Differential Post-Translational Modifications of Proteins in Bladder Ischemia.

Clinical and basic research suggests that bladder ischemia may be an independent variable in the development of lower urinary tract symptoms (LUTS). We have reported that ischemic changes in the bladder involve differential expression and post-translational modifications (PTMs) of the protein's functional domains. In the present study, we performed in-depth analysis of a previously reported proteomic dataset to further characterize proteins PTMs in bladder ischemia. Our proteomic analysis of proteins in bladder ischemia detected differential formation of non-coded amino acids (ncAAs) that might have resulted from PTMs. In-depth analysis revealed that three groups of proteins in the bladder proteome, including contractile proteins and their associated proteins, stress response proteins, and cell signaling-related proteins, are conspicuously impacted by ischemia. Differential PTMs of proteins by ischemia seemed to affect important signaling pathways in the bladder and provoke critical changes in the post-translational structural integrity of the stress response, contractile, and cell signaling-related proteins. Our data suggest that differential PTMs of proteins may play a role in the development of cellular stress, sensitization of smooth muscle cells to contractile stimuli, and deferential cell signaling in bladder ischemia. These observations may provide the foundation for future research to validate and define clinical translation of the modified biomarkers for precise diagnosis of bladder dysfunction and the development of new therapeutic targets against LUTS.

Molecular Regulation of Concomitant Lower Urinary Tract Symptoms and Erectile Dysfunction in Pelvic Ischemia.

Aging correlates with greater incidence of lower urinary tract symptoms (LUTS) and erectile dysfunction (ED) in the male population where the pathophysiological link remains elusive. The incidence of LUTS and ED correlates with the prevalence of vascular risk factors, implying potential role of arterial disorders in concomitant development of the two conditions. Human studies have revealed lower bladder and prostate blood flow in patients with LUTS suggesting that the severity of LUTS and ED correlates with the severity of vascular disorders. A close link between increased prostatic vascular resistance and greater incidence of LUTS and ED has been documented. Experimental models of atherosclerosis-induced chronic pelvic ischemia (CPI) showed increased contractile reactivity of prostatic and bladder tissues, impairment of penile erectile tissue relaxation, and simultaneous development of detrusor overactivity and ED. In the bladder, short-term ischemia caused overactive contractions while prolonged ischemia provoked degenerative responses and led to underactivity. CPI compromised structural integrity of the bladder, prostatic, and penile erectile tissues. Downstream molecular mechanisms appear to involve cellular stress and survival signaling, receptor modifications, upregulation of cytokines, and impairment of the nitric oxide pathway in cavernosal tissue. These observations may suggest pelvic ischemia as an important contributing factor in LUTS-associated ED. The aim of this narrative review is to discuss the current evidence on CPI as a possible etiologic mechanism underlying LUTS-associated ED.

Formation of Double Stranded RNA Provokes Smooth Muscle Contractions and Structural Modifications in Bladder Ischemia.

Purpose: Growing evidence suggests that ischemia provokes detrusor overactivity and degenerative responses in the bladder. Underlying mechanisms appear to involve modification of smooth muscle contractile rudiments by hypoxia, redox, cellular stress and cell survival signaling. Downstream pathways of cellular stress and stress response molecules eliciting bladder dysfunction in ischemia remain largely elusive. Our goal was to define the role of double stranded RNA (dsRNA), a stress response molecule provoked by redox, in ischemia mediated bladder dysfunction. Methods: A rat model of pelvic ischemia along with a cell culture hypoxia model were used to investigate the expression levels, functional consequences, structural aspects, and regulatory mechanisms of dsRNA in the bladder. Gene and protein expression were examined by reverse transcription polymerase chain reaction (RT-PCR), dot blot, and Western blotting, respectively. Tissue structure and function were assessed using histological staining and organ bath. Regulatory mechanisms were analyzed in cultured bladder smooth muscle cells. Results: The data presented here provide the first evidence of the formation of dsRNA in the overactive bladder. dsRNA is a cellular stress response molecule that sensitizes smooth muscle and regulates inflammatory and degenerative rejoinders. Our data suggest that the production of dsRNA in the bladder is provoked by ischemia. Formation of dsRNA appears to augment bladder smooth muscle contractions and provoke fibrotic and apoptotic responses. Downstream actions of dsRNA in the bladder may involve upregulation of dsRNA-activated protein kinase R (PKR) and caspase-3, the executioner of apoptosis. Conclusion: Activation of dsRNA/PKR pathway may play a role in sensitization of bladder smooth muscle cells to contractile stimuli, whereas dsRNA and caspase-3 crosstalk appear to modulate cellular stress and instigate degenerative responses in bladder ischemia. These observations suggest the role of dsRNA in bladder dysfunction and may open new perspectives to overcome overactive smooth muscle contractions and structural damage in the bladder.

