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PubMed2025

Chaperone-mediated autophagy regulates neuronal activity by sex-specific remodelling of the synaptic proteome.

Chaperone-mediated autophagy (CMA) declines in ageing and neurodegenerative diseases. Loss of CMA in neurons leads to neurodegeneration and behavioural changes in mice but the role of CMA in neuronal physiology is largely unknown. Here we show that CMA deficiency causes neuronal hyperactivity, increased seizure susceptibility and disrupted calcium homeostasis. Pre-synaptic neurotransmitter release and NMDA receptor-mediated transmission were enhanced in CMA-deficient females, whereas males exhibited elevated post-synaptic AMPA-receptor activity. Comparative quantitative proteomics revealed sexual dimorphism in the synaptic proteins degraded by CMA, with preferential remodelling of the pre-synaptic proteome in females and the post-synaptic proteome in males. We demonstrate that genetic or pharmacological CMA activation in old mice and an Alzheimer's disease mouse model restores synaptic protein levels, reduces neuronal hyperexcitability and seizure susceptibility, and normalizes neurotransmission. Our findings unveil a role for CMA in regulating neuronal excitability and highlight this pathway as a potential target for mitigating age-related neuronal decline.

Animals↗
PubMed2001

Genome of the apes.

The Human Genome Project has generated both the information and technological infrastructure needed to accelerate genetic comparisons between humans and the African great apes (chimpanzees and gorillas). Sequence and chromosomal organization differences between these highly related genomes will provide clues to the genetic basis for recently evolved, specifically human traits such as bipedal gait and advanced cognitive function. Recent studies comparing the primate genomes have the potential to affect many aspects of human biomedical research and could benefit primate conservation efforts.

Animals↗
PubMed2001

Comparative analysis of two-dimensional protein patterns in malignant and normal human breast tissue.

Malignant and normal human breast tissue were compared by evaluating two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) maps of frozen tissue samples. Image analyzing software was used to scan and process 34 gels. Eight (8/34) of these gels (4 malignant breast tumor samples, 4 normal tissue samples) were selected on the basis of gel and image quality to build a database to identify and measure the expression of a previously unidentified proteome. Growth factor receptor proteins (GFRs), including ERBB2 (HER2) and ERBB3 (HER3), were expressed in the malignant tissue samples. Growth factor receptor proteins were not expressed in the normal tissue. Also, expression of PS2-protein (pS2) was detected in neither malignant nor normal tissue. In benign breast samples a higher intensity of protein expression could be observed for maspin, desmoglein 3 and keratin 8 than in malignant samples. Other proteins expressed in malignant breast tissue include mitogen-activated protein kinase 3 (MK03), heat shock protein 27 kDa (HS27), growth factor receptor-bound protein (GRB2), cathepsin D, G1/S specific cyclin E1 (CGEI), glucose transporter type 5 (GTR5), and a number of as yet unidentified proteins.

Breast↗
PubMed2006

Lactate dehydrogenase-B is silenced by promoter hypermethylation in human prostate cancer.

In order to identify novel candidates associated with prostate cancer metastasis, we compared the proteomic profile of the poorly metastatic human prostate cancer cell line LNCaP, with its highly metastatic variant LNCaP-LN3, by two-dimensional gel electrophoresis. A major protein spot (pI of 5.9 and molecular weight of 37 kDa) was seen in LNCaP cells, but not in LNCaP-LN3 cells and was identified as lactate dehydrogenase-B (LDHB), by tandem mass spectrometry. Furthermore, enzyme kinetic assays and zymography showed a higher LDH enzyme activity in LNCaP cells compared with LNCaP-LN3. Bisulphite-modified DNA sequencing showed promoter hypermethylation in LNCaP-LN3 cells but not in LNCaP, Du145, PC3, CWR22 or BPH45 cells. Treatment of LNCaP-LN3 cells with 5'-azacytidine caused re-expression of LDHB transcripts. In tissues, LDHB promoter hypermethylation occurred at a higher frequency in prostate cancer, 14/ 31 (45%), compared to adjacent nonmalignant or benign tissue, 2/19 (11%) (P < 0.025). Immunohistochemistry showed a higher frequency of LDHB expression in benign or non-malignant tissues, 59/ 73 (81%), compared to cancer cases, 3/53 (6%) (P < 0.001). Absent LDHB expression was also seen in 7/7 (100%) cases of metastatic cancer in bone. Our data are the first to show loss of LDHB expression in prostate cancer, the mechanism of which appears to involve promoter hypermethylation.

Amino Acid Sequence↗
PubMed2006

Analysis of the effects of cryopreservation on rat hepatocytes using SELDI-TOF mass spectrometry.

Successful cryopreservation of hepatocytes is essential to the future of hepatocyte transplantation as a treatment for liver disease, and also for the improved in vitro use of hepatocytes for research. However, hepatocyte function is adversely affected by even the best cryopreservation protocols. To investigate possible mechanisms for these changes, total cell lysates were prepared from fresh and cryopreserved rat hepatocytes and the proteome profiles compared using SELDI-TOF-MS ProteinChip technology. In addition, in vitro functional assays (viability, attachment efficiency, and lactate dehydrogenase leakage) were performed on the corresponding fresh and cryopreserved hepatocytes. Sixty-one peptides were identified as being significantly changed after cryopreservation. Thirty-seven peaks were significantly increased and 24 were significantly decreased after cryopreservation. The peak intensity of a number of these peptides was found to correlate with the in vitro function of the hepatocytes. Seven peptides correlated with in vitro function after cryopreservation and 10 peptides correlated with both fresh and cryopreserved function. The peptides significantly decreased after cryopreservation could include cytosolic enzymes or cofactors, which leaked out of the cells due to cryopreservation-induced membrane damage. The peptides significantly increased after cryopreservation could be retained products of cleavage of larger intracellular polypeptides and proteins or the result of aggregation of peptides caused by physical changes in the cell due to the cryopreservation process. Proteome profiling using SELDI-TOF-MS could be a useful tool to assess the effects of isolation and cryopreservation of hepatocytes, particularly if the findings are extended to human hepatocytes.

Animals↗
PubMed2004

Mitochondria of protists.

Over the past several decades, our knowledge of the origin and evolution of mitochondria has been greatly advanced by determination of complete mitochondrial genome sequences. Among the most informative mitochondrial genomes have been those of protists (primarily unicellular eukaryotes), some of which harbor the most gene-rich and most eubacteria-like mitochondrial DNAs (mtDNAs) known. Comparison of mtDNA sequence data has provided insights into the radically diverse trends in mitochondrial genome evolution exhibited by different phylogenetically coherent groupings of eukaryotes, and has allowed us to pinpoint specific protist relatives of the multicellular eukaryotic lineages (animals, plants, and fungi). This comparative genomics approach has also revealed unique and fascinating aspects of mitochondrial gene expression, highlighting the mitochondrion as an evolutionary playground par excellence.

Animals↗
PubMed2005

Alterations in human vitreous humour following cataract extraction.

Cataract extraction is associated with the risk of posterior vitreous detachment, macular edema and retinal detachment possibly as a result of a disturbance to the vitreous body during surgery. While it is common for lens cortical fiber debris to leak into the vitreous humour during cataract extraction, the extent to which the vitreous humour is altered post-surgery is unknown. The current study examines the integrity of the vitreous humour of pseudophakic and phakic human donor eyes by comparing the proteome, the viscosity and the size distribution of macromolecules in different regions of the vitreous humour from human pseudophakic and phakic donor eyes. Major differences between the proteomes of anterior and posterior vitreous humour were observed in phakic and pseudophakic donor eyes. Seventeen spots identified as complete, modified or cleaved forms of alphaA-, alphaB-, betaA4-, betaB2, and gammaS-crystallins were present in the anterior vitreous humour of all pseudophakic eyes studied. Crystallins were not detected in the posterior vitreous humour of the pseudophakic eye or the vitreous humour of the phakic eye. Significant alterations in abundance and/or modification of transthyretin, alpha antitrypsin, and retinoic acid binding protein were observed in all locations of pseudophakic vitreous humour as compared to phakic samples. In addition, a significant decrease in the number and intensity of protein spots was observed for the posterior vitreous humour of pseudophakic eyes when compared to posterior vitreous humour of phakic eyes. Proteins which were affected include antioxidant proteins and enzymes such as carbonic anhydrase and trisephosphate isomerase. A reversal of the viscosity gradient, anterior to posterior, in the vitreous humour of pseudophakic eyes was observed concomitant with alterations in the distribution of 50 nm particles. These particles are likely primarily composed of hyaluronan. While varying degrees of vitreous degradation may have existed prior to surgery and may have contributed to the cataract formation, in no case did the phakic donor eyes exhibit the same alterations in the vitreous humour proteome, viscosity or particle sizes as did the pseudophakic donor eyes. The examination of phakic/pseudophakic donor eye pairs confirmed that the vitreous humour proteome and structural integrity were very similar in the matched phakic donor eye to eyes from donors with no history of cataract. Even though the number of samples for this study was limited, the observed changes support the hypothesis that alterations in the vitreous humour proteome occur in psuedophakic eyes with concurrent alterations in the structure of the vitreous humor. These modifications of the microenvironment of the retina may contribute to the development of retinal complications following cataract surgery.

