia could involve membrane damage and subsequent modification of the kernel sugar composition by cell wall invertase.

The Yersinia pestis YscY Protein Directly Binds YscX, a Secreted Component of the Type III Secretion Machinery

ABSTRACT Human pathogenic yersiniae organisms export and translocate the Yop virulence proteins and V antigen upon contact with a eukaryotic cell.Yersinia pestis mutants defective for production of YscX or YscY were unable to export the Yops and V antigen. YscX and YscY were both present in the Y. pestis cell pellet fraction; however, YscX was also found in the culture supernatant. YscY showed structural and amino acid sequence similarities to the Syc family of proteins. YscY specifically recognized and bound to a region of YscX that included a predicted coiled-coil region. These data suggest that YscY may function as a chaperone for YscX in Y. pestis.

Subcellular distribution analysis of the cucumber mosaic virus 2b protein

The cucumoviral 2b protein is a viral counterdefence factor that interferes with the establishment of virus-induced gene silencing in plants. Synthetic peptides were used to generate an antibody to the 2b protein encoded by the Fny strain of cucumber mosaic virus (Fny-CMV). This polyclonal antibody was able to recognize the Fny-CMV 2b protein in a 10000 g pellet fraction of infected tobacco. No protein of equivalent size was detected in mock-inoculated or tobacco mosaic virus-infected samples. This represents the first demonstration of 2b protein expression by a subgroup I strain of CMV. Subcellular fractionation experiments on CMV-infected tobacco leaf tissue showed that the Fny-CMV 2b protein accumulated within a fraction that sedimented at forces of less than 5000 g and that the 2b protein was solubilized only by treatment with urea or SDS. These results suggested that the 2b protein associates either with the nucleus or cytoskeleton of the host cell. Further analysis showed that the 2b protein was enriched in a fraction that sedimented through a 2·2 M sucrose cushion. This fraction was also enriched in histones, suggesting that the CMV 2b protein associates preferentially with the host cell nucleus.

Production and Distribution of Endoglucanase, Cellobiohydrolase, and β-Glucosidase Components of the Cellulolytic System of Volvariella volvacea, the Edible Straw Mushroom

ABSTRACT The edible straw mushroom, Volvariella volvacea, produces a multicomponent enzyme system consisting of endo-1,4-β-glucanase, cellobiohydrolase, and β-glucosidase for the conversion of cellulose to glucose. The highest levels of endoglucanase and cellobiohydrolase were recorded in cultures containing microcrystalline cellulose (Avicel) or filter paper, while lower but detectable levels of activity were also produced on carboxymethyl cellulose, cotton wool, xylitol, or salicin. Biochemical analyses of different culture fractions in cultures exhibiting peak enzyme production revealed that most of the endoglucase was present either in the culture filtrate (45.8% of the total) or associated with the insoluble pellet fraction remaining after centrifugation of homogenized mycelia (32.6%). Cellobiohydrolase exhibited a similar distribution pattern, with 58.9% of the total enzyme present in culture filtrates and 31.0% associated with the pellet fraction. Conversely, most β-glucosidase activity (63.9% of the total) was present in extracts of fungal mycelia whereas only 9.4% was detected in culture filtrates. The endoglucanase and β-glucosidase distribution patterns were confirmed by confocal laser scanning microscopy combined with immunolabelling. Endoglucanase was shown to be largely cell wall associated or located extracellularly, with the highest concentrations being present in a region 1 to 2 μm wide immediately adjacent to the outer surface of (and possibly including) the hyphal wall and extending 60 to 70 μm from the hyphal tip. Immunofluorescence patterns indicated little if any intracellular endoglucanase. Most β-glucosidase was located intracellularly in the apical area extending 60 to 70 μm below the hyphal tip, although enzyme was also evident in the extracellular region extending approximately 15 μm all around the hyphal tip and trailing back along the length of the hypha. The regions of the hypha located some distance from the apical region appeared to be devoid of intracellular β-glucosidase, and the enzyme appears to be associated almost exclusively with, or located on the outside surface of, the hyphal wall.