Dyslipidemia can be a comorbidity of both insulin-resistance and atherosclerosis. Hypertriglyceridemia is common in hyperglycemia and is associated with hypoalphalipoproteinemia (low HDL) and with altered nucleotide or purinergic signaling. We therefore hypothesized that extracellular nucleotides may affect hepatic lipoprotein metabolism. Our studies confirm this view and show that nucleotides regulate cellular proteolytic pathways in liver cells and thereby control lipoprotein secretion and their metabolism by hepatic lipase (HL). Treatment of liver cells with the nucleotide, adenosine diphosphate (ADP), stimulates VLDL-apoB100 and apoE secretion, but blocks HDL-apoA-I and HL secretion. ADP functions like a proteasomal inhibitor to block proteasomal degradation and stimulate apoB100 secretion. Blocking the proteosome is known to activate autophagic pathways. The nucleotide consequently stimulates autophagic degradation in liver cells and increases cellular levels of the autophagic proteins, LC3 and p62. Confocal studies show that ADP increases cellular LC3 levels and promotes co-localization of LC3 and apoA-I in an autophagosomal degradation compartment. ADP acts through the G-protein coupled receptor, P2Y13, to stimulate autophagy and block both HDL and HL secretion. Overexpression of P2Y13 increases cellular LC3 levels and blocks the induction of both HDL and HL secretion, while P2Y13 siRNA reduce LC3 protein levels and cause up to a ten-fold stimulation in HDL and HL secretion. P2Y13 gene expression regulates autophagy through the insulin receptor (IR-β). A reduction in P2Y13 expression increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, while increasing P2Y13 expression inhibits the activation of IR-β and Akt. Experiments with epitope-labeled apoA-I and HL show that activation of purinergic pathways has no effect on the internalization and degradation of extracellular apoA-I and HL, which confirms the view that nucleotides primarily impact intracellular protein transport and degradation. In conclusion, elevated blood glucose levels may promote dyslipidemia by stimulating purinergic signaling through P2Y13 and IR-β and perturbing the intracellular degradation and secretion of both HDL and VLDL.

Made available in DSpace on 2017-06-01T18:20:30Z (GMT). No. of bitstreams: 1 dissertaca-_marcelo_silva.pdf: 974611 bytes, checksum: 8bf2b65106d8d1d7d04ec3c0dd5827f6 (MD5) Previous issue date: 2011-03-17 / The production of protease and biosurfactant by Bacillus licheniformis UCP-1014 was investigated in this work. The experiments were performed in Erlenmeyer flasks, in triplicate, and inoculum 10% v/v, 150 rpm and 37ºC. A factorial design was conducted to investigate the concentrations of the medium. Metabolic fluid samples were collected, centrifuged and the supernatant used to determine pH, proteolytic activity and surface tension. The liquid was concentrated by ultrafiltration metabolic the stability and proteolytic activity in the retentate was determined for pH and temperature. In making the retentate was used a factorial design, and protease stability was determined during 10, 20 and 30 days at 28ºC. The determination of protease was performed in the presence of azo-casein. The culture of B.licheniformis UCP-1014 produced 112 U/mL protease in the presence of 1% molasses and urea 0,5%, pH 7,5 at 24h of culture. The reduction in surface tension was not significant in these metabolic conditions. The concentration of proteases produced by B. licheniformis UCP-1014 had the highest stability of enzyme activity in the absence of substrate at pH 7 during 60 min of incubation and maximum thermal stability between 40 90ºC for 90 min. The liquid concentrate and formulated metabolic retained about 50% of proteolytic activity whose value decreased during storage at 28ºC. Proteases produced by B. licheniformis UCP-1014 in the presence of nutrients of low cost can be competitive in the market / A produção de proteases e biossurfactantes por Bacillus licheniformis UCP-1014 foi investigada neste trabalho. Os experimentos foram realizados em frascos de Erlenmeyer de 125 mL, em triplicata, inóculo 10% v/v, a 150 rpm e 37ºC. Um planejamento fatorial foi realizado para investigar as concentrações dos componentes do meio de cultivo. Amostras de líquido metabólico foram coletadas, centrifugadas e os sobrenadantes utilizados para determinar pH, atividade proteolítica e tensão superficial. O líquido metabólico foi concentrado por ultrafiltração e a estabilidade da atividade proteolítica no retentado foi determinada quanto ao pH e à temperatura. A estabilidade do retentado foi investigada por planejamento fatorial e a atividade proteolítica determinada com 10, 20 e 30 dias de armazenamento a 28 ºC. A determinação de proteases foi realizada na presença de azo-caseína. A cultura de B. licheniformis UCP-1014 produziu 112 U/mL de proteases na presença de melaço 1% e uréia 0,5%, a pH 7,5 com 24 h de cultivo. A redução da tensão superficial do líquido metabólico não foi significativa nessas condições de trabalho. O líquido metabólico concentrado reteve cerca de 50% da atividade proteolítica inicial. O concentrado de proteases apresentou a maior atividade enzimática em pH 8 durante 30 min de incubação, retendo 97 % da atividade; a estabilidade térmica máxima foi a 50ºC durante 30 min, retendo 98 % da atividade enzimática. O retentado do líquido metabólico após formulado manteve 54 % da atividade com 30 dias de armazenamento a 28ºC. Proteases produzidas por B. licheniformis UCP-1014 na presença de nutrientes de baixo custo podem ser competitivas no mercado