Cellular Stress and Molecular Responses in Bladder Ischemia.

The concept of bladder ischemia as a contributing factor to detrusor overactivity and lower urinary tract symptoms (LUTS) is evolving. Bladder ischemia as a consequence of pelvic arterial atherosclerosis was first documented in experimental models and later in elderly patients with LUTS. It was shown that early-stage moderate ischemia produces detrusor overactivity, while prolonged severe ischemia provokes changes consistent with detrusor underactivity. Recent studies imply a central role of cellular energy sensors, cellular stress sensors, and stress response molecules in bladder responses to ischemia. The cellular energy sensor adenosine monophosphate-activated protein kinase was shown to play a role in detrusor overactivity and neurodegeneration in bladder ischemia. The cellular stress sensors apoptosis signal-regulating kinase 1 and caspase-3 along with heat shock proteins were characterized as important contributing factors to smooth muscle structural modifications and apoptotic responses in bladder ischemia. Downstream pathways seem to involve hypoxia-inducible factor, transforming growth factor beta, vascular endothelial growth factor, and nerve growth factor. Molecular responses to bladder ischemia were associated with differential protein expression, the accumulation of non-coded amino acids, and post-translational modifications of contractile proteins and stress response molecules. Further insight into cellular stress responses in bladder ischemia may provide novel diagnostic and therapeutic targets against LUTS.

Post-Translational Modification Networks of Contractile and Cellular Stress Response Proteins in Bladder Ischemia.

Molecular mechanisms underlying bladder dysfunction in ischemia, particularly at the protein and protein modification levels and downstream pathways, remain largely unknown. Here we describe a comparison of protein sequence variations in the ischemic and normal bladder tissues by measuring the mass differences of the coding amino acids and actual residues crossing the proteome. A large number of nonzero delta masses (11,056) were detected, spanning over 1295 protein residues. Clustering analysis identified 12 delta mass clusters that were significantly dysregulated, involving 30 upregulated (R2 > 0.5, ratio > 2, p < 0.05) and 33 downregulated (R2 > 0.5, ratio < -2, p < 0.05) proteins in bladder ischemia. These protein residues had different mass weights from those of the standard coding amino acids, suggesting the formation of non-coded amino acid (ncAA) residues in bladder ischemia. Pathway, gene ontology, and protein-protein interaction network analyses of these ischemia-associated delta-mass containing proteins indicated that ischemia provoked several amino acid variations, potentially post-translational modifications, in the contractile proteins and stress response molecules in the bladder. Accumulation of ncAAs may be a novel biomarker of smooth muscle dysfunction, with diagnostic potential for bladder dysfunction. Our data suggest that systematic assessment of global protein modifications may be crucial to the characterization of ischemic conditions in general and the pathomechanism of bladder dysfunction in ischemia.

Quantitative Proteomic Analysis Reveals Impaired Axonal Guidance Signaling in Human Postmortem Brain Tissues of Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a distinct neurodegenerative disease that associated with repetitive head trauma. CTE is neuropathologically defined by the perivascular accumulation of abnormally phosphorylated tau protein in the depths of the sulci in the cerebral cortices. In advanced CTE, hyperphosphorylated tau protein deposits are found in widespread regions of brain, however the mechanisms of the progressive neurodegeneration in CTE are not fully understood. In order to identify which proteomic signatures are associated with CTE, we prepared RIPA-soluble fractions and performed quantitative proteomic analysis of postmortem brain tissue from individuals neuropathologically diagnosed with CTE. We found that axonal guidance signaling pathwayrelated proteins were most significantly decreased in CTE. Immunohistochemistry and Western blot analysis showed that axonal signaling pathway-related proteins were down regulated in neurons and oligodendrocytes and neuron-specific cytoskeletal proteins such as TUBB3 and CFL1 were reduced in the neuropils and cell body in CTE. Moreover, oligodendrocyte-specific proteins such as MAG and TUBB4 were decreased in the neuropils in both gray matter and white matter in CTE, which correlated with the degree of axonal injury and degeneration. Our findings indicate that deregulation of axonal guidance proteins in neurons and oligodendrocytes is associated with the neuropathology in CTE. Together, altered axonal guidance proteins may be potential pathological markers for CTE.

