Lorsque l'allaitement est impossible, du lait maternel pasteurisé (LMP) est préférentiellement administré, en particulier aux nouveau-nés hospitalisés. La pasteurisation de Holder (62,5°, 30 min) est appliquée pour des raisons sanitaires mais pourrait réduire l'absorption des lipides via l'inactivation des lipases endogènes du lait. L’homogénéisation du LMP pourrait contrer cet effet négatif en augmentant la surface disponible pour l’adsorption des enzymes. L’objectif de cette thèse était d’étudier l’impact de la pasteurisation de Holder et de l’homogénéisation par ultrasons sur la digestion du LM chez le nouveau-né. Un modèle de digestion in vitro a été mis en place pour évaluer la digestion gastro-intestinale de LM cru (LMC) vs. LMP aux stades « nouveau-né à terme » ou « prématuré ». Une étude clinique a été menée chez des nouveau-nés prématurés pour comparer la digestion gastrique de (A) LMC vs. LMP et (B) LMP vs. LM pasteurisé et homogénéisé (LMPH).La pasteurisation et l’homogénéisation ont modifié la structure initiale du LM, ses cinétiques digestives et sa désintégration structurale. In vitro, la pasteurisation a réduit la lipolyse gastrique au stade à terme, alors qu’aucun impact n'a été observé au stade prématuré. La lipolyse intestinale, in vitro, a été réduite. In vivo, la pasteurisation a accélérée la protéolyse gastrique de la lactoferrine et a réduit celle de l’a-lactalbumine. L'homogénéisation a accéléré la lipolyse et la protéolyse de l'albumine sérique. Concernant les conditions physiologiques, l’activité lipolytique postprandiale était augmentée après adm / When breastfeeding is not possible, pasteurized human milk (PHM) from milk banks is preferentially administered, especially for vulnerable hospitalized newborns. Holder pasteurization (62.5°, 30 min) is applied for sanitary reasons but may reduce fat absorption through inactivation of milk endogenous lipases. This could be counteracted by homogenization of PHM through an increase of the specific surface available for enzyme adsorption. The objective of this thesis was to study the impact of Holder pasteurization and ultrasonic homogenization on the digestion of HM in the newborn. An in vitro dynamic model was used to evaluate the gastrointestinal digestion of raw HM (RHM) vs. PHM at preterm and term stages. A clinical trial was conducted on hospitalized preterm newborns for comparing the gastric digestion of (A) RHM vs. PHM and (B) PHM vs. pasteurized-homogenized HM (PHHM). Pasteurization and homogenization affected the HM initial structure and its digestive kinetics and structural disWhile gastric lipolysis was reduced after pasteurization in term in vitro study, no impact was observed at the preterm stage. Intestinal lipolysis, in vitro, was reduced by pasteurization. Gastric proteolysis was selectively affected by pasteurization, being, in vivo, faster for lactoferrin and slower for a-lactalbumin. Homogenization increased lipolysis and proteolysis of serum albumin. Some physiological gastric conditions were affected by treatments: RHM had enhanced postprandial lipolytic activity and PHHM had a reduced gastric emptying time. The in vivo data described here may help to i

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Orientadores: Adriane Elisabete Antunes de Moraes, Izildinha Moreno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-24T08:36:22Z (GMT). No. of bitstreams: 1 Ferreira_NataliaChinellatodeAzambuja_M.pdf: 4159232 bytes, checksum: 2e7450f8e5d25cd46199b110960d9b48 (MD5) Previous issue date: 2013 / Resumo: A utilização de culturas adjuntas é uma das novas alternativas tecnológicas que vem sendo estudada por pesquisadores de diversos países visando a melhoria da qualidade geral de produtos alimentícios, em especial produtos lácteos como queijos, leites fermentados, iogurte e outros. Outra vantagem é a obtenção de novas variedades de produtos que passam a apresentar sabor, aroma e outras características diferenciadas. O presente trabalho teve como objetivo a avaliação do papel da cultura de L. helveticus (LH-B02 / CHR-Hansen) na acidificação, proteólise, redução do