This work was initiated to acquire a better understanding of the mechanisms, regulations, and significances of deamidation, as well as its role in the aging process.

No description available.

Graduation date: 1995

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

Glucose phosphate isomerase (GPI) occurs in different bovine tissues as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis and isoelectric focusing. GPI from bovine heart was purified to homogeneity and each of the isozymes was resolved. Four of the five isozymes were characterized with regard to their physical, chemical and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118 Kd) and subunit (59 Kd) molecular weights but had different apparent pi values of 7.2, 7.0, 6.8 and 6.6. Structural analyses showed that the amino terminus was blocked and the carboxyl terminal sequence was -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes (lower pi values) at pH extremes, at high ionic strength, in the presence of denaturants or upon exposure to proteases. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes. Identical amino acid composition and peptide mapping by chemical cleavage at methionine and cysteine residues of the isozymes suggest a postsynthetic modification rather then a genetic origin for the in vivo isozymes. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with electrophoretic properties identical to those found in vivo. The simultaneous release in ammonia along with the spontaneous shift to more acidic isozymes and changes in the specific cleavage of the Asn-Gly bonds by hydroxylamine of the acidic isozyme indicates deamidation as the probable molecular basis. In summary the isozymes appear to be the result of spontaneous, postsynthetic modifications involving the addition of an equal number of negative charges and are consistent with the deamidation process.

An 18.5-kb human DNA segment was selected from a human XCharon-4A library by hybridization to mammalian valine tRNAiAc and found to encompass a cluster of three tRNA genes. Two valine tRNA genes with anticodons of AAC and CAC, encoding the major and minor cytoplasmic valine tRNA isoacceptors, respectively, and a lysine tRNAcuu gene were identified by Southern blot hybridization and DNA sequence analysis of a 7.1-kb region of the human DNA insert. At least nine Alu family members were found interspersed throughout the human DNA fragment. The tRNA genes are accurately transcribed by RNA polymerase III in a HeLa cell extract, since the RNase Ti fingerprints of the mature-sized tRNA transcription products are consistent with the DNA sequences of the structural genes. Three members of the chimpanzee triosephosphate isomerase (TPI) gene family, the functional transcription unit and two processed pseudogenes, were characterized by genomic blotting and DNA sequence analysis. The bona fide TPI gene spans 3.5 kb with seven exons and six introns, and is the first complete hominoid TPI gene sequenced. The gene exhibits a very high identity with the human and rhesus TPI genes. In particular, the polypeptides of 248 amino acids encoded by the chimpanzee and human TPI genes are identical, although the two coding regions differ in the third codon wobble positions for five amino acids. An Alu member occurs upstream from one of the processed pseudogenes, whereas an isolated endogenous retroviral long terminal repeat (HERV-K) occurs within the structural region of the other processed pseudogene. The ages of the processed pseudogenes were estimated to be 2.6 and 10.4 million years, implying that one was inserted into the genome before the divergence of the chimpanzee and human lineages, and the other inserted into the chimpanzee genome after the divergence.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1981. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Vita. / Includes bibliographical references. / by Thomas Clifford Alber. / Ph.D.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 186-198).

Structural properties of phosphoglucose isomerases isolated from a variety of species have been compared by peptide fingerprinting, predicted amino acid sequence homologies and by denaturation and renaturation studies. The enzymes are more readily denatured in guanidinium chloride than in urea, and the isomerase isolated from yeast is more stable toward acid pH than the rabbit muscle enzyme. The rates of guanidinium chloride-induced denaturation are markedly increased by ionic strength and decreased by substrates, competitive inhibitors or glycerol. The enzyme can be renatured, but only in the presence of glycerol. The renaturation process is dependent on protein concentration and temperature and provides a method for the formation of mixed species heterodimers.

