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PubMed2001

Gaucher disease: understanding the molecular pathogenesis of sphingolipidoses.

Gaucher disease is a typical lysosomal storage disease, resulting from an inborn deficiency of glucocerebrosidase. This leads to the accumulation of glycolipids in macrophages, particularly those in the liver, bone marrow, spleen and lung. In addition, disease of the nervous system can arise as a result of the accumulation of endogenous glycosphingolipid metabolites in brain tissue. About 150 mutations of the glucocerebrosidase gene have been identified in patients with Gaucher disease, some of which are predictive of phenotype. However, even patients and siblings with the same mutation, including monozygotic twins, may exhibit marked variability in disease expression and severity, illustrating our lack of understanding of the phenotype-genotype relationship in the sphingolipidoses. Massive organomegaly, particularly of the spleen, is a frequent feature of the disease. Although the liver and spleen may increase greatly in size, the amount of pathological lipid stored in the affected macrophages (Gaucher cells) accounts for less than 2% of the additional tissue mass. It is therefore clear that an inflammatory response occurs in affected individuals and that the clinical phenotype is due to an effect of macrophage storage beyond the physical presence of the Gaucher cells. Factors released by Gaucher cells, including pro-inflammatory cytokines and perhaps cathepsins, provide a mechanistic link between lysosomal storage and the diverse clinical manifestations of Gaucher disease. Emerging proteomic technology and gene expression profiling should not only improve our understanding of pathogenesis but also offer the prospect of identifying novel biomarkers that can be used as surrogate measures of disease activity and responses to treatment.

Gaucher Disease↗

PubMed2002

Tektin B1 demonstrates flagellar localization in human sperm.

The human flagellar protein tektin B1 (h-tekB1) in human sperm was cloned, and its sequence and subcellular location were determined. Human sperm proteins were separated by 2-dimensional electrophoresis, and a resolved protein spot of 54 kDa with an isoelectric point (pI) of 5.3 was removed from the gel, trypsinized, and microsequenced by tandem mass spectrometry. The resulting peptides did not match any protein in the (then current) protein databases. Degenerate oligonucleotides based on the microsequences were used with a polymerase chain reaction to amplify a partial cDNA clone from human testis poly(A)(+) mRNA, and subsequently a full-length 1.5-kilobase (kb) clone (GenBank AF054910) was obtained from a testis cDNA library. The open reading frame encoded a 430-amino acid protein with 47% homology to the sea urchin tektin B1. Hybridization of labeled h-tekB1 cDNA to a multiple-tissue Northern blot demonstrated a transcript of 1.7 kb in human testis, and a multiple tissue dot-blot demonstrated high levels of expression in testis, trachea, and lung, intermediate levels in fetal brain and appendix, and low levels in ovary, pituitary, and fetal kidney. Rat polyclonal serum generated against a recombinant h-tekB1 demonstrated 3 h-tekB1 isoforms of pI 5.25, 5.5, and 5.35 at 53.5 kDa on a 2-dimensional Western blot of human sperm proteins. Immunofluorescent studies localized h-tekB1 to the principal piece of human sperm, but the endpiece was unstained.

Amino Acid Sequence↗

PubMed2005

Exercise-related novel gene is involved in myoblast differentiation.

In this study we tried to identify new genes or proteins in skeletal muscle induced by exercise. We analyzed alterations of protein expression in mouse gastrocnemius muscles induced by swim-exercise using two dimensional gel electrophoresis and mass spectrometry. Nine spots were significantly altered between control and swim groups. One of the four protein spots whose expression was decreased was identified as functionally unknown "expressed sequence AI854635" gene. The AI854635 gene has C2H2 type zinc finger motif, and is considered to be a transcription factor. The mRNA of AI854635 gene was expressed in skeletal muscle, brain, kidney, and thymus. To elucidate the function of the AI854635 gene we analyzed mRNA expression levels during C2C12 myoblast differentiation. C2C12 myoblast began to form myotube around 20 h after the initiation of differentiation. The mRNA expression levels of AI854635 gene was significantly induced around 6 h and increased till 48 h, indicating a pivotal role in myoblast differentiation. Although the significance of decreased expression of AI854635 gene induced by swim-exercise is not clear, we found that this gene is involved in myoblast differentiation.

