Implications for oxidative stress and astrocytes following 26S proteasomal depletion in mouse forebrain neurones.

Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system, but the underlying mechanisms involved in neuroprotection and neurodegeneration remain unclear. Dysfunction of the ubiquitin proteasome system is one of the proposed hypotheses for the cause and progression of neuronal loss. We have performed quantitative two-dimensional fluorescence difference in-gel electrophoresis combined with peptide mass fingerprinting to reveal proteome changes associated with neurodegeneration following 26S proteasomal depletion in mouse forebrain neurones. Differentially expressed proteins were validated by Western blotting, biochemical assays and immunohistochemistry. Of significance was increased expression of the antioxidant enzyme peroxiredoxin 6 (PRDX6) in astrocytes, associated with oxidative stress. Interestingly, PRDX6 is a bifunctional enzyme with antioxidant peroxidase and phospholipase A2 (PLA2) activities. The PLA2 activity of PRDX6 was also increased following 26S proteasomal depletion and may be involved in neuroprotective or neurodegenerative mechanisms. This is the first in vivo report of oxidative stress caused directly by neuronal proteasome dysfunction in the mammalian brain. The results contribute to understanding neuronal-glial interactions in disease pathogenesis, provide an in vivo link between prominent disease hypotheses and importantly, are of relevance to a heterogeneous spectrum of neurodegenerative diseases.

Hydroxylase inhibition attenuates colonic epithelial secretory function and ameliorates experimental diarrhea.

Hydroxylases are oxygen-sensing enzymes that regulate cellular responses to hypoxia. Transepithelial Cl(-) secretion, the driving force for fluid secretion, is dependent on O(2) availability for generation of cellular energy. Here, we investigated the role of hydroxylases in regulating epithelial secretion and the potential for targeting these enzymes in treatment of diarrheal disorders. Ion transport was measured as short-circuit current changes across voltage-clamped monolayers of T(84) cells and mouse colon. The antidiarrheal efficacy of dimethyloxallyl glycine (DMOG) was tested in a mouse model of allergic disease. Hydroxylase inhibition with DMOG attenuated Ca(2+)- and cAMP-dependent secretory responses in voltage-clamped T(84) cells to 20.2 ± 2.6 and 38.8 ± 6.7% (n=16; P≤0.001) of those in control cells, respectively. Antisecretory actions of DMOG were time and concentration dependent, being maximal after 18 h of DMOG (1 mM) treatment. DMOG specifically inhibited Na(+)/K(+)-ATPase pump activity without altering its expression or membrane localization. In mice, DMOG inhibited agonist-induced secretory responses ex vivo and prevented allergic diarrhea in vivo. In conclusion, hydroxylases are important regulators of epithelial Cl(-) and fluid secretion and present a promising target for development of new drugs to treat transport disorders.

Isotypic analysis of anti-p53 serum autoantibodies and p53 protein tissue phenotypes in colorectal cancer.

The presence of IgA- and IgM-specific autoantibody (AAb) isotypes and their relationship to p53 tissue expression patterns are not well understood. This study aims to investigate the clinical utility of the anti-p53 AAb isotypes and tissue positivity in colorectal cancer (CRC). We analyzed anti-p53 IgG, IgM, and IgA AAbs in sera of 99 CRC patients and 99 non-cancer control subjects. Corresponding tissue expression of the p53 protein was evaluated by immunohistochemistry (IHC). Anti-p53 AAbs of the IgG isotype were present in the sera of 21 out of 99 patients (21%), whereas IgM AAbs were observed in 9 (9%) and IgA in 2 (2%) CRC patients. Anti-p53 AAbs of all 3 isotypes were generally associated with IHC staining indicative of mutated TP53. Seropositive anti-p53 IgM cases in the absence of anti-p53 IgG were linked to wild-type p53. Anti-p53 IgA in the absence of IgG AAbs was detected in 2 non-cancer controls indicating a potential p53 epitope mimicry. Although seropositivity was not associated with patient survival (P = .650), mutant-pattern p53 tissue expression was associated with reduced 5-year overall survival (P = .032); however, it was not an independent prognostic marker (multivariate Cox regression, P = .193). In conclusion, immunoglobulin isotyping revealed that anti-p53 IgM and IgA AAbs were predominantly concurrent with anti-p53 serum IgG and the mutant-pattern p53 tissue phenotype. IgM and IgA seropositive cases in absence of anti-p53 IgG were linked to wild-type p53 tissue phenotype indicating early anti-p53 immune responses preceding isotype class-switch (IgM) or p53 antigen mimicry (IgA).