amargor, determinação do perfil de aminoácidos das amostras de queijo Prato e possível liberação de peptídeos bioativos. No estudo os queijos foram fabricados com cultura CHN-22 (cultura mesofílica aromática / CHR-Hansen) com e sem adição da cultura adjunta L. helveticus (LH) de modo a verificar se a presença da cultura adjunta influencia na qualidade da maturação do queijo Prato e se esta cultura é possível produtora de peptídeo bioativo inibidores da Enzima Conversora de Angiotensina (ECA). Para obtenção dos queijos Prato foram realizados três processamentos em planta piloto, com dois tratamentos diferentes, com e sem adição da cultura adjunta de L. helveticus. Dentro do período de maturação, ou tempo de estocagem, dos queijos, foram realizadas as análises microbiológicas, físico-químicas, eletroforéticas, cromatográficas e análise sensorial. Os queijos elaborados apresentaram composição típica de queijo Prato e atenderam aos padrões microbiológicos exigidos para o consumo do produto. Em relação ao perfil eletroforético observou-se aumento no desdobramento da fração ?s1-caseína pela ação do coalho, com surgimento de uma banda de maior intensidade, correspondente à ?s1-I-caseína, mais evidente no queijo com a adição da cultura de L. helveticus. Essa cultura também intensificou a degradação da ?-caseína com aparecimento das bandas de ?3-, ?1- e ?2-, já visíveis com 10 dias de maturação. A amostra de queijo adicionado da cultura adjunta foi mais bem aceita sensorialmente do que a amostra controle (apenas adicionado de CHN-22) e queijo comercial diferindo estatisticamente (p?0,05) destes em alguns parâmetros, tais como aceitação global, aroma, sabor e intenção de compra. Os índices de extensão de proteólise (IEP) e profundidade da proteólise (IPP) dos queijos Prato com e sem adição de cultura adjunta, praticamente não diferiram entre si, embora em todos os casos tenham sido observados aumento dos índices ao longo do tempo de maturação. Através da análise de aminoácidos livres, as amostras de queijo adicionadas de L. helveticus apresentaram teores maiores de aminoácidos comparadas com as amostras sem adição desta cultura. Pelo teste estatístico apenas os aminoácidos histidina, cistina e triptofano não diferiram quantitativamente entre os tratamentos (p?0,05). Na eletroforese capilar (CE) foi encontrado apenas para a amostra adicionada da cultura adjunta aos 31 dias de maturação um pico com tempo de retenção na faixa de 9 a 12 minutos, indicando a possível presença dos tripeptídeos de interesse na amostra / Abstract: The use of adjunct cultures is a new alternative technology that have been studied by researchers from several countries in order to improve the overall quality of food products , especially dairy products such as cheese , fermented milk , yogurt and others. Another advantage is to obtain new varieties of products with new flavours and texture. This study aimed to evaluate the role of culture L. helveticus (LH-B02 / CHR- Hansen) acidification, proteolysis, reducing bitterness, determination of the amino acid profile of Prato cheese and possible release of bioactive peptides. In the present study the cheeses were manufactured with culture CHN-22 (mesophilic aromatic culture / CHR-Hansen) with and without adjunct culture of L. helveticus (LH) in order to verify if the presence of the adjunct culture influences the quality of the cheese ripening and if this culture can produce bioactive peptide that may couse the inhibition of Angiotensin Converting Enzyme (ACE). To obtain the Prato cheese three processins in pilot plant were done, with two different treatments, with and without addition of adjunct culture of L. helveticus. Within the period of maturation, and storage time it was conducted microbiological, physicochemical, electrophoretic, chromatographic and sensory analysis. The cheeses produced presented composition typical of Prato cheese and attended the microbiological standards required for the consumption of the product. Regarding the electrophoretic profile it was observed an increase in a break on the fraction ?s1-casein by the action of rennet, with the appearance of a band of greater intensity, corresponding to ?s1-I-casein, most evident in the cheese with the addition of the culture of L. helveticus. This culture also intensified the degradation of ?