Animals↗

PubMed2025

A cellular model of TDP-43 induces phosphorylated TDP-43 aggregation with distinct changes in solubility and autophagy dysregulation.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that affects neurons in the brain and spinal cord, causing loss of muscle control, and eventually leads to death. Phosphorylated transactive response DNA binding protein-43 (TDP-43) is the major pathological protein in both sporadic and familial ALS, forming cytoplasmic aggregates in over 95% of cases. Of the 10-15% of ALS cases that are familial, mutations in TDP-43 represent about 5% of those with a family history. We have developed an in vitro overexpression model by introducing three familial ALS mutations (A315T, M337V, and S379P) in the TDP-43 (TARDBP) gene which we define as 3X-TDP-43. This overexpression model TDP-43 shows deficits in autophagy flux and colocalization of TDP-43 with stress granules. We also observe a progressive shift of TDP-43 to the cytoplasm in this model. This overexpression model shows a reduction in solubility of phosphorylated TDP-43 from RIPA to urea soluble. Four glycolytic enzymes, phosphoglycerate kinase one (PGK1), aldolase A (ALDOA), enolase 1 (ENO1), and pyruvate dehydrogenase kinase 1 (PDK1) show significant time-dependent decreases in 3X-TDP-43 expressing cells. Shotgun proteomic analysis shows global changes in the importin subunit alpha-1 (KPNA2), heat shock 70 kDa protein 1A (HSPA1A), and protein disulfide-isomerase A3 (PDIA3) expression levels and coimmunoprecipitation reveals that these proteins complex with TDP-43. Overall, these results suggest that the 3X-TDP-43 model may provide new insights into pathophysiology and an avenue for drug screening in vitro for those suffering from ALS and related TDP-43 proteinopathies.

Autophagy↗

PubMed2002

Biochemical engineering of cell surface sialic acids stimulates axonal growth.

Sialylation is essential for development and regeneration in mammals. Using N-propanoylmannosamine, a novel precursor of sialic acid, we were able to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into cell surface glycoconjugates. Here we report that this biochemical engineering of sialic acid leads to a stimulation of neuronal cells. Both PC12 cells and cerebellar neurons showed a significant increase in neurite outgrowth after treatment with this novel sialic acid precursor. Furthermore, also the reestablishment of the perforant pathway was stimulated in brain slices. In addition, we surprisingly identified several cytosolic proteins with regulatory functions, which are differentially expressed after treatment with N-propanoylmannosamine. Because sialic acid is the only monosaccharide that is activated in the nucleus, we hypothesize that transcription could be modulated by the unnatural CMP-N-propanoylneuraminic acid and that sialic acid activation might be a general tool to regulate cellular functions, such as neurite outgrowth.

Animals↗

PubMed2006

Regulation of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase (ACC) catalyses the formation of malonyl-CoA, an essential substrate for fatty acid synthesis in lipogenic tissues and a key regulatory molecule in muscle, brain and other tissues. ACC contributes importantly to the overall control of energy metabolism and has provided an important model to explore mechanisms of enzyme control and hormone action. Mammalian ACCs are multifunctional dimeric proteins (530-560 kDa) with the potential to further polymerize and engage in multiprotein complexes. The enzymatic properties of ACC are complex, especially considering the two active sites, essential catalytic biotin, the three-substrate reaction and effects of allosteric ligands. The expression of the two major isoforms and splice variants of mammalian ACC is tissue-specific and responsive to hormones and nutritional status. Key regulatory elements and cognate transcription factors are still being defined. ACC specific activity is also rapidly modulated, being increased in response to insulin and decreased following exposure of cells to catabolic hormones or environmental stress. The acute control of ACC activity is the product of integrated changes in substrate supply, allosteric ligands, the phosphorylation of multiple serine residues and interactions with other proteins. This review traces the path and implications of studies initiated with Dick Denton in Bristol in the late 1970s, through to current proteomic and other approaches that have been consistently challenging and immensely rewarding.

Acetyl-CoA Carboxylase↗

PubMed2006

Does isoform diversity explain functional differences in the 14-3-3 protein family?

The 14-3-3 family of proteins was originally identified in 1967 as simply an abundant brain protein. However it took almost 25 years before the ubiquitous role of 14-3-3 in cell biology was recognized when it was found to interact with several signalling and proto-oncogene proteins. Subsequently 14-3-3 proteins were the first protein recognized to bind a discrete phosphoserine/threonine-binding motifs. In mammals the 14-3-3 protein family is comprised of seven homologous isoforms. The 14-3-3 family members are expressed in all eukaryotes and although no single conserved function of the 14-3-3s is apparent, their ability to bind other proteins seems a crucial characteristic. To date more than 300 binding partners have been identified, of which most are phosphoproteins. Consequently, it has become clear that 14-3-3 proteins are involved in the regulation of most cellular processes, including several metabolic pathways, redox-regulation, transcription, RNA processing, protein synthesis, protein folding and degradation, cell cycle, cytoskeletal organization and cellular trafficking. In this review we include recent reports on the regulation of 14-3-3 by phosphorylation, and discuss the possible functional significance of the existence of distinct 14-3-3 isoforms in light of recent proteomics studies. In addition we discuss 14-3-3 interaction as a possible drug target.