A Dissulfeto Isomerase Proteica (PDI) é uma chaperona ditiol-dissulfeto oxidoredutase da superfamília tiorredoxina que catalisa o enovelamento de proteínas secretadas ou de membrana por meio da introdução, redução ou isomerização de pontes dissulfeto. A PDI é primariamente localizada no lúmen do retículo endoplasmático, no entanto a presença de uma pequena fração da PDI na superfície celular e no meio extracelular tem sido documentada em diversos tipos celulares. Particularmente em plaquetas e células endoteliais, a PDI epi/pericelular (pecPDI) está envolvida em diversos processos incluindo ativação de plaquetas/trombose, infecções virais e remodelamento vascular. A ausência de PDI (e outras tiol isomerases) na circulação tem sido proposta como mecanismo para prevenir trombose na ausência de lesão vascular. No entanto, esta questão permanece obscura e existe pouca informação sobre a concentração circulante da PDI e outras tiol isomerases vasculares. Neste estudo, investigamos a ocorrência e implicações fisiológicas de um pool circulante de PDI em indivíduos saudáveis e validamos um ensaio para detecção da PDI. Os resultados mostraram um pool detectável de PDI no plasma por ELISA, confirmados por imunoprecipitação e ensaio de atividade (inibição da redução da sonda di-eosina-GSSG pela rutina, um inibidor específico da PDI). A concentração de PDI no plasma (mediana=330 pg/mL) indicam uma alta variabibilidade interindividual, com valores muito baixos/indetectáveis (plasmas pobres em PDI [PP-PDI], definidos como <= 330 pg/mL) até valores superiores a 1000 pg/mL (plasmas ricos em PDI [PR-PDI], designados como > 330 pg/mL). Por outro lado, um resultado importante foi o fato de que valores de PDI mostraram variabilidade intraindividual muito baixa ao longo do tempo, detectada através de medidas repetidas em diferentes ocasiões e/ou condições. A fração de PDI presente em micropartículas plasmáticas foi variável, mas em geral pequena em relação ao pool total de PDI. O pool da PDI no plasma está majoritariamente reduzido (60-80%) sem diferenças entre os grupos PP-PDI e PR-PDI. Importante, os valores de PDI associaram-se a distintos perfis proteômicos plasmáticos. Enquanto os PR-PDI se associaram preferencialmente a proteínas relacionadas a diferenciação celular, processamento de proteínas, funções housekeepings, entre outras, os PP-PDI mostraram expressão diferencial de proteínas associadas a coagulação, respostas inflamatórias e imunoativação. A atividade de plaquetas medida por agregação foi semelhante entre os indivíduos com PP-PDI vs. PR-PDI. No entanto, a PDI solúvel foi diminuída após agregação plaquetária na maioria dos indivíduos em ambos os grupos, sugerindo captura devida a exposição de moléculas adesivas. Em outras séries de experimentos, mostramos que tais perfis proteômicos plasmáticos se correlacionaram ao fenótipo e função endotelial. Células endoteliais em cultura incubadas com PP-PDI ou PR-PDI recapitularam padrões de expressão gênica e de secreção de proteínas similares aos perfis plasmáticos correspondentes. Além disso, as assinaturas proteômicas identificadas em ambos os tipos de plasma traduziram-se em distintas respostas funcionais endoteliais. Os PP-PDI promoveram comprometimento da adesão de células endoteliais à fibronectina e perturbaram o padrão de migração celular associado à reparação de lesão endotelial. Em contraste, os PR-PDI não afetaram significativamente a adesão celular e sustentaram um padrão de migração organizado. Em outra população de pacientes com eventos cardiovasculares, os valores de PDI no plasma (mediana= 35 pg/mL) foram significativamente inferiores aos de indivíduos saudáveis. Em conclusão, o pool detectável de PDI presente no plasma se associou a distintos perfis proteômicos e parece se comportar como um indicador/ marcador de assinaturas proteômicas relacionadas à função e sinalização endotelial. Este é o primeiro estudo descrevendo valores circulantes de PDI diretamente relacionados a distintos fenótipos endoteliais / Protein disulfide isomerase (PDI) is a dithiol-disulfide oxidoreductase chaperone from thioredoxin superfamily which catalyzes introduction, reduction or isomerization of disulfide bonds in nascent proteins, typically destined to extracellular secretion or membrane insertion. PDI is primarily located into the endoplasmic reticulum; however, there are clear evidences for the presence of a small PDI fraction at the cell surface and extracellular milieu in several cell types. Particularly in platelets and endothelial cells, such peri/epicellular pool of PDIA1 (pecPDI) is involved in distinct processes including platelet activation/thrombosis, viral infection and vascular remodeling. The absence of PDI (and other thiol isomerases) from circulating plasma has been proposed as a mechanism to prevent thrombogenesis in the absence of vascular injury. However, this question remains unclear, as there is little information on the circulating levels of PDI and other vascular thiol isomerases. Here we investigated the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. The results showed a detectable pool of plasma PDI by ELISA, confirmed by immunoprecipitation and activity assay (dieosin-GSSG inhibitable by rutin, a specific PDI inhibitor). PDI levels (median= 330 pg/mL) exhibited high interindividual variability, ranging from undetectable/low (PDI-poor plasma, defined as <= 330 pg/mL) until 1000 pg/mL (PDI-rich plasma, > 330 pg/mL). Remarkably, opposite to interindividual variability, the intra-individual variability was quite low, so that values assessed under distinct conditions over time were close and reproducible. The majority (60-80%) of plasma PDI is in the reduced state, without any difference among individuals with PDIpoor and PDI-rich plasma. Importantly, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet activity assessed by aggregation was similar between PDI-poor vs. PDI-rich plasma. However, soluble PDI was decreased after platelet activation in both groups, suggesting sequestration of plateletderived PDI by its potential substrates. In other set of experiments, we showed that such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. In contrast, PDI-rich plasma did not significantly affect cell adhesion and supported organized endothelial migration. In another dataset, patients with cardiovascular events had lower PDI levels (median= 35 pg/mL) vs. healthy individuals. In conclusion, a PDI pool detectable in plasma from healthy individuals is associated with distinct proteomic profiles and seems to behave as an indicator/marker of proteomic signatures related with endothelial function and signaling. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses