Intracellular Protein Fraction Research Articles

Streptococcus thermophilus: A Source of Postbiotics Displaying Anti-Inflammatory Effects in THP 1 Macrophages.

In addition to traditional use in fermented dairy products, S. thermophilus also exhibits anti-inflammatory properties both in live and heat-inactivated form. Recent studies have highlighted that some hydrolysates from surface proteins of S. thermophilus could be responsible partially for overall anti-inflammatory activity of this bacterium. It was hypothesized that anti-inflammatory activity could also be attributed to peptides resulting from the digestion of intracellular proteins of S. thermophilus. Therefore, total intracellular proteins (TIP) from two phenotypically different strains, LMD-9 and CNRZ-21N, were recovered by sonication followed by ammonium sulphate precipitation. The molecular masses of the TIP of both strains were very close to each other as observed by SDS-PAGE. The TIP were fractionated by size exclusion fast protein liquid chromatography to obtain a 3-10 kDa intracellular protein (IP) fraction, which was then hydrolysed with pancreatic enzyme preparation, Corolase PP. The hydrolysed IP fraction from each strain exhibited anti-inflammatory activity by modulating pro-inflammatory mediators, particularly IL-1β in LPS-stimulated THP-1 macrophages. However, a decrease in IL-8 secretion was only observed with hydrolysed IP fraction from CNRZ-21N, indicating that strain could be an important parameter in obtaining active hydrolysates. Results showed that peptides from the 3-10 kDa IP fraction of S. thermophilus could therefore be considered as postbiotics with potential beneficial effects on human health. Thus, it can be used as a promising bioactive ingredient for the development of functional foods to prevent low-grade inflammation.

Proteolytic Maturation of the Outer Membrane c-Type Cytochrome OmcZ by a Subtilisin-Like Serine Protease Is Essential for Optimal Current Production by Geobacter sulfurreducens.

An outer membrane c-type cytochrome (OmcZ) in Geobacter sulfurreducens is essential for optimal current production in microbial fuel cells. OmcZ exists in two forms, small and large, designated OmcZS and OmcZL, respectively. However, it is still not known how these two structures are formed. A mutant with a disruption of the GSU2075 gene encoding a subtilisin-like serine protease (designated ozpA for the OmcZprotease), which is located downstream of omcZ, produced low currents at a level similar to that of the omcZ-deficient mutant strain. Biochemical analyses revealed that the ozpA mutant accumulated OmcZL and did not produce OmcZS, which is thought to be a mature form that is essential for the extracellular electron transfer to the electrode. A heterologous expression system cell lysate from an Escherichia coli strain producing OzpA cleaved OmcZL and generated OmcZS as the proteolytic product. Among the culture supernatant, loosely bound outer surface, and intracellular protein fractions from wild-type G. sulfurreducens, only the culture supernatant protein fraction showed OmcZL cleavage activity, indicating that the mature form of OmcZ, OmcZS, can be produced outside the cells. These results indicate that OzpA is an essential protease for current production via the maturation of OmcZ, and OmcZS is the key to the extracellular electron transfer to electrodes. This proteolytic maturation of OmcZ is a unique regulation among known c-type cytochromes in G. sulfurreducens. IMPORTANCE Microbial fuel cells are a promising technology for energy generation from various waste types. However, the molecular mechanisms of microbial extracellular electron transfer to the electrode need to be elucidated. G. sulfurreducens is a common key player in electricity generation in mixed-culture microbial fuel cell systems and a model microorganism for the study of extracellular electron transfer. Outer membrane c-type cytochrome OmcZ is essential for an optimal current production by G. sulfurreducens. OmcZ proteolytic cleavage occurs during maturation, but the underlying mechanism is unknown. This study identifies a subtilisin-like protease, OzpA, which plays a role in cleaving OmcZ and generating the mature form of OmcZ (OmcZS). OzpA is essential for current production and, thus, the proteolytic maturation of OmcZ. This is a novel regulation of the c-type cytochrome for G. sulfurreducens extracellular electron transfer. This study also provides new insights into the design strategy and development of microbial extracellular electron transfer for an efficient energy conversion from chemical energy to electricity.

Integrated Transcriptomic and Proteomic Analyses Reveal the Role of NprR in Bacillus anthracis Extracellular Protease Expression Regulation and Oxidative Stress Responses.