Adolescent↗
PubMed2002

Alterations of stress related proteins in genetically altered mice revealed by two-dimensional differential in-gel electrophoresis analysis.

Transgenic, knockout and knockin mice are useful tools for linking specific genes with behaviour and other complex biological processes. However, complications arising due to compensatory changes, genetic background differences and other factors could lead to difficulty in interpreting the resulting changes in phenotype. We have used fluorescence two-dimensional differential in-gel electrophoresis in combination with matrix-assisted laser desorption/ionization-time of flight mass fingerprinting to investigate the possibility that distinct genetic alterations can lead to common protein expression changes in genetically modified mice. Brain proteomes were compared from two transgenic mouse strains (Tg2576 x TgPS1 and Tg2576), two knockout mouse strains (5-HT(7)R -/- and GABA(A)Ralpha5 -/-) and one knockin mouse strain (GABA(A)Ralpha1-H101R). Both of the transgenic models showed an isoform change in the heat shock 70 related protein, mortalin. The knockout and knockin models showed similar changes in mortalin expression along with an alteration of the anti-oxidant protein 2. The observed proteomic alterations indicate that stress-responsive protein pathways may be altered artefactually in all of the mouse models used in this study and highlights an area where caution is needed in interpreting proteomic changes in genetically modified mice.

Animals↗
PubMed2007

Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts.

Many key processes central to bone formation and homeostasis require the involvement of osteoblasts, cells responsible for accumulation and mineralization of the extracellular matrix (ECM). During this complex and only partially understood process, osteoblasts generate and secrete matrix vesicles (MVs) into the ECM to initiate mineralization. Although they are considered an important component of mineralization process, MVs still remain a mystery. To better understand their function and biogenesis, a proteomic analysis of MVs has been conducted. MVs were harvested by two sample preparation approaches and mass spectrometry was utilized for protein identification. A total of 133 proteins were identified in common from the two MV preparations, among which were previously known proteins, such as annexins and peptidases, along with many novel proteins including a variety of enzymes, osteoblast-specific factors, ion channels, and signal transduction molecules, such as 14-3-3 family members and Rab-related proteins. To compare the proteome of MV with that of the ECM we conducted a large-scale proteomic analysis of collagenase digested mineralizing osteoblast matrix. This analysis resulted in the identification of 1,327 unique proteins. A comparison of the proteins identified from the two MV preparations with the ECM analysis revealed 83 unique, non-redundant proteins identified in all three samples. This investigation represents the first systematic proteomic analysis of MVs and provides insights into both the function and origin of these important mineralization-regulating vesicles.

Animals↗
PubMed2005

Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model.

With advancements in the analytical technologies and methodologies in proteomics, there is great interest in biomarker discovery in biofluids such as serum and plasma. Current hypotheses suggest that the low molecular weight (LMW) serum proteome possesses an archive of clipped and cleaved protein fragments that may provide insight into disease development. Though these biofluids represent attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. Mice are one of the most extensively used animal models for studying human disease because they represent a highly controllable experimental model system. In this study, the LMW serum proteome was compared between xenografted tumor-bearing mice and control mice by differential labeling utilizing trypsin-mediated incorporation of the stable isotope of oxygen, 18O. The digestates were combined, fractionated by strong cation exchange chromatography, and analyzed by nanoflow reversed-phase liquid chromatography coupled online with tandem mass spectrometry, resulting in the identification of 6003 proteins identified by at least a single, fully tryptic peptide. Almost 1650 proteins were identified and quantitated by two or more fully tryptic peptides. The methodology adopted in this work provides the means for future quantitative measurements in comparative animal models of disease and in human disease cohorts.