Made available in DSpace on 2017-06-01T18:20:37Z (GMT). No. of bitstreams: 1 dissertacao_joao_caitano_alves_neto.pdf: 777326 bytes, checksum: 2e15b6afc4e315a3f94d61e55ec5b9ce (MD5) Previous issue date: 2012-09-04 / The reuse of agro-industrial waste as sources of carbon and nitrogen has been investigated in biotechnology for the production of enzymes by microorganisms. Among the microbial enzymes imported into Brazil, the proteases are applied in technological processes in the fields of detergents, pharmaceuticals, cosmetics, food, among others. The aim of this work was to produce proteases by submerged cultivation in the presence of agro-industrial waste. The determination of proteolytic activity was in the presence of 0.2 % azocasein. Submerged cultivation of Bacillus sp. strains isolated were carried out in Erlermeyer flasks. The maximum protease activity was determined in the presence of corn steep liquor. The concentration of the liquid metabolic by ultrafiltration of the metabolic liquid with proteolytic activity retained 80% of the activity. A factorial experimental design was carried out to investigate the stability of the metabolic liquid. The maximum proteolitic activity of the liquid metabolic cell-free (196 U/mL) was determined in the presence of 0.5 % sodium sorbate, 0.5 % calcium chloride, and 7.5 % of glycerol and polyethyleneglycol-200. The enzyme extract formulated retained 68 % of the proteolytic activity after 10 days at storage at room temperature (28 ˚C). The retentate with proteolytic activity showed optimum pH 9 and 11 and retention 90 - 100 % of the activity for 90 min at optimum pH; the optimum temperature was 50 ˚C e the maximum thermal stability was at 40 ˚C for 30 min at pH 11. The formulation of metabolic liquid concentrate with proteolytic activity which has thermal stability at alkaline pH is a bioproduct which can be used as an additive in detergents. / O reaproveitamento de resíduos agroindustriais como fontes de carbono e de nitrogênio tem sido investigado na área de biotecnologia para produção de enzimas por micro-organismos. Dentre as enzimas microbianas importadas no Brasil, as proteases são aplicadas no processamento tecnológico de detergentes, fármacos, cosméticos, alimentos, dentre outros. O objetivo deste trabalho foi produzir proteases por cultivo submerso utilizando resíduos agroindustriais. A determinação da atividade proteolítica ocorreu na presença de azocaseína a 0,2 %. Cultivos submersos de culturas isoladas de Bacillus sp. foram realizados em frascos de Erlermeyer. A atividade máxima de proteases foi determinada na presença de milhocina. A concentração do líquido metabólico com atividade proteolítica por ultrafiltração reteve cerca de 80 % da atividade inicial. Um planejamento experimental fatorial foi realizado para investigar a estabilidade do líquido metabólico. A maior atividade proteolítica média do líquido metabólico livre de células (196 U/mL) foi determinada na presença de sorbato de sódio a 0,5 %, cloreto de cálcio a 0,5 %, glicerol a 7,5 % e polietilenoglicol-200 a 0,5 %. O extrato enzimático formulado reteve 68 % da atividade proteolítica com 10 dias de armazenamento à temperatura ambiente (28 ˚C). O retentado com atividade proteolítica apresentou pH ótimo 9 e 11 e retenção de 90 - 100 % da atividade durante 90 min em pH ótimo; a temperatura ótima foi 50 ˚C e a estabilidade térmica máxima a 40 ˚C durante 30 min a pH 11. A formulação de líquido metabólico concentrado com atividade proteolítica que apresenta estabilidade térmica em pH alcalino é um bioproduto que pode ser utilizado como aditivo em detergentes.