Post-Translational Modification of Human Histone by Wide Tolerance of Acetylation.

Histone acetylation adds an acetyl group on the lysine residue commonly found within the N-terminal tail protruding from the histone core of the nucleosome, and is important for chromosome structure and function in gene transcription and chromatin remodeling. Acetylation may also occur on other residues additional to lysine, but have not been thoroughly investigated at the proteomics level. Here we report a wide tolerance acetylation study mimicking the addition of 42 ± 0.5 Da delta mass modification on undefined amino acid residues of histones by shotgun proteomics using liquid chromatography-tandem mass spectrometry. A multi-blind spectral alignment algorithm with a wide peptide tolerance revealed frequent occurrence of 42 ± 0.5 Da modifications at lysine (K), serine (S) and threonine (T) residues in human histones from kidney tissues. Precision delta mass analysis identified acetylation (42.011 ± 0.004 Da) and trimethylation (42.047 ± 0.002 Da) modifications within the delta mass range. A specific antibody was produced to validate the acetylated T22 of human histone H3 (H3T22ac) by immune assays. Thus, we demonstrated that the wide tolerance acetylation approach identified histone acetylation as well as modification variants commonly associated with acetylation at undefined residues additional to lysine.

LC-MS/MS Analysis Unravels Deep Oxidation of Manganese Superoxide Dismutase in Kidney Cancer.

Manganese superoxide dismutase (MNSOD) is one of the major scavengers of reactive oxygen species (ROS) in mitochondria with pivotal regulatory role in ischemic disorders, inflammation and cancer. Here we report oxidative modification of MNSOD in human renal cell carcinoma (RCC) by the shotgun method using data-dependent liquid chromatography tandem mass spectrometry (LC-MS/MS). While 5816 and 5571 proteins were identified in cancer and adjacent tissues, respectively, 208 proteins were found to be up- or down-regulated (p < 0.05). Ontological category, interaction network and Western blotting suggested a close correlation between RCC-mediated proteins and oxidoreductases such as MNSOD. Markedly, oxidative modifications of MNSOD were identified at histidine (H54 and H55), tyrosine (Y58), tryptophan (W147, W149, W205 and W210) and asparagine (N206 and N209) residues additional to methionine. These oxidative insults were located at three hotspots near the hydrophobic pocket of the manganese binding site, of which the oxidation of Y58, W147 and W149 was up-regulated around three folds and the oxidation of H54 and H55 was detected in the cancer tissues only (p < 0.05). When normalized to MNSOD expression levels, relative MNSOD enzymatic activity was decreased in cancer tissues, suggesting impairment of MNSOD enzymatic activity in kidney cancer due to modifications. Thus, LC-MS/MS analysis revealed multiple oxidative modifications of MNSOD at different amino acid residues that might mediate the regulation of the superoxide radicals, mitochondrial ROS scavenging and MNSOD activity in kidney cancer.

Quantitative Proteomic Analysis of Differentially Expressed Proteins and Downstream Signaling Pathways in Chronic Bladder Ischemia.

PURPOSE: Growing evidence suggests that ischemia may contribute to aging associated bladder dysfunction and lower urinary tract symptoms. Our goal was to determine the effects of chronic ischemia on bladder proteomic profiles and characterize downstream signaling pathways. MATERIALS AND METHODS: Bilateral iliac artery atherosclerosis and chronic bladder ischemia were created in male Sprague Dawley® rats. At 8 weeks cystometrograms were obtained. Ischemic and control bladder tissues were then processed for label-free quantitative proteomic analysis. GO (Gene Ontology) and IPA (Ingenuity® Pathway Analysis) software were used to classify altered proteins in bladder ischemia. Western blot was done to confirm differentially expressed proteins. Tissue structure was examined by transmission electron microscopy. RESULTS: Chronic ischemia resulted in detrusor instability and noncompliance. Proteomic analysis revealed a total of 4,277 proteins in ischemic and 4,602 in control bladder tissues. In ischemic bladders 359 and 66 proteins were differentially expressed with a greater than twofold and fivefold change, respectively. On GO analysis differentially expressed proteins were associated with molecular signaling mechanisms underlying proteolysis and degenerative processes. Pathway and network analysis of ischemic tissues suggested that altered proteins are involved in ubiquitination, Nrf2 mediated oxidative stress response, cell death, glucose metabolism and cytoskeleton remodeling. Western blot verified changes in 4 representative proteins, including Nedd4l, Mpo, Ca3 and Fkbp5. Altered proteomic profile of the bladder was associated with widespread ultrastructural damage. CONCLUSIONS: Alterations of bladder proteomic profiles in ischemia may provide new insight into molecular pathways underlying bladder dysfunction and lower urinary tract symptoms in pelvic atherosclerosis.