Animals↗
PubMed2004

Proteomic analysis of the TGF-beta signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 expression.

Smad4 is a tumor-suppressor gene that is lost or mutated in 50% of pancreatic carcinomas. Smad4 is also an intracellular transmitter of transforming growth factor-beta (TGF-beta) signals. Although its tumor-suppressor function is presumed to reside in its capacity to mediate TGF-beta-induced growth inhibition, there seems to be a Smad4-independent TGF-beta signaling pathway. Here, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using stable RNA interference. Smad4 protein expression and TGF-beta-Smad4 signaling were impaired in S4KD cells, and we compared the proteomic changes with TGF-beta stimulation using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. We identified five proteins that were up-regulated and seven proteins that were down-regulated; 10 of them were novel targets for TGF-beta. These proteins function in processes such as cytoskeletal regulation, cell cycle, and oxidative stress. Introducing siRNA-mediated gene silencing into proteomics revealed a novel TGF-beta signal pathway that did not involve Smad4.

Blotting, Western↗
PubMed2002

A comparison of murine and human immunoproteomes of Helicobacter pylori validates the preclinical murine infection model for antigen screening.

Preclinical mouse infection models are widely used for Helicobacter vaccine development, but how well such models mimic important aspects of human infections is unknown. A comparison of Helicobacter pylori immunoproteomes of infected mice with previously reported patient data reveals a high agreement in the antigens recognized, suggesting that H. pylori in vivo protein composition and recognition by the host immune system are comparable in mice and humans. Murine Helicobacter models may thus be valid to screen antigens for human vaccination.

Animals↗
PubMed2006

Protein expression in Escherichia coli S17-1 biofilms: impact of indole.

Bacteria undergo significant changes during adherence to surfaces and biofilm development. Cell-to-cell signalling molecules are known to be involved in these phenotypic adaptations to the sessile mode of life. We demonstrated previously that indole can act as an extracellular signal to regulate biofilm formation in E. coli. To identify proteins over- or under-expressed in response to E. coli biofilm formation and indole signalling, we compared the proteomes of the E. coli S17-1 wild-type and 3714 (S17-1 tnaA::Tn5) tryptophanase-negative mutant cells (which don't produce indole) grown as suspensions or biofilms in the presence or absence of exogenous indole. From computer-assisted image analysis, 407 spots were discriminated on two-dimensional electropherograms. Principal component analysis (PCA) of the electropherograms did not discriminate between the proteomes of the wild-type and mutant cells grown as suspensions indicating that indole has a limited impact onto protein expression of planktonic cells. The first principal component extracted by PCA, after standardization of the observations, opposed planktonic and biofilm cells confirming the existence of changes in protein expression during E. coli biofilm formation. Among proteins over- or under-expressed by both sessile wild-type and mutant cells, we identified metabolic enzymes, transporters, proteins involved in the translation and transcription machinery, stress response and regulation, and signalling proteins. The wild-type and mutant strains grown as biofilms in the presence of indole were discriminated by the second component. The role of some proteins whose expression was altered in biofilm bacteria compared to suspended counterparts is discussed.

Amino Acid Sequence↗
PubMed2006

Protein expression profiles of Chlamydia pneumoniae in models of persistence versus those of heat shock stress response.