-casein with the appearance of bands ?3, ?1 and ?2-casein, already visible at 10 days of ripening. The sample of the cheese added adjunct culture was more acceptable sensory than the control sample (just added CHN-22) and was statistically different (p?0.05) of commercial cheese in some of these parameters, such as overall aceptability, aroma, taste and purchase intent. The extension rates of proteolysis (IEP) and depth of proteolysis (IPP) of the Prato cheese with and without addition of adjunct culture were different. In all cases it was observed increased rates over time for maturation. By analyzing the free amino acid, samples of cheese added L. helveticus presented higher levels of amino acids compared with the samples without the addition of this culture. By the statistical test only the amino acid histidine, cysteine and tryptophan did not differ between treatments (p?0.05). In capillary electrophoresis (CE) a peak with retention time in the range of 9 to 12 minutes, indicating the possible presence of tripeptides of interest was found in the sample added by the adjunct culture after 31 days of ripening / Mestrado / Nutrição / Mestra em Ciências da Nutrição e do Esporte e Metabolismo

O pH final (pHf) no músculo post mortem é amplamente utilizado como um indicador potencial de maciez e é um fator importante associado à qualidade de carne. O Brasil é líder mundial nas exportações de carne bovina, porém não são conhecidos os valores de pHf do músculo pós-abate e seu impacto na qualidade da carne de animais machos inteiros Bos taurus indicus. Baseado no exposto, objetivou-se com este trabalho identificar o atual \"status\" dos valores de pHf 24 h post mortem apresentados no frigorífico comercial, e a influência do pHf na proteólise muscular e parâmetros de qualidade como perda por gotejamento, cor e maciez de machos inteiros avaliados aos 0, 7, 14, 21 e 28 dias de maturação. Foi realizado um estudo preliminar, com 399 carcaças em abatedouro comercial, para conhecer a ocôrrencia pHf de animais machos inteiros abatidos no Brasil, podendo-se identificar três faixas de pHf: pHf 5,5 até 5,8 (baixo-normal); pHf 5,81 até 6,3 (intermediário); pHf > 6,3. Músculos Longissimus dorsi (n=12) foram porcionados em bifes, embalados a vácuo, classificados nos três diferentes grupos de pHf e maturados a 2°C por 0 (48h), 7, 14, 21 e 28 dias. Foram avaliadas características de perda por gotejamento, cor, força de cisalhamento, índice de fragmentação miofibrilar, colágeno total e solúvel, assim como proteólise miofibrilar. Carnes pertencentes ao grupo de pHf alto, apresentaram maior maciez (P<0,05) desde o início do experimento, quando comparado aos outros grupos do estudo. Bifes de pHf intermediário apresentaram os valores de força de cisalhamento mais elevados (P<0,05), o que indica um processo de maciez mais lento quando comparado com os outros grupos de pHf. Perdas por gotejamento, diminuem com o aumento nos valores de pHf (P< 0,05). Valores de L*, a*, b*, não apresentam diferenças entre os grupos de pH. O índice de fragmentação miofibrilar (MFI) foi maior em carne de pHf alto, seguido pelo grupo de pHf baixo-normal, e os menores valores pertenceram ao grupo de pHf intermediário (P<0,05) através do tempo de maturação. A degradação das proteínas como desmina e troponina-T foi maior e mais visível no pHf alto e baixo-normal desde as 48 h post mortem em comparação com o pHf intermediário, onde foram observadas as bandas de sua degradação quase ao final do período experimental. Proteínas chave como filamina e nebulina apresentaram maior degradação desde as 48 horas post mortem no pHf alto e mais lenta no pHf intermediário. Não foram observadas diferenças (P> 0,05) nos valores de colágeno total e solúvel nos diferentes grupos de pHf e nem através do tempo de maturação. Em geral, o grupo de pHf intermediário (5,8-6,3) apresentou maior inconsistência quanto às características