The hard tick Ixodes ricinus is an important blood-feeding parasite that transmits tick- borne diseases, such as tick-borne encephalitis and Lyme disease. Ticks employ a battery of proteolytic enzymes, including cathepsins, to digest their bloodmeal. These proteins are potential targets for the development of anti-tick vaccines. This work is focused on cathepsin L from I. ricinus (IrCL), namely its isoenzymes IrCL1 and IrCL3. IrCL1 was expressed in Pichia pastoris and chromatographically purified. Its substrate specifity was determined by the cleavage of (a) peptide fluorogenic substrates and (b) protein substrates analyzed by mass spectrometry. The proteolytic activity of IrCL1 was modulated by its interaction with glycosaminoglycans, which affected the pH optimum value. Futhermore, a proteolytically active mutant of IrCL1 with reduced number of N-glycosylation sites was prepared; this form will be used for crystallization experiments. IrCL3 was expressed in Escherichia coli, refolded and activated to its active form. The proteolytic activity of IrCL3 is in many ascpects similar to that of IrCL1, including substrate specifity, acidic pH optimum and activity modulation by glycosaminoglycans. Key words: cysteine proteases, cathepsin L, hard tick I. ricinus, substrate specifity, proteolytic activity...

Equistatin from the sea anemone Actinia equina contains a protein domain Eqd2 which inhibits aspartic peptidases and has not been characterized in detail. Recombinant Eqd2 was produced in the yeast expression system, and a protocol for its chromatographic purification was designed. The inhibitory specificity of Eqd2 was determined using a fluorescence inhibition assay, showing that Eqd2 is a highly selective inhibitor of cathepsin D-like and pepsin-like aspartic peptidases of family A1. Furthermore, size exclusion chromatography was used to analyze the Eqd2-peptidase complex and Eqd2 oligomerization in solution. Initial screening of crystallization conditions for Eqd2 was performed towards its structural analysis. This work provides important new information about Eqd2 as a unique type of natural inhibitors of aspartic peptidases. Its interaction mechanism can be exploited in the development of synthetic mimetics for regulation of medically important peptidases. (In Czech) Key words: peptidase inhibitors, proteolytic enzymes, activity and inhibition of enzymes, recombinant expression, protein purification, protein crystallization, equistatin

Die Dicklegung der Milch, ausgehend von der hydrolytischen Spaltung des κ-Caseins, stellt den ersten essentiellen Schritt in der Käseherstellung dar. Dabei finden Gerinnungsenzyme verschiedener Herkunft Anwendung, deren neueste Varianten auf Grundlage des aktuellen Forschungsstandes umfassend charakterisiert werden. Verschiedene Kälberlabpräparate, mikrobielle Gerinnungsenzyme aus Rhizomucor miehei und mit Hilfe von gentechnisch modifizierten Mikroorganismen gewonnenes Chymosin (FPC) aus Rind und Kamel werden mittels HPLC und Elektrophorese hinsichtlich ihrer Zusammensetzung analysiert. Die neueste Generation mikrobieller Enzyme weist im Gegensatz zur herkömmlichen Variante keine Nebenkomponenten und damit einen höheren Aufreinigungsgrad auf. Die unspezifische proteolytische Aktivität wird durch fluorimetrische Quantifizierung der in 12 % TCA löslichen Stickstoffkomponenten bestimmt, die nach Inkubation rekonstituierter Magermilch bei 32 °C und pH 6,5 über 24 h mit den Enzymen freigesetzt werden. Mikrobielle Gerinnungsenzyme besitzen eine signifikant höhere unspezifische proteolytische Aktivität gegenüber chymosinbasierten Präparaten, deren Auswirkung sich bei Erhöhung der zugegebenen Enzymmenge besonders ausgeprägt darstellen. Oszillationsrheometrische Analysen lassen bei gleicher Enzymaktivität eine geringere Gelfestigkeit nach 40 min bei Einsatz von mikrobiellen Präparaten im Vergleich zu Kälberlab und bovinem FPC erkennen. Zusätzlich wird eine Abhängigkeit der Flockungszeit und der Gelfestigkeit vom eingesetzten Substrat beobachtet, die für Chymosin aus Kamel besonders stark ausgeprägt ist. Die Substratspezifität dieses Enzyms ist weder mit der des bovinen Chymosins noch mit der der mikrobiellen Gerinnungsenzyme identisch. Im Rahmen von Käsungsversuchen im Labor-, Pilot- und Industriemaßstab wird eine signifikant höhere Käseausbeute (0,50 - 1,19 %) bei Verwendung vom traditionellem Kälberlab im Vergleich zur neuesten Generation der kommerziellen mikrobiellen Substitute ermittelt. Im Verlaufe der Reifung von Schnittkäse wird durch mikrobielles Gerinnungsenzym gegenüber Kälberlab eine signifikant höhere Menge an Nichtproteinstickstoff freigesetzt sowie ein unterschiedliches Profil an Proteinabbauprodukten gebildet. Die höhere proteolytische Aktivität resultiert in einer signifikant stärker ausgeprägten Bitterkeit der mit mikrobiellem Gerinnungsenzym hergestellten Käse nach 12 Wochen Reifungszeit. / Clotting of milk caused by hydrolytic cleavage of κ-casein is the first important step in cheese milk processing. This cleavage is caused by clotting enzymes of different origin, which are comprehensively characterized by considering results of latest investigations. The composition of selected calf rennets, microbial coagulants derived from Rhizomucor miehei and genetically engineered chymosin (FPC) derived from cow and camel is analyzed by HPLC and electrophoresis. In contrast to conventional products, the latest generation of microbial coagulants does not show minor components in a detectable amount because of a sufficient purification. The unspecific proteolytic activity is determined by fluorimetric quantification of 12 % tricloric-acid-soluble nitrogen, which is released by the enzymes from reconstituted skim milk, pH 6.5, after incubation at 32 °C for 24 h. Microbial coagulants show a significantly higher unspecific proteolysis as compared to chymosin-based clotting enzymes, especially when the enzymes are added in amount higher than used during cheese-making. Small amplitude oscillation rheometry analysis showed a lower gel firmness after 40 min of gelling when microbial coagulants were applied instead of calf rennet or FPC. Furthermore, flocculation time, gel formation time and gel firmness additionally depends on the test substrate, and this dependency is exceptionally pronounced when camel chymosin was used. The substrate specificity of this enzyme is neither identical to that of bovine chymosin nor to that of microbial coagulants. Cheese making experiments in laboratory-, pilot- and commercial-scale revealed a significantly higher cheese yield (0.50 - 1.19 %) when using calf rennet instead of microbial coagulant of the latest generation. During ripening of semi-hard cheese a higher amount of non-protein-nitrogen was released and a different electrophoretic casein degradation profile was generated when using microbial enzymes. Enhanced proteolysis is responsible for a significantly higher pronounced bitterness of microbial produced cheese after 12 weeks of maturation.