14-3-3 Proteins↗

PubMed2006

Identification of sites of mannose 6-phosphorylation on lysosomal proteins.

Most newly synthesized soluble lysosomal proteins contain mannose 6-phosphate (Man-6-P), a specific carbohydrate modification that is recognized by Man-6-P receptors (MPRs) that direct targeting to the lysosome. A number of proteomic studies have focused on lysosomal proteins, exploiting the fact that Man-6-P-containing forms can be purified by affinity chromatography on immobilized MPRs. These studies have identified many known lysosomal proteins as well as many proteins not previously classified as lysosomal. The latter are of considerable biological interest with potential implications for lysosomal function and as candidates for lysosomal storage diseases of unknown etiology. However, a significant problem in interpreting the biological relevance of such proteins has been in distinguishing true Man-6-P glycoproteins from simple contaminants and from proteins associated with true Man-6-P glycoproteins (e.g. protease inhibitors and lectins). In this report, we describe a mass spectrometric approach to the verification of Man-6-phosphorylation based upon LC-MS of MPR-purified proteolytic glycopeptides. This provided a useful tool in validating novel MPR-purified proteins as true Man-6-P glycoproteins and also allowed identification of low abundance components not observed in the analysis of the total Man-6-P glycoprotein mixture. In addition, this approach allowed the global mapping of 99 Man-6-phosphorylation sites from 44 known lysosomal proteins purified from mouse and human brain. This information is likely to provide useful insights into protein determinants for this modification and may be of significant value in protein engineering approaches designed to optimize protein delivery to the lysosome in therapeutic applications such as gene and enzyme replacement therapies.

Amino Acid Sequence↗

PubMed2004

Metabolic profiles to define the genome: can we hear the phenotypes?

There is an increased reliance on genetically modified organisms as a functional genomic tool to elucidate the role of genes and their protein products. Despite this, many models do not express the expected phenotype thought to be associated with the gene or protein. There is thus an increased need to further define the phenotype resultant from a genetic modification to understand how the transcriptional or proteomic network may conspire to alter the expected phenotype. This is best typified by the description of the silent phenotype in genetic manipulations of yeast. High-resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy provides an ideal mechanism for the profiling of metabolites within biofluids, tissue extracts or, with recent advances, intact tissues. These metabolic datasets can be readily mined using a range of pattern recognition techniques, including hierarchical cluster analysis, principal components analysis, partial least squares and neural networks, with the combined approach being termed metabolomics. This review describes the application of NMR-based metabolomics or metabonomics to genetic and chemical interventions in a number of different species, demonstrating the versatility of such an approach, as well as suggesting how it may be integrated with other "omic" technologies.

Animals↗

PubMed2006

Multifunctional drugs with different CNS targets for neuropsychiatric disorders.

The multiple disease etiologies that lead to neuropsychiatric disorders, such as Parkinson's and Alzheimer's disease, amyotrophic lateral sclerosis, Huntington disease, schizophrenia, depressive illness and stroke, offer significant challenges to drug discovery efforts aimed at preventing or even reversing the progression of these disorders. Transcriptomic tools and proteomic profiling have clearly indicated that such diseases are multifactorial in origin. Further, they are thought to be initiated by a cascade of molecular events that involve several neurotransmitter systems. In response to this complexity, a new paradigm has recently emerged that challenges the widely held assumption that 'silver bullet' agents are superior to 'dirty drugs' in therapeutic approaches aimed at the prevention or treatment of neuropsychiatric diseases. A similar pattern of drug development has occurred in strategies for the treatment of cancer, AIDS and cardiovascular diseases. In this review, we offer an overview of therapeutic strategies and novel investigative drugs discovered or developed in our own and other laboratories, that address multiple CNS etiological targets associated with an array of neuropsychiatric disorders.

Animals↗

PubMed2006

Testis-specific lactate dehydrogenase (LDH-C4; Ldh3) in murine oocytes and preimplantation embryos.