NprR is a protein of Bacillus anthracis that exhibits moonlighting functions as either a phosphatase or a neutral protease regulator that belongs to the RNPP family. We previously observed that the extracellular protease activity of an nprR deletion mutant significantly decreased within in vitro cultures. To identify the genes within the regulatory network of nprR that contribute to its protease activity, integrated transcriptomic and proteomic analyses were conducted here by comparing the nprR deletion mutant and parent strains. A total of 366 differentially expressed genes (DEGs) between the strains were observed via RNA-seq analysis. In addition, label-free LC-MS/MS analysis revealed 503 differentially expressed proteins (DEPs) within the intracellular protein fraction and 213 extracellular DEPs with significant expressional differences between the strains. The majority of DEGs and DEPs were involved in environmental information processing and metabolism. Integrated transcriptomic and proteomic analyses indicated that oxidation-reduction-related GO terms for intracellular DEPs and endopeptidase-related GO terms for extracellular DEPs were significantly enriched in the mutant strain. Notably, many genes involved in protease activity were largely downregulated in the nprR deletion mutant cultures. Moreover, western blot analysis revealed that the major extracellular neutral protease Npr599 was barely expressed in the nprR deletion mutant strain. The mutant also exhibited impaired degradation of protective antigen, which is a major B. anthracis toxin component, thereby resulting in higher protein yields. Concomitantly, another global transcriptional regulator, SpxA1, was also dramatically downregulated in the nprR deletion mutant, resulting in higher sensitivity to oxidative and disulfide stress. These data consequently indicate that NprR is a transcriptional regulator that controls genes whose products function as extracellular proteases and also is involved in oxidative stress responses. This study thus contributes to a more comprehensive understanding of the biological function of NprR, and especially in the middle growth stages of B. anthracis.

Anti-dengue potential of bioactive protein from exophytic bacteria that are symbiotic with brown algae Sargassum sp.

Symbiont bacteria of algae are bioactive metabolite sources with potential as medicinal raw materials. This study aims to find out the anti-dengue potential of a protein fraction isolated from Enterobacter agglomerans SB 5(1) as the symbiont of brown algae Sargassum binderi collected from Lae-Lae island, South Sulawesi. These extracellular and intracellular fractions were isolated by ammonium sulphate fractionation at saturation levels of 0-20 %, 20-40 %, 40-60 %, and 60-80 %. The protein was purified by dialysis method using cellophane bag. Toxicity was tested by BSLT method using shrimp larvae of Artemia salina, Leach. Cytotoxicity test against vero cells infected with dengue virus DEVN-2 was performed by MTT method. Study findings indicate that intracellular protein fraction from E. agglomerans SB 5(1), a symbiont of brown algae Sargassum binderi, showed the presence of bioactive protein having strong toxicity with LC50 of 48.67 µg/mL. Anti-dengue activity toward vero cells indicates inhibition percentage and CC50 value of 70% and 260.37 µg./mL, respectively, therefore it had no potential as anti-virus dengue agent. In future studies, it is recommended to perform hydrolysis of protein compound from symbiont bacteria of Sargassum sp. to explore other peptide compounds with more potential as anti-dengue agents.

Clathrin-Mediated Endocytosis Delivers Proteolytically Active Phytaspases Into Plant Cells.

Phytaspases belong to the family of plant subtilisin-like proteases and are distinct from other family members, as they have strict and rarely occurring aspartate cleavage specificity and unusual localization dynamics. After being secreted into the apoplast of healthy plant tissues, phytaspases are able to return back into cells that have been committed to cell death due to a variety of biotic and abiotic stresses. It was recently discovered that retrograde transport of phytaspases involves clathrin-mediated endocytosis. Here, consequences of phytaspase internalization were studied. Proteolytic activity of phytaspases in the apoplast and intracellular protein fractions obtained from Nicotiana benthamiana leaves containing either endogenous phytaspase only or transiently producing Nicotiana tabacum phytaspase-EGFP protein (NtPhyt-EGFP) was determined. We demonstrated that triggering phytaspase internalization by antimycin A-induced oxidative stress is accompanied by re-distribution of phytaspase activity from the apoplast to the cell interior. Inhibition of clathrin-mediated endocytosis by co-production of the Hub protein prevented phytaspase internalization and phytaspase activity re-localization. Specificity of endocytic uptake of phytaspases was demonstrated by the co-production of an apoplast-targeted mRFP protein marker, which retained its apoplastic localization when phytaspase internalization was essentially complete. Overproduction of NtPhyt-EGFP, but not of the proteolytically inactive phytaspase mutant, per se caused moderate damage in young Nicotiana benthamiana seedlings, whereas antimycin A treatment induced a pronounced loss of cell viability independent of the NtPhyt-EGFP overproduction. Interestingly, inhibition of clathrin-mediated endocytosis abrogated cell death symptoms in both cases. In contrast to stress-induced internalization of tobacco phytaspase, Arabidopsis thaliana phytaspase-EGFP protein (AtPhyt-EGFP) was spontaneously internalized when transiently produced in N. benthamiana leaves. The AtPhyt-EGFP uptake was dependent on clathrin-mediated endocytosis as well, the internalized protein being initially visualized within the membranous vesicles. At later time points, the EGFP tag was cleaved off from AtPhyt, though the elevated level of intracellular AtPhyt proteolytic activity persisted. Our data, therefore, point to clathrin-mediated endocytosis as a means to deliver proteolytically active phytaspases into plant cells. It would be interesting to learn whether or not phytaspases are unique among the large family of plant subtilisin-like proteases in their ability to utilize retrograde trafficking.