The aim of this study was to better understand the role of conditioned medium (CM) in Spodoptera frugiperda Sf9 insect cell proliferation and recombinant protein production using the baculovirus expression system. CM was found to stimulate cell proliferation. Addition of CM and 10 kDa CM filtrate to an Sf9 culture decreased the lagphase and the maximum cell density was reached earlier than for cultures in fresh medium. The positive effect of 10 kDa CM filtrate showed that CM contains at least one small growth promoting factor. The effect was not eliminated by trypsin treatment. Addition of CM or 10 kDa CM filtrate to Sf9 cultures was found to have a negative effect on the recombinant protein production. The effect was thought to be indirect and most probably via the impact of CM on cell physiology. CM was also found to contain proteinase activity. The proteinase was identified as Sf9 cathepsin L. A proform with a molecular mass about 49 kDa and two active forms at about 39 and 22 kDa were found. The role of cathepsin L in Sf9 cultures is not yet clear. However, the knowledge of the presence of this proteinase in CM can be of great value for improving product quality and yield. Further, CM was found to have other properties as well: a concentrated fraction of CM exhibited strong antibacterial activity towards Bacillus megaterium and a weaker activity towards Escherichia coli. B. megaterium lysed rapidly after incubation in the CM fraction. Repeated subculturing of Sf9 cells provoked a switch in growth kinetics. After 30-45 passages the cells started to proliferate earlier after inoculation and addition of CM had no longer a growth stimulating effect. However, CM still stimulated growth of a culture with low passage (LP) number (up to 45 passages). High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. This synchronisation was more pronounced for HP cells than for LP cells, which correlated to a higher recombinant protein production in baculovirus infected HP cells than in LP cells. Synchronisation of cells in G2/M by yeastolate-limitation before infection with baculoviruses suggested that the degree of synchronisation is connected to the cell density dependent decrease in recombinant protein production of Sf9 cultures. / QC 20101222