LDH-C(4) (Ldh3) is a member of the lactate dehydrogenase family of isozymes that catalyze the terminal reaction in the glycolytic pathway. In mammals, 3 genes, ldha, ldhb, and Ldhc, encode the subunits that assemble into catalytically active homo- and heterotetramers. Differential expression of these genes determines the lactate dehydrogenase (LDH) isozyme composition of tissues, and, as is well known, A subunits predominate in skeletal muscle and B subunits are abundantly produced in brain and heart, with the Ldh2 isozyme the most abundant form in oocytes. The C peptide can be detected first in pachytene spermatocytes and constitutes the primary LDH of spermatozoa. Originally the Ldhc gene (Ldh3 in terminology applied to murine cells) was considered to be testis specific on the basis of immunochemical, enzymatic, and molecular analyses. Here we report the detection of this isozyme in the murine oocyte and early embryo. Our results indicate that Ldh3 mRNA is transcribed in oocytes and cannot be detected in fertilized eggs. Ldh3 protein, however, persists to the blastocyst stage of embryonic development localizing mainly to the cortex region of oocytes, eggs, zygotes, and embryonic blastomeres.

Amino Acid Sequence↗

PubMed2000

Molecular biology and neurosurgery in the third millennium.

The application of techniques in molecular biology to human neurosurgical conditions has led to an increased understanding of disease processes that affect the brain and to novel forms of therapy that favorably modify the natural history of many of these conditions. Molecular strategies are currently being either used or sought for brain tumors, stroke, neurodegenerative diseases, vascular malformations, spinal degenerative diseases, and congenital malformations of the central nervous system. Considering that the structure of deoxyribonucleic acid was ascertained by Watson and Crick as recently as 1953, the progress that has been made to implement molecular medicine in clinical practice has been meteoric. More than 2000 patients have been treated in approved gene therapy trials throughout the world. Many of these patients have been treated for neurological diseases for which conventional medical therapies have been of limited utility. As part of this continuing series on advances in neurosurgery in the third millennium, we first reflect on the history of the nascent field of molecular biology. We then describe the powerful techniques that have evolved from knowledge in this field and have been used in many publications in Neurosurgery, particularly within the past decade. These methods include commonly used techniques such as advanced cytogenetics, differential display, microarray technology, molecular cell imaging, yeast two-hybrid assays, gene therapy, and stem cell utilization. We conclude with a description of the rapidly growing field of bioinformatics. Because the Human Genome Project will be completed within 5 years, providing a virtual blueprint of the human race, the next frontier (and perhaps our greatest challenge) will involve the development of the field of "proteomics," in which protein structure and function are determined from the deoxyribonucleic acid blueprint. It is our conviction that neurosurgeons will continue to be at the forefront of the treatment of patients with neurological diseases using molecular strategies, by performing essential research leading to increased understanding of diseases, by conducting carefully controlled studies to test the effects of treatments on disease processes, and by directly administering (by neurosurgical, endovascular, endoscopic, or stereotactic means) the treatments to patients.

Cytogenetics↗

PubMed2003

Cardiac natriuretic peptides:a proteomic window to cardiac function and clinical management.

Congestive heart failure (CHF) is a leading cause of adult hospitalization in the United States, and despite advancements in treatment, the disease remains a major clinical challenge. The chief symptom of CHF is dyspnea, but in the urgent-care setting, it is often difficult to distinguish between cardiac and pulmonary causes of this symptom. B-type natriuretic peptide (BNP) is mainly synthesized, stored, and released in the ventricular myocardium and is strongly induced during ventricular-wall tension or stretch. It can be measured rapidly at the point of care and can be used to differentiate cardiac from pulmonary etiologies of dyspnea. In addition to its diagnostic utility, it also has prognostic value and may help guide the treatment of patients with CHF. Thus, it is likely that future algorithms incorporating BNP levels and other clinical indicators will become available to guide critical-care physicians in making management decisions for their CHF patients.

Biomarkers↗

PubMed2000

Identification of beta-tubulin isoforms as tumor antigens in neuroblastoma.

There is currently substantial interest in the identification of human tumor antigens for diagnosis and immunotherapy of cancer. We have implemented a proteomic approach for the identification of tumor proteins that elicit a humoral response in cancer patients, which we have applied to neuroblastoma. Proteins from neuroblastoma tumors and cell lines were separated by two-dimensional PAGE and transferred to poly(vinylidene difluoride) membranes. Sera from 23 newly diagnosed patients with neuroblastoma, from 12 newly diagnosed children with other solid tumors, and from 13 normal individuals were screened for IgG and IgM autoantibodies against neuroblastoma proteins by means of Western blot analysis. Sera from 11 patients with neuroblastoma and from 1 patient with a primitive neuroectodermal tumor, but none of the other controls exhibited IgG-based reactivity against a protein constellation with an estimated Mr 50,000. NH2-terminal sequence and mass spectrometric analysis identified the major constituents of this constellation as beta-tubulin isoforms I and III. The IgG antibodies were additionally characterized to be of the subclass IgG1. Neuroblastoma patient sera that contained anti-beta-tubulin IgG antibodies also contained IgM antibodies specific against the full-length beta-tubulin molecule and against COOH-terminal beta-tubulin cleavage products. Neuroblastoma patient sera that reacted with beta-tubulin I and III isoforms in neuroblastoma tissues did not react with beta-tubulin I and III isoforms found in normal brain tissue. Our findings indicate the occurrence of beta-tubulin peptides in neuroblastoma, which are immunogenic. The occurrence of immunogenic peptides in neuroblastoma may have utility in diagnosis and in immunotherapy of this aggressive childhood tumor.