Microparticles Decorated with Cell-Instructive Surface Chemistries Actively Promote Wound Healing.

Wound healing is a complex biological process involving close crosstalk between various cell types. Dysregulation in any of these processes, such as in diabetic wounds, results in chronic nonhealing wounds. Fibroblasts are a critical cell type involved in the formation of granulation tissue, essential for effective wound healing. 315 different polymer surfaces are screened to identify candidates which actively drive fibroblasts toward either pro- or antiproliferative functional phenotypes. Fibroblast-instructive chemistries are identified, which are synthesized into surfactants to fabricate easy to administer microparticles for direct application to diabetic wounds. The pro-proliferative microfluidic derived particles are able to successfully promote neovascularization, granulation tissue formation, and wound closure after a single application to the wound bed. These active novel bio-instructive microparticles show great potential as a route to reducing the burden of chronic wounds.

IgM and IgA augmented autoantibody signatures improve early-stage detection of colorectal cancer prior to nodal and distant spread.

OBJECTIVES: Tumor-associated autoantibodies (AAbs) in individuals with cancer can precede clinical diagnosis by several months to years. The objective of this study was to determine whether the primary immune response in form of IgM and gut mucosa-associated IgA can aid IgG AAbs in the detection of early-stage colorectal cancer (CRC). METHODS: We developed a novel protein array comprising 492 antigens seropositive in CRC. The array was used to profile IgG, IgM and IgA antibody signatures in 99 CRC patients and 99 sex- and age-matched non-cancer controls. A receiver operating curve (ROC), Kaplan-Meier survival analysis and univariate and multivariate Cox regression analyses were conducted. RESULTS: We identified a panel of 16 multi-isotype AAbs with a cumulative sensitivity of 91% and specificity of 74% (AUC 0.90, 95% CI: 0.850-0.940) across all CRC stages. IgM and IgG isotypes were conversely associated with disease stage with IgM contributing significantly to improved stage I and II sensitivity of 96% at 78% specificity (AUC 0.928, 95% CI: 0.884-0.973). A single identified IgA AAb reached an overall sensitivity of 5% at 99% specificity (AUC 0.520, 95% CI: 0.440-0.601) balanced across all CRC stages. Kaplan-Meier analysis revealed that se33-1 (ZNF638) IgG AAbs were associated with reduced 5-year overall survival (log-rank test, P = 0.012), whereas cumulative IgM isotype signatures were associated with improved 5-year overall survival (log-rank test, P = 0.024). CONCLUSION: IgM AAbs are associated with early-stage colorectal cancer. Combining IgG, IgM and IgA AAbs is a novel strategy to improve early diagnosis of cancers.

Shear stress induces internalization of E-cadherin and invasiveness in metastatic oesophageal cancer cells by a Src-dependent pathway.

Metastatic disease is dependent on tumor cell migration through the venous and lymphatic systems and requires dynamic rearrangement of adherens junctions. Endocytosis of cadherins is a key mechanism to dynamically arrange adherens junctions, signaling, and motility in tumor cells; however, the role of shear in regulating this process in metastatic cells is unknown. In this study, the role of shear in regulating cell surface expression of E-cadherin was investigated. We found that exposure to venous shear (shear rate, 200/s) induced internalization of E-cadherin in adherent metastatic oesophageal tumor cells (OC-1 tumor cell line). Internalized E-cadherin was found localized to Rab5-positive endosomes and was not present in lysosomes. As the Src family of tyrosine kinase have been implicated in regulating cadherin expression, we investigated the role of shear in regulating E-cadherin through Src activity. Pretreatment of OC-1 cells with the specific Src kinase inhibitor 4-amino-5- (4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) prevented shear-induced internalization of E-cadherin. Direct measurement of Src activity (phosphorylation on Y416) showed that Src is activated in sheared OC-1 cells and that the shear-induced increase in phospho-Src is inhibited by the presence of PP1. Moreover, we show that shear stress significantly increased the invasive capacity of OC-1 cells (P < 0.001), a process inhibited by the presence of PP1. These results indicate a novel role for shear in regulating the endocytosis of E-cadherin and invasiveness in metastatic cells.