Gene cloning and expression of MAP30 in Pichia pastoris

MAP30, a single-stranded type-I ribosome inactivating protein found in Momordica charantia, shows anti-HIV and anti-tumour activity. It could significantly inhibit the HIV-1 and herpes simplex virus infection. In this study, we tried a safe and convenient expression system supplying MAP30 protein for medical practice. The gene encoding MAP30 was cloned into pMD18-T vector. The pMD18-MAP30 plasmid was transformed into competent Escherichia coli JM109 by a chemical method. The MAP30 gene was obtained from the pMD18-MAP30 plasmid digested with NotI and SnaBI and the MAP30 gene was ligated into pGAPHα. Then, pGAPHα-MAP30 was transformed into Pichia pastoris GS115 by electroporation. GS115 transformants were analysed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and Western blot. SDS-PAGE revealed an extra band of approximately 32 kDa in the supernatant protein of the GS115 transformants and in their intracellular protein fraction. The result of Western-blot analysis showed that the supernatant and the cell pellet from GS115 with pGAPHα-MAP30 could specially bind to monoclonal antibodies against His in the 32 kDa site. These results demonstrated that the expression of MAP30 in P. pastoris was successful; the process of the expression did not need methanol induction or introduction of an antibiotic-resistance gene. The study may provide a new way for MAP30 synthesis. Owing to its safety, this new approach is expected to be widely used in the medical field.

Protein identification in two phases of 1,3-propanediol production by proteomic analysis

Proteomic analysis by two-dimensional electrophoresis (2D)-mass spectrometry was used to identify differentially expressed proteins in the Clostridium sp. native strain (IBUN 158B) in two phases of the 1,3-propanediol (1,3-PD) production (lag phase and exponential growth phase). Intracellular protein fraction extraction conditions were standardised, as well as the 2D electrophoresis. Differences were found between both of the growth phases evaluated here. Thirty-two of the differentially expressed proteins were chosen to be identified by tandem mass spectrometry (MALDI TOF/TOF). The presence of four enzymes implicated in the 1,3-PD metabolic pathway was recorded: one from the reductive route (1,3-propanediol dehydrogenase) and three from the oxidative route (3-hydroxybutyryl-CoA dehydrogenase, NADPH-dependent butanol dehydrogenase and phosphate butyryl transferase). The following enzymes which have not been previously reported for Clostridium sp., were also identified: phosphoglycerate kinase, glucose 6-phosphate isomerase, deoxyribose phosphate aldolase, transketolase, cysteine synthetase, O-acetylhomoserine sulphhydrylase, glycyl-tRNA ligase, aspartate-β-semialdehyde dehydrogenase, inosine-5-monophosphate dehydrogenase, aconitate hydratase and the PrsA protein. The foregoing provides a novel contribution towards knowledge of the native strain for the purpose of designing genetic manipulation strategies to obtain strains with high production of 1,3-PD. The article "Protein identification in two phases of 1,3-propanediol production by proteomic analysis" provides a novel contribution towards knowledge regarding the Colombian Clostridium sp. native strain (IBUN 158B) because this is a new approximation in comparative proteomics in two phases of the bacterial growth and 1,3-propanediol (1,3-PD) production conditions. The proteomic studies are very important to identify the enzymes that are expressed at different stages of production and therefore genes of interest in the genetic manipulation strategies; the results can be taken into account in future studies in metabolic engineering when optimising 1,3-PD production, in a cost-effective process having direct industrial applications.