de maciez e fragmentacão miofibrilar, consequência de uma proteólise tardia. / The ultimate pH (pHu) of the postmortem muscle is broadly use as a potential meat tenderness indicator and is an important factor related to meat quality. Brazil is the bovine meat exporter world leader; nevertheless the values of the postmortem pHu of the muscle and its impact on the meat quality of Bos indicus taurus bulls, are unknown. Therefore, the objective of this research was to establish the values of pHu after 48 hours postmortem and its impact over the meat quality through the characterization of the biochemical processes that occurs in the muscle Longissimus dorsi of Bos taurus indicus bulls. In order to know the actual pHu of the Brazilian bulls, a preliminary study were made, using 399 carcasses of a commercial slaughter house and resulting three levels of pHu: pHu 5,5 to 5,8 (low-normal); pHu 5,81 to 6,3 (intermediate); pHu > 6,3 (high). Twelve muscles Longissimus dorsi were cut into steaks, classified into the three groups of pHu, vacuum packed up and matured at 2°C for 0 (48h), 7, 14, 21 and 28 days. Drip loss, color, shear force, myofibril fragmentation index, total and soluble collagen and miofibrillar proteolysis were evaluated. The high pHu meat presented more tenderness from the beginning of the experiment (P<0,05) when compared with the other groups. The medium pHu steaks presented the highest shear force values (P<0,05), indicating a slower tenderization process in relation to the other pHu groups. The drip loss values diminished as the value of pHu rised (P< 0,05). The values of L*, a*, b* did not show significant differences within the groups of pHu. The highest miofibrillar fragmentation index (MFI) through the maturation time, was found in the high pHu meat, followed by the low pHu group and then by the intermediate pHu group (P<0,05). At 48 hours postmortem, degradation of proteins, like desmin and troponin-T, was higher and more evident in the high and low-normal pHu when compared to the intermediate pHu, where the bands and its degradation were only observed by the end of the experiment. Key proteins like filamin and nebulin, showed higher degradation rate from the 48 h postmortem in the high pHu group, and a slower degradation in the intermediate pHu group. No differences were observed through the pHu groups, nor through the maturation time (P<0,05), for total and soluble collagen values. Then, it is possible to say that the meat quality of Bos taurus indicus bulls, especially the tenderness, is related to the pHu, and can be affected by the proteolytic systems activity. In general, the tenderness and the MFI were more inconsistent in the medium pHu group (5,8-6,3) than in the two other pHu groups, as a consequence of a late proteolysis.

Rhomboid intramembrane serine proteases cleave polypeptide chains within lipid bilayer. Rhomboid proteases were originally discovered in Drosophila melanogaster where they regulate ontogenesis of the fly, but they are present in all domains of life. Nowadays, various diseases, such as malaria, amoebiasis, Parkinson's disease, various tumour malignancies, and diabetes, have been linked with rhomboid proteases. However, natural substrates and function of most rhomboids remain elusive. Cell biology tools are needed for unravelling functions of rhomboids, as well as for potential pharmacological applications, and this together fuels the effort to develop specific rhomboid inhibitors. The inhibitors known to date always bear an electrophilic warhead attacking the nucleophilic serine of the atypical serine-histidine catalytic dyad of rhomboid. From the various developed inhibitors, peptidyl -ketoamides substituted at the ketoamide nitrogen by hydrophobic groups, discovered in our laboratory, hold the biggest potential. They are potent, reversible, selective, tunable, and are built around a pharmacophore already approved for medical use. Here, I set out to improve peptidyl -ketoamides by exploring the chemical space in the active site of rhomboid and testing substituents of the ketoamide nitrogen of increasing...