Dynamic metabolic patterns tracking neurodegeneration and gliosis following 26S proteasome dysfunction in mouse forebrain neurons.

Metabolite profiling is an important tool that may better capture the multiple features of neurodegeneration. With the considerable parallels between mouse and human metabolism, the use of metabolomics in mouse models with neurodegenerative pathology provides mechanistic insight and ready translation into aspects of human disease. Using 400 MHz nuclear magnetic resonance spectroscopy we have carried out a temporal region-specific investigation of the metabolome of neuron-specific 26S proteasome knockout mice characterised by progressive neurodegeneration and Lewy-like inclusion formation in the forebrain. An early significant decrease in N-acetyl aspartate revealed evidence of neuronal dysfunction before cell death that may be associated with changes in brain neuroenergetics, underpinning the use of this metabolite to track neuronal health. Importantly, we show early and extensive activation of astrocytes and microglia in response to targeted neuronal dysfunction in this context, but only late changes in myo-inositol; the best established glial cell marker in magnetic resonance spectroscopy studies, supporting recent evidence that additional early neuroinflammatory markers are needed. Our results extend the limited understanding of metabolite changes associated with gliosis and provide evidence that changes in glutamate homeostasis and lactate may correlate with astrocyte activation and have biomarker potential for tracking neuroinflammation.

Continued 26S proteasome dysfunction in mouse brain cortical neurons impairs autophagy and the Keap1-Nrf2 oxidative defence pathway.

The ubiquitin-proteasome system (UPS) and macroautophagy (autophagy) are central to normal proteostasis and interdependent in that autophagy is known to compensate for the UPS to alleviate ensuing proteotoxic stress that impairs cell function. UPS and autophagy dysfunctions are believed to have a major role in the pathomechanisms of neurodegenerative disease. Here we show that continued 26S proteasome dysfunction in mouse brain cortical neurons causes paranuclear accumulation of fragmented dysfunctional mitochondria, associated with earlier recruitment of Parkin and lysine 48-linked ubiquitination of mitochondrial outer membrane (MOM) proteins, including Mitofusin-2. Early events also include phosphorylation of p62/SQSTM1 (p62) and increased optineurin, as well as autophagosomal LC3B and removal of some mitochondria, supporting the induction of selective autophagy. Inhibition of the degradation of ubiquitinated MOM proteins with continued 26S proteasome dysfunction at later stages may impede efficient mitophagy. However, continued 26S proteasome dysfunction also decreases the levels of essential autophagy proteins ATG9 and LC3B, which is characterised by decreases in their gene expression, ultimately leading to impaired autophagy. Intriguingly, serine 351 phosphorylation of p62 did not enhance its binding to Keap1 or stabilise the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor in this neuronal context. Nrf2 protein levels were markedly decreased despite transcriptional activation of the Nrf2 gene. Our study reveals novel insights into the interplay between the UPS and autophagy in neurons and is imperative to understanding neurodegenerative disease where long-term proteasome inhibition has been implicated.

Analysis of the Sam50 translocase of excavate organisms supports evolution of divergent organelles from a common endosymbiotic event.

As free-living organisms the ancestors of mitochondria and plastids encoded complete genomes, proteomes and metabolomes. As these symbionts became organelles all these aspects were reduced - genomes have degenerated with the host nucleus now encoding the most of the remaining endosymbiont proteome, while the metabolic processes of the symbiont have been streamlined to the functions of the emerging organelle. By contrast, the topology of the endosymbiont membrane has been preserved, necessitating the development of complex pathways for membrane insertion and translocation. In this study, we examine the characteristics of the endosymbiont-derived ß-barrel insertase Sam501 in the excavate super-group. A candidate is further characterized in Trichomonas vaginalis, an unusual eukaryote possessing degenerate hydrogen-producing mitochondria called hydrogenosomes. This information supports a mitochondriate eukaryotic common ancestor with a similarly evolved ß-barrel insertase, which has continued to be conserved in degenerate mitochondria.