Chondrogenic cell proliferation and signaling pathways induced by bioassayable growth hormone

Bioassayable growth hormone (BGH) was previously shown to stimulate growth of the tibial epiphyseal plate in rats in vivo, but the mechanisms contributing to this response have not been elucidated. The objective of this study, therefore, was to investigate the ability of BGH to induce cartilage proliferation in vitro, and examine the cellular signaling pathways responsible for this effect. The RCJ3.1C5.18 chondrogenic cell line was cultured to 10 days post‐confluence and stimulated with BGH‐containing plasma or conditioned media samples. Proliferation was measured by immunocytochemical staining for bromodeoxyuridine incorporation and quantified by cell counting. Intracellular protein fractions from control and stimulated cultures were isolated and subjected to Western blotting for phosphotyrosine, phosphoERK‐1/2 and phosphoMEK‐1/2. RCJ3.1C5.18 cell proliferation was significantly and dose‐dependently increased by BGH. BGH also increased the overall level of protein tyrosine phosphorylation, and elevated the cellular concentration of phosphorylated ERK‐1 and ERK‐2 in a dose‐dependent fashion. In addition, BGH enhanced the Ser/Thr phosphorylation of MEK‐2. The results demonstrate that BGH directly stimulates cartilage proliferation in vitro, and may utilize multiple signaling molecules to achieve this effect.Support: NIH/NCRR 5G12 RR008124, NIH/NIGMS 5R25GM0490, NSF DBI‐0353887

Peptide Aptamer-mediated Inhibition of Target Proteins by Sequestration into Aggresomes

Peptide aptamers (PAs) can be employed to block the intracellular function of target proteins. Little is known about the mechanism of PA-mediated protein inhibition. Here, we generated PAs that specifically bound to the duck hepatitis B virus (HBV) core protein. Among them, PA34 strongly blocked duck HBV replication by inhibiting viral capsid formation. We found that PA34 led to a dramatic intracellular redistribution of its target protein into perinuclear inclusion bodies, which exhibit the typical characteristics of aggresomes. As a result, the core protein is efficiently removed from the viral life cycle. Corresponding findings were obtained for bioactive PAs that bind to the HBV core protein or to the human papillomavirus-16 (HPV16) E6 protein, respectively. The observation that PAs induce the specific sequestration of bound proteins into aggresomes defines a novel mechanism as to how this new class of intracellular inhibitors blocks the function of their target proteins.

Immobilization of free ionic gold and L-asparagine-complexed ionic gold by Sporosarcina ureae: The importance of organo-gold complexes in gold mobility

Sporosarcina ureae was able to grow in the presence of up to 10-ppm L-asparagine complexed gold. However, 10 ppm Au was found to be highly toxic to Sporosarcina ureae, killing 109 bacteria/mL within a few seconds. To compare the effects of these responses on gold mobility, an in vitro bacterial biofilm model was developed to examine the interaction between Sporosarcina ureae and ionic gold or L-asparagine gold complexes in gravity-fed columns. The immobilization of ionic gold was between 80% and 90% over a period of one week. But, as the available cellular reactive sites became saturated, immobilization decreased to 14% within two weeks. In the L-asparagine-gold system, gold immobilization in the cytoplasm occurred at a high rate (80% to 90%) throughout the experiment, with limited toxicity towards Sporosarcina ureae. The bacterial immobilization and detoxification of L-asparagine-complexed ionic gold was associated with a low molecular weight, i.e, <5,000 dalton (atomic mass unit), intracellular protein fraction. This peptide-complexed gold would allow for the continued biogeochemical cycling of gold under ambient (<100°C) surface or near-surface conditions.

Assessment of the protein quality of the smooth muscle myofibrillar and connective tissue proteins of chicken gizzard

The objective of this study was to assess the protein quality of the myofibrillar and connective tissue proteins of chicken gizzard. Protein fractions were isolated from White Leghorn chicken gizzards and quantified by detailed amino acid analysis. This quantification involved repeated extractions of ground gizzards first with Triton X-100, then with low ionic strength imidazole-buffered saline (pH 7.1), followed by either 2% SDS or by 5 M guanidine hydrochloride. The total soluble intracellular protein fraction averaged 86.3% of the total protein and the insoluble extracellular connective tissue proteins comprised the remaining 13.7%. These fractions differed significantly in their essential amino acid (EAA) profiles, with the soluble intracellular fraction having the highest percentage EAA9 (48.6 to 49.0%) and the insoluble connective tissue fraction varying from 20.8 to 23%, compared to the FAO/WHO reference pattern value of 33.9% for a 2- to 5-yr-old child. Calculated protein efficiency ratios (PER) for intracellular proteins averaged 3.02 compared with a value of 1.65 for the extracellular matrix proteins. These results provide an accurate assessment of the protein quality of smooth muscle proteins of chicken gizzard and may prove valuable for industrial control of the amount of connective tissue added to formulations of meats and poultry products.