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDA) is a deadly disease due to early metastasis and resistance to chemotherapy. PDA is commonly associated with a dense desmoplastic stroma, which forms a protective niche for cancer cells. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme, is abundantly expressed in pancreatic cancer cells and crosslinks proteins through acyl-transfer transamidation between glutamine and lysine residues. The objective of the study was to determine the functions of TG2 in the pancreatic stroma. Orthotopic pancreatic xenografts and co-culture systems tested the mechanisms by which the enzyme modulates tumor-stroma interactions. We showed that TG2 secreted by cancer cells is enzymatically active and renders the stroma denser by crosslinking collagen, which in turn activates fibroblasts and stimulates their proliferation. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are transcription factors involved in mechanotransduction. The TG2-mediated fibrosis-rich, stiff microenvironment conveys mechanical cues to cancer cells leading to activation of YAP and TAZ, promoting cell proliferation and tumor growth. Stable knockdown of TG2 in pancreatic cancer cells led to decreased size of pancreatic xenografts and increased sensitivity of xenografts to gemcitabine. Taken together, our results demonstrate that TG2 secreted in the tumor microenvironment orchestrates the crosstalk between cancer cells and the stroma, fundamentally impacting tumor growth and response to chemotherapy. Our study supports TG2 inhibition in the pancreatic stroma as a novel strategy to block pancreatic cancer progression.

Presented diploma thesis is focused on testing encapsulation methods of enzymes and probiotic bacteria. In the theoretical part a summary of different encapsulation techniques used in food industry is given. Further, materials for encapsulation, above all polysaccharides are presented. Next, some procedures of encapsulation of biopolymers and microorganisms – mainly enzymes and probiotic cultures are discussed. In the experimental part methods for preparation of several types of particles based on polysaccharides and liposomes are introduced. Particles were used for encapsulation of selected hydrolytic enzymes and probiotic strains Bifidobacterium breve a Lactobacillus acidophilus. The encapsulation effectiveness was evaluated by analysis of total proteins and enzyme activities. Particles sizes and their stability in water, in selected model foods and model body fluids were observed, too. According to results obtained in this work it was found that encapsulation of enzymes into polysaccharide particles were succesfull in all types of particles (encapsulation effectivness was more than 50 %). Polysaccharide particles showed a very good stability in body fluids as well as in model foods. As the most suitable materials for enzymes encapsulation chitosan and liposomes were found. Polysaccharide particles were used also for the encapsulation of microorganisms. The stability of particles with lactic acid bacteria was similar to particles containig enzymes, very good stability was verified aslo in model foods and model body fluids. Encapsulation enables long-term stabilization of biologically active compounds as well as posibility of their transport and controlled releasing in gastrointestinal tract. Encapsulation of probiotic bacteria could preserve their viability and long-term survival until the product expiration date. Thus, encapsulation is one of the most promissing procedures for production of foods and food suplements of great quality and high additional value.

Indiana University-Purdue University Indianapolis (IUPUI) / Patients with the genetic disorder tuberous sclerosis complex (TSC) suffer from neoplastic growths in multiple organ systems. These growths are the result of inactivating mutations in either the TSC1 or TSC2 tumor suppressor genes, which negatively regulate the activity of mammalian target of rapamycin complex 1(mTORC1). There is currently no cure for this disease; however, my research has found that cells harboring TSC2-inactivating mutations derived from a rat model of TSC are sensitive to apoptosis induced by the clinically approved proteasome inhibitor, bortezomib, in a manner dependent on their high levels of mTORC1 activation. We see that bortezomib induces the unfolded protein response (UPR) in our cell model of TSC, resulting in cell death via apoptosis. The UPR is induced by accumulation of unfolded protein in the endoplasmic reticulum (ER) which activates the three branches of this pathway: Activating transcription factor 6 (ATF6) cleavage, phosphorylation of eukaryotic initiation factor 2α (eIF2α), and the splicing of X-box binding protein1 (XBP1) mRNA. Phosphorylation of eIF2α leads to global inhibition of protein synthesis, preventing more unfolded protein from accumulating in the ER. This phosphorylation also induces the transcription and translation of ATF4 and CCAAT-enhancer binding protein homologous protein (CHOP). Blocking mTORC1 activity in these cells using the mTORC1 inhibitor, rapamycin, prevented the expression of ATF4 and CHOP at both the mRNA and protein level during bortezomib treatment. Rapamycin treatment also reduced apoptosis induced by bortezomib; however, it did not affect bortezomib-induced eIF2α phosphorylation or ATF6 cleavage. These data indicate that rapamycin can repress the induction of UPR-dependent apoptosis by suppressing the transcription of ATF4 and CHOP mRNAs. In addition to these findings, we find that a TSC2-null angiomyolipoma cell line forms vacuoles when treated with the proteasome inhibitor MG-132. We found these vacuoles to be derived from the ER and that rapamycin blocked their formation. Rapamycin also enhanced expansion of the ER during MG-132 stress and restored its degradation by autophagy. Taken together these findings suggest that bortezomib might be used to treat neoplastic growths associated with TSC. However, they also caution against combining specific cell death inducing agents with rapamycin during chemotherapy.