Pale body-like inclusion formation and neurodegeneration following depletion of 26S proteasomes in mouse brain neurones are independent of α-synuclein.

Parkinson's disease (PD) is characterized by the progressive degeneration of substantia nigra pars compacta (SNpc) dopaminergic neurones and the formation of Lewy bodies (LB) in a proportion of the remaining neurones. α-synuclein is the main component of LB, but the pathological mechanisms that lead to neurodegeneration associated with LB formation remain unclear. Three pivotal elements have emerged in the development of PD: α-synuclein, mitochondria and protein degradation systems. We previously reported a unique model, created by conditional genetic depletion of 26S proteasomes in the SNpc of mice, which mechanistically links these three elements with the neuropathology of PD: progressive neurodegeneration and intraneuronal inclusion formation. Using this model, we tested the hypothesis that α-synuclein was essential for the formation of inclusions and neurodegeneration caused by 26S proteasomal depletion. We found that both of these processes were independent of α-synuclein. This provides an important insight into the relationship between the proteasome, α-synuclein, inclusion formation and neurodegeneration. We also show that the autophagy-lysosomal pathway is not activated in 26S proteasome-depleted neurones. This leads us to suggest that the paranuclear accumulation of mitochondria in inclusions in our model may reflect a role for the ubiquitin proteasome system in mitochondrial homeostasis and that neurodegeneration may be mediated through mitochondrial factors linked to inclusion biogenesis.

Heterozygosity for the proteasomal Psmc1 ATPase is insufficient to cause neuropathology in mouse brain, but causes cell cycle defects in mouse embryonic fibroblasts.

The ubiquitin proteasome system (UPS) is a fundamental cellular pathway, degrading most unwanted intracellular soluble proteins. Dysfunction of the UPS has been associated with normal aging as well as various age-related pathological conditions, including chronic human neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, leading to a significant interest in the involvement of this degradative system in neurones. We previously reported that the 26S proteasome was essential for neuronal homeostasis and survival in mouse brains following conditional genetic homozygous knockout of a key subunit of the multi-meric 26S proteasome (19S ATPase Psmc1). Here, we investigated the effects of Psmc1 heterozygosity in the mouse brain and primary mouse embryonic fibroblasts. Neuropathologically and biochemically, Psmc1 heterozygous (Psmc1(+/-)) knockout mice were indistinguishable from wild-type mice. However, we report a novel age-related accumulation of intraneuronal lysine 48-specific polyubiquitin-positive granular staining in both wild-type and heterozygous Psmc1 knockout mouse brain. In Psmc1(+/-) MEFs, we found a significant decrease in PSMC1 levels, altered 26S proteasome assembly and a notable G2/M cell cycle arrest that was not associated with an increase in the cell cycle regulatory protein p21. The disturbance in cell cycle progression may be responsible for the growth inhibitory effects in Psmc1(+/-) MEFs.

Localisation of a family of complex-forming ß-barrels in the T. vaginalis hydrogenosomal membrane.

Crucial to organellogenesis was the development of membrane translocases responsible for delivering proteins to new cellular compartments. This investigation examines the Trichomonas vaginalis hydrogenosome, a mitochondrially derived organelle. We identify an expanded family of putative ß-barrel proteins (THOM A-I) comprising nine related sequences. Sub-cellular localisation by immunofluorescence and biochemical fractionation is consistent with THOMs being localised to the hydrogenosomal membrane. Native gel electrophoresis and chemical cross-linking support the ability of THOM proteins to be components of membrane-bound oligomeric protein complexes, consistent with a role in protein translocation.

The ATP-binding cassette proteins of the deep-branching protozoan parasite Trichomonas vaginalis.

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters.