Physiologically Based Pharmacokinetic Modeling: Principles and Applications

Concentrations of bromophenol blue (I) in plasma, urine, and bile were determined spectrophotometrically after intravenous bolus injections and infusions in rats. The plasma concentrations were found to decrease monoexponentially after all doses except the highest, where the decrease was biexponential. Although the disposition kinetics of I were apparently first-order at all doses, the half-life increased with in- creasing dose. The area under the plasma concentration-time curve (AUCs,) increased disproportionately with increasing dose. The binding of I to rat plasma proteins, as determined by equilibrium dialysis, showed that the fraction bound (96%) remained constant in the concentration range of 10-300 pg/ml. Plasma concentrations were determined at time zero after intravenous administration and after a second dose adminis- tered 20 min later when plasma concentrations from the first dose were minimal. The apparent first-order elimination rate constant for the plasma concentration decline following the second dose was significantly less than after the first dose, indicating that the residual dye in the liver altered the elimination of I after the second dose. The fraction of the dose in the liver decreased with increasing dose, indicating a saturable uptake process. The biliary excretion profile reflected the uptake saturation that occurred in the liver and demonstrated that the biliary excretion of I depended on the amount present in the liver. When liver damage was induced by exposure to carbon tetrachloride, dye concentrations in the plasma, liver, and kidney increased markedly. Keyphrases 0 Bromophenol blue-in rat urine, plasma, and bile, dose-dependent pharmacokinetics, biliary excretion 0 Pharmacoki- netics-bromophenol blue, rat plasma, urine, and bile, biliary excretion I I Biliary excretion-bromophenol blue, rat urine, plasma, and bile, dose-dependent pharmacokinetics Bromophenol blue (I) is a high molecular weight anionic sulfonephthalein dye (670 g/mole, pK, 4.0). Several re- ports (1-3) have shown that I is extensively excreted in the bile and is not metabolized in a variety of species including the rat. Takada et al. (4, 5 ) conducted studies aimed at char- acterizing the role of the intracellular protein fractions Y and Z in the uptake and transport of I in rat hepatocytes, and also developed a linear pharmacokinetic model for the biliary excretion of I after intravenous administration in rats (6). The model adequately described a single low-dose plasma and bile profile, but could not describe the ob- served nonlinearity a t higher doses. The overall aim of these authors was to evaluate I as a model organic anionic compound by characterizing the pharmacokinetics at three different doses. Their report did not fully characterize the pharmacokinetics at any dose. Liver concentrations and plasma protein binding were not measured, and the ob- served nonlinearity was not addressed. Studies on the pharmacokinetics of drugs excreted in the bile have increased in recent years as investigators have become more interested in the effects of liver disease on drug clearance, the effects of first-pass liver clearance on drug bioavailability, the effects of enterohepatic recircu- lation on drug disposition, and the nonlinear excretion of drugs (7-11). Usually these studies have not considered the extent of protein binding, liver-to-plasma and bile- to-liver drug concentration ratios, hepatic blood flow, and bile flow. These factors contribute to the interpretation of hepatobiliary elimination. In addition, the interpreta- tion has been complicated by the use of compounds such as sulfobromophthalein that undergo metabolism. Ideally, a model compound used in studying hepatobiliary elimi- nation would not undergo metabolism, nor enterohepatic recirculation, and would not be pharmacologically ac- tive. The organic anion, bromophenol blue (I), possesses these qualities (2, 4). Therefore, the present investigation was undertaken to: (a ) determine the pharmacokinetic profile 0022-3549/83/ 1000-1 127$01.00/0 @ 1983, American Pharmaceutical Association Journal of Pharmaceutical Sciences I 1127 Vol. 72, No. 10. October 1983

Subcellular electrical characteristics of amphibian muscle

Proteins, nucleic acids and SH containing substances migrated to the cathode indicating a net positive sign of the charge. Increase of amino-acids in the cathode half indicated the possibility of their electromigration or increased proteolysis. However, the total amino-acid content did not increase in experimental muscle indicating that the increase in amino-acid content in the cathode half was due to electromigration only. The electromigration of the protein fraction was smooth and steady in the muscle. The migration of the protein fraction towards the cathode was due to the net positive sign of the charge on them which was dependent of the intracellular pH. Protein estimations of the tissue fluids squeezed out of the muscle account for the intracellular and intercellular protein content. The protein content in the intercellular spaces had a net negative sign of the charge and migrated to the anode. The intracellular protein fraction had a net positive sign of the charge and migrated to the cathode. It is suggested that the intracellular pH is different from that of the intercellular pH, which is assumed to be identical to the physiological pH.