Proteomic analysis reveals differentially regulated protein acetylation in human amyotrophic lateral sclerosis spinal cord.

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that primarily affects motor neurons in the brain and spinal cord. Histone deacetylase (HDAC) inhibitors have neuroprotective effects potentially useful for the treatment of neurodegenerative diseases including ALS; however, the molecular mechanisms underlying their potential efficacy is not well understood. Here we report that protein acetylation in urea-soluble proteins is differently regulated in post-mortem ALS spinal cord. Two-dimensional electrophoresis (2-DE) analysis reveals several protein clusters with similar molecular weight but different charge status. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identifies glial fibrillary acidic protein (GFAP) as the dominant component in the protein clusters. Further analysis indicates six heavily acetylated lysine residues at positions 89, 153, 189, 218, 259 and 331 of GFAP. Immunoprecipitation followed by Western blotting confirms that the larger form of GFAP fragments are acetylated and upregulated in ALS spinal cord. Further studies demonstrate that acetylation of the proteins additional to GFAP is differently regulated, suggesting that acetylation and/or deacetylation play an important role in pathogenesis of ALS.

Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori.

The functional characterization of Open Reading Frames (ORFs) from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from Helicobacter pylori ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate using derivatized nano-particles, and subsequent identification by mass spectrometry. Candidate genes encoding these proteins were cloned and expressed in Escherichia coli, and the recombinant proteins were purified and characterized biochemically and their biochemical parameters compared with the native ones. These proteins include a guanosine triphosphate (GTP) cyclohydrolase (HP0959), an ATPase (HP1079), an adenosine deaminase (HP0267), a phosphodiesterase (HP1042), an aminopeptidase (HP1037), and new substrates were characterized for a peptidoglycan deacetylase (HP0310). Generally, characterized enzymes were active at acidic to neutral pH (4.0-7.5) with temperature optima ranging from 35 to 55°C, although some exhibited outstanding characteristics.

COMBREX: a project to accelerate the functional annotation of prokaryotic genomes.

COMBREX (http://combrex.bu.edu) is a project to increase the speed of the functional annotation of new bacterial and archaeal genomes. It consists of a database of functional predictions produced by computational biologists and a mechanism for experimental biochemists to bid for the validation of those predictions. Small grants are available to support successful bids.

Targeting of the GTPase Irgm1 to the phagosomal membrane via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) promotes immunity to mycobacteria.

Vertebrate immunity to infection enlists a newly identified family of 47-kilodalton immunity-related GTPases (IRGs). One IRG in particular, Irgm1, is essential for macrophage host defense against phagosomal pathogens, including Mycobacterium tuberculosis (Mtb). Here we show that Irgm1 targets the mycobacterial phagosome through lipid-mediated interactions with phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and PtdIns(3,4,5)P(3). An isolated Irgm1 amphipathic helix conferred lipid binding in vitro and in vivo. Substitutions in this region blocked phagosome recruitment and failed to complement the antimicrobial defect in Irgm1(-/-) macrophages. Removal of PtdIns(3,4,5)P(3) or inhibition of class I phosphatidylinositol-3-OH kinase (PI(3)K) mimicked this effect in wild-type cells. Cooperation between Irgm1 and PI(3)K further facilitated the engagement of Irgm1 with its fusogenic effectors at the site of infection, thereby ensuring pathogen-directed responses during innate immunity.

Extracellular 5-aminolevulinic acid production by Escherichia coli containing the Rhodopseudomonas palustris KUGB306 hemA gene.

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'- phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).

Emerging themes in IFN-gamma-induced macrophage immunity by the p47 and p65 GTPase families.

Vertebrates have evolved complex immune specificity repertoires beyond the primordial components found in lower multi-cellular organisms to combat microbial infections. The type II interferon (IFN-gamma) pathway represents one such system, bridging innate and acquired immunity and providing host protection in a cell-autonomous manner. Recent large-scale transcriptome analyses of IFN-gamma-dependent gene expression in effector cells such as macrophages have highlighted the prominence of two families of GTPases -- p47 IRGs and p65 GBPs -- that are now beginning to emerge as major determinants of antimicrobial resistance. Here we discuss the recent clarification of known family members, their cellular biochemistry and host defense functions as a means to understanding the complex innate immune response engendered in higher vertebrates such as humans and mice.

Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306.

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.