Chlamydia pneumoniae is an obligate intracellular pathogen that causes both acute and chronic human disease. Several in vitro models of chlamydial persistence have been established to mimic chlamydial persistence in vivo. We determined the expression patterns of 52 C. pneumoniae proteins, representing nine functional subgroups, from the gamma interferon (IFN-gamma) treatment (primarily tryptophan limitation) and iron limitation (IL) models of persistence compared to those following heat shock (HS) at 42 degrees C. Protein expression patterns of C. pneumoniae persistence indicates a strong stress component, as evidenced by the upregulation of proteins involved in protein folding, assembly, and modification. However, it is clearly more than just a stress response. In IFN persistence, but not IL or HS, amino acid and/or nucleotide biosynthesis proteins were found to be significantly upregulated. In contrast, proteins involved in the biosynthesis of cofactors, cellular processes, energy metabolism, transcription, and translation showed an increased in expression in only the IL model of persistence. These data represent the most extensive protein expression study of C. pneumoniae comparing the chlamydial heat shock stress response to two models of persistence and identifying the common and unique protein level responses during persistence.

Cell Line, Tumor↗
PubMed2006

Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid.

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.

Biomarkers↗
PubMed2006

Construction of phylogenetic trees by kernel-based comparative analysis of metabolic networks.

BACKGROUND: To infer the tree of life requires knowledge of the common characteristics of each species descended from a common ancestor as the measuring criteria and a method to calculate the distance between the resulting values of each measure. Conventional phylogenetic analysis based on genomic sequences provides information about the genetic relationships between different organisms. In contrast, comparative analysis of metabolic pathways in different organisms can yield insights into their functional relationships under different physiological conditions. However, evaluating the similarities or differences between metabolic networks is a computationally challenging problem, and systematic methods of doing this are desirable. Here we introduce a graph-kernel method for computing the similarity between metabolic networks in polynomial time, and use it to profile metabolic pathways and to construct phylogenetic trees. RESULTS: To compare the structures of metabolic networks in organisms, we adopted the exponential graph kernel, which is a kernel-based approach with a labeled graph that includes a label matrix and an adjacency matrix. To construct the phylogenetic trees, we used an unweighted pair-group method with arithmetic mean, i.e., a hierarchical clustering algorithm. We applied the kernel-based network profiling method in a comparative analysis of nine carbohydrate metabolic networks from 81 biological species encompassing Archaea, Eukaryota, and Eubacteria. The resulting phylogenetic hierarchies generally support the tripartite scheme of three domains rather than the two domains of prokaryotes and eukaryotes. CONCLUSION: By combining the kernel machines with metabolic information, the method infers the context of biosphere development that covers physiological events required for adaptation by genetic reconstruction. The results show that one may obtain a global view of the tree of life by comparing the metabolic pathway structures using meta-level information rather than sequence information. This method may yield further information about biological evolution, such as the history of horizontal transfer of each gene, by studying the detailed structure of the phylogenetic tree constructed by the kernel-based method.

Archaeal Proteins↗
PubMed2005

The utility of accurate mass and LC elution time information in the analysis of complex proteomes.

The combination of mass and normalized elution time (NET) of a peptide identified by liquid chromatography-mass spectrometry (LC-MS) measurements can serve as a unique signature for that peptide. However, the specificity of an LC-MS measurement depends upon the complexity of the proteome (i.e., the number of possible peptides) and the accuracy of the LC-MS measurements. In this work, theoretical tryptic digests of all predicted proteins from the genomes of three organisms of varying complexity were evaluated for specificity. Accuracy of the LC-MS measurement of mass-NET pairs (on a 0 to 1.0 NET scale) was described by bivariate normal sampling distributions centered on the peptide signatures. Measurement accuracy (i.e., mass and NET standard deviations of +/-0.1, 1, 5, and 10 ppm, and +/-0.01 and 0.05, respectively) was varied to evaluate improvements in process quality. The spatially localized confidence score, a conditional probability of peptide uniqueness, formed the basis for the peptide identification. Application of this approach to organisms with comparatively small proteomes, such as Deinococcus radiodurans, shows that modest mass and elution time accuracies are generally adequate for confidently identifying most peptides. For more complex proteomes, more accurate measurements are required. However, the study suggests that the majority of proteins for even the human proteome should be identifiable with reasonable confidence by using LC-MS measurements with mass accuracies within +/-1 ppm and high efficiency separations having elution time measurements within +/-0.01 NET.

Animals↗
PubMedDate not supplied

Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis.

The ability to identify and quantitate integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and facilitate derivatization of these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified, of which 40 were integral membrane proteins containing from one to nine mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g., ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g., pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.

Affinity Labels↗