Surface expression of recombinant proteins has attracted a lot of attention due to its potential in applications such as enzyme production, vaccine delivery and bioremediation. Autotransporters have been used for surface expression of a variety of proteins, but the expression systems reported in literature have typically been inflexible and incapable of detecting proteolysis, thereby limiting surface expression yield. In this thesis, a modular surface expression system, utilizing dual tag detection, was therefore created. It was based on the adhesin involved in diffuse adherence (AIDA-I) autotransporter, and was here used to express the model proteins SefA and H:gm on the cell surface of Escherichia coli. Due to the dual tag detection system, proteolysed H:gm could be successfully verified on the cell surface. By optimizing cultivation conditions, surface expression yield of SefA was increased by 300 %, and proteolysis reduced by 33 %. While proteolysis could not be eliminated completely, the work presented in this thesis is a major step towards a general system for surface expression of a wide range of proteins in varied applications. / <p>QC 20130506</p>

A biochemical characterization of ubiquitin (Ub)-proteasome pathway activity was conducted in <I>Zinnia</I> mesophyll cell cultures to examine potential differences between differentiating cells of tracheary element (TE) cultures and non-differentiating cells of control cultures. The pathway is highly active throughout development of differentiating TEs, a programmed cell death (PCD) process during which the majority of cellular proteins and biochemical processes are expected to be down-regulated in activity and/or expression. Addition of the proteasome inhibitors <I>clasto</I>-lactacystin Beta-lactone (LAC) and carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) at culture initiation prevented TE differentiation in this system. Proteasome inhibition at 48h did not alter the final percentage of TEs compared to controls. However, proteasome inhibition at 48 h delayed the differentiation program by approximately 24 h, as indicated by examination of morphological markers and the expression of putative autolytic cysteine proteases.These results suggest that proteasome activity is required both for induction of TE differentiation and for progression of the TE program in committed cells. Treatment at 48 h with LLL resulted in partial uncoupling of autolysis from differentiation. Results of protease activity gel analysis suggest that incomplete autolysis was due to the ability of LLL to inhibit TE cysteine proteases. A characterization of phytohormone-stimulated growth of non-differentiating cultured <I>Zinnia</I> cells is also presented. Differential effects on radial cell expansion versus cell elongation were observed for the four plant growth regulators examined. Auxin (naphthaleneacetic acid, NAA) and a brassinosteroid (2,4-epibrassinolide, BI) stimulate only cell elongation. Cytokinin (N-6-benzyladenine, BA) has a greater effect on growth in cell girth rather than length. Gibberellic acid (GA₃) has equivalent effects on expansion in both dimensions. These results demonstrate that radial cell expansion and cell elongation can be uncoupled, and therefore, may be controlled by different mechanisms. Additionally, this study establishes the utility of <I>Zinnia</I> suspension cultures as a valuable model for studies of cell expansion. Finally, we modified <I>Arabidopsis</I> plant growth conditions to promote proliferation of secondary tissues, permitting the separation of secondary xylem from bark (phloem plus nonvascular) tissues using hypocotyl-root segments. Dissected tissues were used for semi-quantitative and quantitative RT-PCR and for the construction of bark and xylem cDNA libraries for PCR-based screening of several Ub pathway components, including Ub-conjugating enzymes (<I>UBCs</I>), deubiquitinating enzymes (DUBs), and an Alpha (<I>PAF1</I>) and Beta (<I>PAF1</I>) subunit of the proteasome. All targeted <I>UBC</I> families, candidate <I>UBCs</I> and DUBs, and proteasome subunits are expressed in secondary xylem and bark in this system. / Ph. D.

We also investigated whether BDE-47 exposure could worsen PD situation by applying transgenic Drosophila (fly) model in which human α-synuclein (α-syn) was overexpressed in wide-type fly to simulate PD. BDE-47 (0, 2, 10 and 50 µM) was fed to flies continuously for 30 days. Integrated LC-MS and GC-MS profiling indicated metabolic changes in tryptophan, phenylalanine, purine, and alanine, aspartate and glutamate pathways, similar to those from mouse experiment. After quantified metabolites of interest by LC-triple quadrupole MS, we confirmed the slowed-down formation of KYNA (kynurenic acid, a neuro-protector) and speeded-up formation of 3HKYN (3-hydroxykynurenine, a neurotoxin) in all BDE-47 exposed groups on the 20th exposure day. The levels of SAM/SAH (methylation biomarker) and GSH/GSSG (oxidative stress biomarker) were found to decrease on the 30th exposure day. Collectively, we propose that BDE-47 could induce imbalance of kynurenine metabolism, insufficient methylation and oxidative stress, which might contribute to the PD progression. To further explore the underlying mechanism of 6-OH-BDE-47 induced neurotoxicity, we conducted omics study of metabolic changes induced by 6-OH-BDE-47 on N2a cells. Cells were exposed to 6-OH-BDE-47 (0, 0.5 and 1 μM) for 24 hours. Considerable