Heterologous expression of a membrane-spanning auxin importer: implications for functional analyses of auxin transporters.

Biochemical studies of plant auxin transporters in vivo are made difficult by the presence of multiple auxin transporters and auxin-interacting proteins. Furthermore, the expression level of most such transporters in plants is likely to be too low for purification and downstream functional analysis. Heterologous expression systems should address both of these issues. We have examined a number of such systems for their efficiency in expressing AUX1 from Arabidopsis thaliana. We find that a eukaryotic system based upon infection of insect cells with recombinant baculovirus provides a high level, easily scalable expression system capable of delivering a functional assay for AUX1. Furthermore, a transient transfection system in mammalian cells enables localization of AUX1 and AUX1-mediated transport of auxin to be investigated. In contrast, we were unable to utilise P. pastoris or L. lactis expression systems to reliably express AUX1.

Mobility and invasiveness of metastatic esophageal cancer are potentiated by shear stress in a ROCK- and Ras-dependent manner.

To metastasize, tumor cells must adopt different morphological responses to resist shear forces encountered in circulating blood and invade through basement membranes. The Rho and Ras GTPases play a critical role in regulating this dynamic behavior. Recently, we demonstrated shear-induced activation of adherent esophageal metastatic cells, characterized by formation of dynamic membrane blebs. Although membrane blebbing has only recently been characterized as a rounded mode of cellular invasion promoted through Rho kinase (ROCK), the role of shear forces in modulating membrane blebbing activity is unknown. To further characterize membrane blebbing in esophageal metastatic cells (OC-1 cell line), we investigated the role of shear in cytoskeletal remodeling and signaling through ROCK and Ras. Our results show that actin and tubulin colocalize to the cortical ring of the OC-1 cell under static conditions. However, under shear, actin acquires a punctuate distribution and tubulin localizes to the leading edge of the OC-1 cell. We show for the first time that dynamic bleb formation is induced by shear alone independent of integrin-mediated adhesion (P < 0.001, compared with OC-1 cells). Y-27632, a specific inhibitor of ROCK, causes a significant reduction in shear-induced bleb formation and inhibits integrin alpha(v)beta(3)-Ras colocalization at the leading edge of the cell. Direct measurement of Ras activation shows that the level of GTP-bound Ras is elevated in sheared OC-1 cells and that the shear-induced increase in Ras activity is inhibited by Y-27632. Finally, we show that shear stress significantly increases OC-1 cell invasion (P < 0.007), an effect negated by the presence of Y-27632. Together our findings suggest a novel physiological role for ROCK and Ras in metastatic cell behavior.

Shear stress modulates the interaction of platelet-secreted matrix proteins with tumor cells through the integrin alphavbeta3.

Interaction of tumor cells with the vascular wall is required for metastasis from the bloodstream. The precise interaction among metastatic cells, circulating platelets, the vessel wall, and physiological flow conditions remains to be determined. In this study, we investigated the interaction of shear on metastatic cell lines adherent to lipopolysaccharide (LPS)-treated endothelium. Tumor cells were perfused over LPS-treated human umbilical vein endothelial cells (HUVECs) at incremental venous shear rates from 50 to 800 s(-1). At a venous shear rate of 400 s(-1), 3% of adherent tumor cells formed pseudopodia under shear, a process we termed shear-induced activation. Because platelets promote tumor dissemination, we then investigated the effect of pretreating tumor cells with platelet releasate collected from activated platelet concentrate. We found that in the presence of platelet releasate, the number of tumor cells adhering to HUVECs increased and tumor "activation" occurred at a significantly lower shear rate of 50 s(-1). This was inhibited with acetylsalicylic acid. Depletion of fibronectin or vitronectin from the platelet releasate resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s(-1). The integrin alphavbeta3 has been shown to mediate cell adhesion primarily through vitronectin and fibronectin proteins. Inhibition of alphavbeta3, followed by the addition of platelet releasate to the tumor cells, resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s(-1). Collectively, our data suggest that alphavbeta3 promotes the metastatic phenotype of tumor cells through interactions with the secreted platelet proteins vitronectin and fibronectin under venous shear conditions.