Adolescent↗

PubMed2005

Autoantibodies against the amino terminal of alpha-enolase are a useful diagnostic marker of Hashimoto's encephalopathy.

We investigated autoantibodies and their epitope(s) in Hashimoto's encephalopathy associated with Hashimoto's thyroiditis. In a proteomic analysis, they proved to recognize alpha-enolase. We further searched the epitope region in alpha-enolase using different regions of recombinant proteins expressed in cultured human cells. The amino terminal region was recognized by autobodies from a much higher proportion of patients with Hashimoto's encephalopathy (83.3%; 5/6) than from patients with Hashimoto's thyroiditis (11.8%; 2/17), and not at all by sera from controls (25 healthy individuals and 25 controls with other neurological disorders) (0%; 0/50). Neither the carboxyl terminal nor the mid-region of alpha-enolase showed specificity for Hashimoto's encephalopathy. Autoantibodies against the amino terminal of alpha-enolase are a useful diagnostic marker for Hashimoto's encephalopathy.

Adult↗

PubMed2005

Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex.

Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-alpha as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-alpha complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Neuropeptide processing profile in mice lacking prohormone convertase-1.

Prohormone convertase 1 (PC1; also known as PC3) is believed to be responsible for the processing of many neuropeptide precursors. To look at the role PC1 plays in neuropeptide processing in brain and pituitary, we used radioimmunoassays (RIA) as well as quantitative peptidomic methods and examined changes in the levels of multiple neuropeptide products in PC1 knockout (KO) mice. The processing of proenkephalin was impaired in PC1 KO mouse brains with a decrease in the level of Met-Enkephalin immunoreactivity (ir-Met-Enk) and an accumulation of higher molecular weight processing intermediates containing ir-Met-Enk. Processing of the neuropeptide precursor VGF was also affected in PC1 KO mouse brains with a decrease in the level of an endogenous 3 kDa C-terminal peptide. In contrast, the processing of proSAAS into PEN was not altered in PC1 KO mouse brains. Quantitative mass spectrometry was used to analyze a number of peptides derived from proopiomelanocortin (POMC), provasopressin, prooxytocin, chromogranin A, chromogranin B, and secretogranin II. Among them, the levels of oxytocin and peptides derived from chromogranin A and B dramatically decreased in the PC1 KO mouse pituitaries, while the levels of peptides derived from proopiomelanocortin and provasopressin did not show substantial changes. In conclusion, these results support the notion that PC1 plays a key role in the processing of multiple neuroendocrine peptide precursors and also reveal the presence of a redundant system in the processing of a number of physiologically important bioactive peptides.

Amino Acid Sequence↗

PubMed2001

Metabolic profiling of genetic disorders: a multitissue (1)H nuclear magnetic resonance spectroscopic and pattern recognition study into dystrophic tissue.

A principal problem in understanding the functional genomics of a pathology is the wide-reaching biochemical effects that occur when the expression of a given protein is altered. To complement the information available to bioinformatics through genomic and proteomic approaches, a novel method of providing metabolite profiles for a disease is suggested, using pattern recognition coupled with (1)H NMR spectroscopy. Using this technique the mdx mouse, a model of Duchenne muscular dystrophy (DMD) was examined. Dystrophic tissue had distinct metabolic profiles not only for cardiac and other muscle tissues, but also in the cerebral cortex and cerebellum, where the role of dystrophin is still controversial. These metabolic ratios were expressed crudely as biomarker ratios to demonstrate the effectiveness of the approach at separating dystrophic from control tissue (cardiac (taurine/creatine): mdx = 2.08 +/- 0.04, control 1.55 +/- 0.04, P < 0.005; cortex (phosphocholine/taurine): mdx = 1.28 +/- 0.12, control = 0.83 +/- 0.05, P < 0.01; cerebellum (glutamate/creatine): mdx = 0.49 +/- 0.03, control = 0.34 +/- 0.03, P < 0.01). This technique produced new metabolic biomarkers for following disease progression but also demonstrated that many metabolic pathways are perturbed in dystrophic tissue.

Animals↗