metabolic changes in pyrimidine and purine metabolism were observed in high exposure condition while oxidative stress was appeared under low exposure condition. Moreover, 6-OH-BDE-47 was found to affect the dopamine production. iTRAQ proteomics was carried out and pinpointed the dysregulation of ribosome, proteasome, RNA metabolism, aminoacyl-tRNA biosynthesis, vesicular trafficking, purine pathway, and mitochondria electron transport. Immunocytochemistry and Western blot analysis further confirmed that 6-OH-BDE-47 could inhibit autophagy flux, which might result in the aberrant protein aggregation, a pathological hallmark of PD. We further investigated whether 6-OH-BDE-47 exposure could directly induce PD pathology in Sprague Dawley rat. 6-OH-BDE-47 (0.1, 1 and 10 µg) was stereotaxically injected into the right VTA and SNc regions in the midbrain of rat where there are abundant dopaminergic neurons. The apomorphine-induced rotation test indicated significant deterioration in motor function in the group receiving injection of 10 µg. Striatal dopamine was found to decline in a dose-dependent manner. Notably, 6-OH-BDE-47 also promoted the formation of α-syn aggregate, an important pathological hallmark of PD. Proteomics study revealed that protein degradation processes were crucial rather than oxidative stress in 6-OH-BDE-47 induced neurotoxicity in vivo. Mechanistic study based on Western blot further confirmed that 6-OH-BDE-47 could inhibit ubiquitination and autophagy. Collectively, the rat experiment demonstrated that 6-OH-BDE-47 administration could induce motor defect by impairing dopaminergic system and promote α-syn aggregation by inhibiting ubiquitination and autophagy, suggesting that 6-OH-BDE-47 could be a novel risk factor of PD.;Polybrominated diphenyl ethers (PBDEs), as one typical persistent organic pollutants (POPs), are widely spread in the environment and pose potential adverse impacts on human health. As a predominant congener of PBDEs, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) has been reported to affect habituation capability, synaptic plasticity, and vesicular neurotransmitter release. As an important in vivo metabolite derived from BDE-47, 6-hydroxy-BDE-47 (6-OH-BDE-47) was also reported as a neurotoxin. However, the possible linkages between BDE-47/6-OH-BDE-47 exposure and typical neurodegenerative diseases such as Parkinson's disease (PD) are still unclear. Mass spectrometry (MS) based omics integrated with bioinformatics is emerging as a powerful tool to evaluate metabolic changes occurred after different exposures. Here we developed non-targeted metabolomics, lipidomics, and isobaric tag for relative and absolute quantitation (iTRAQ) proteomics methods based on liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) to depict BDE-47/6-OH-BDE-47 induced metabolic changes and to explore the possible contribution of their exposure to PD pathology/pathogenesis. BDE-47 dissolved in corn oil (0, 1, 10 and 100 mg/kg bwt) was orally administered to adult male C57BL/6 mice for 30 consecutive days. Results of global metabolomics and lipidomics studies of PD-related brain regions based on LC-orbitrap MS revealed significant metabolite changes between the exposed and control groups in purine pathway, glutathione pathway, tryptophan pathway, phenylalanine pathway, alanine, aspartate and glutamate pathway, and lipid composition, mainly involved in oxidative stress and neurotransmitter production. By further quantifying metabolites involved in tryptophan and phenylalanine pathways in mice serum, colon and brain samples by using LC-triple quadrupole MS, dysregulation of PD linked neurotransmitters dopamine and serotonin were confirmed. iTRAQ proteomics study of the striatum, the part of the brain that is most intensively studied in PD pathogenesis, revealed that BDE-47 could induce neurotransmitter system disturbance, mitochondrial dysfunction, oxidative stress and abnormal phosphorylation. Oxygen consumption rate after BDE-47 treatment (0, 1 and 10 μM) in mouse neuroblastoma (N2a) cells was measured for the confirmation. BDE-47 was demonstrated to impair mitochondrial function.

Protein degradation by ATP dependent proteases is a universally conserved process. Recognition of substrates by such proteases commonly occurs via direct interaction or with the aid of a regulatory adaptor protein. An example of this regulation is found in Caulobacter crescentus, where key regulatory proteins are proteolysed in a cell-cycle dependent fashion. Substrates include essential transcription factors, structural proteins, and second messenger metabolism components. In this study, we explore sequence and structural requirements for regulated adaptor mediated degradation of PdeA, an important regulator of cyclic-di-GMP levels. Robust degradation of PdeA is dependent on the response regulator CpdR in vivo and in vitro. Here, I structurally identify a novel PAS domain in PdeA that is necessary and sufficient for CpdR mediated PdeA degradation. The PAS domain was found to contain a unique dimerization element that is associated with PdeA function. I show specifically that PdeA engages ClpXP through C-terminal recognition motifs. Finally, we present evidence that PdeA contains cryptic ClpXP recognition sites that are revealed during partial processing. Due to these uncommon degradation characteristics of PdeA, unique proteolytic insights may be gained by investigating this model system.

<p>The ubiquitin-proteasome system is responsible for cellular protein recycling, and it is a crucial system to maintain proper protein balances in cells. Proteasome is the main component of the system, and the system is tightly related to multiple cellular processes. Malfunction of the proteasome could lead to various diseases including cancer, neurodegenerative diseases and autoimmune diseases. As a result, researchers have been developing small molecules to target the proteasome to regulate its function. Currently, three small molecules have been approved by FDA as proteasome inhibitors to treat hematological cancer multiple myeloma. However, these small molecules inhibit the same enzymatic subunit on the proteasome and drug resistance has been observed among patients administrating these proteasome inhibitors. To develop new small molecules to target the proteasome, we started to investigate the 19S regulatory particle of the proteasome. In this work, we presented a workflow of discovering a small molecule selective binder, TXS-8, to 19S regulatory particle subunit Rpn-6. We also developed a series of assays to investigate the impact of small molecule on proteasome activity. At last, we introduced the binding site study of TXS-8, development of TXS-8-based PROTAC and new proteasome probe development.</p> <p>We first developed a one-bead-one-compound (OBOC) library to screen with Rpn-6 to discover potential binders to Rpn-6. After careful evaluation and validation, TXS-8 was discovered as the best hit from the screening. Our covalent pull-down experiment with cell lysate later confirmed TXS-8 as a selective binder of Rpn-6 and proteomic analysis of the pulled down protein also validated Rpn-6 as the major target of TXS-8.</p> <p>We then investigated the impact of TXS-8 in Rpn-6 overexpressed cancer cells like Ramos B-cell and multiple myeloma. TXS-8 was four-fold more toxic in these cells comparing to our control HEK-293T cells. To understand the cause of cell death when dosed with TXS-8, we began to investigate the impact of TXS-8 on proteasome activity, but some preliminary results were inconsistent. By the same time, there is also lack of a general workflow to investigate the impact of small molecules on proteasome activity. Therefore, we developed a three-step process to illustrate the general workflow using TXS-8 as an example. We first knocked down Rpn-6 in HEK-293T cells and monitored proteasome activity changes with a cell permeable probe our lab developed. We then transfected HEK-293T cells with a full-length foreign protein and knocked down Rpn-6 in these cells. We later monitored the degradation of the foreign protein when dosed with TXS-8. In the last step, we monitored the proteasome activity changes in primary cell lines when dosed with TXS-8. From these three steps, we successfully demonstrated a general workflow to investigate if a small molecule can affect proteasome activity. We also concluded that TXS-8 was unable to affect proteasome activity at non-lethal concentration.</p> <p> To further investigate TXS-8 and provide guidance for future structural optimization to improve potency, we proposed two methods on investigating the general binding site of TXS-8 on Rpn-6 using cross-linking techniques that is currently ongoing. We also modified TXS-8 into proteolysis targeting chimeras (PROTACs) to investigate if TXS-8-based PROTAC can improve toxicity and selectively induce Rpn-6 degradation in cells. However, no significant cell toxicity or Rpn-6 degradation was observed when dosed with TXS-8-based PROTACs.</p> Finally, Due to limitation of cell permeable probes, we were unable to investigate the impact of TXS-8 on the caspase-like β1 and trypsin-like β2 subunit of the proteasome in our previous studies. Although TXS-8 did not alter the chymotrypsin-like activity at non-lethal concentration, examining the effect of TXS-8 on the caspase-like and trypsin-like activity could still benefit our research. Besides, we also desire to expand our proteasome activity toolbox by developing more sensitive proteasome probes. Therefore, by analyzing and combing the commercially available proteasome probes and LLVY-Rh probes, we decided to develop selective proteasome probes for the β1 and β2 subunit to provide useful tools for future potential small molecule proteasome regulator characterization.