kDa Fragments Research Articles

Calpain-Mediated Cleavage of Calcineurin in Puromycin Aminonucleoside-Induced Podocyte Injury.

The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus are widely used in the treatment of proteinuria diseases. As the direct target of these drugs, calcineurin has previously been demonstrated to play a role in proteinuria diseases. However, aside from its immune-related effects, the local status of calcineurin in renal inherent cells has not been fully explored in the settings of proteinuria disease and podocyte injury. In this study, calcineurin activity and protein expression in the well-known puromycin aminonucleoside (PAN)-induced podocyte injury model were examined. Interestingly, we found that calcineurin activity was abnormally increased in PAN-treated podocytes, whereas the expression of the full-length 60-kDa calcineurin protein was decreased. This result suggests that there may be another activated form of calcineurin that is independent of the full-length phosphatase. To investigate whether calpain is involved in regulating calcineurin, we exposed PAN-treated podocytes to both pharmacological inhibitors of calpain and specific siRNAs against calpain. Calpain blockade reduced the enhanced calcineurin activity and restored the down-regulated expression of 60-kDa calcineurin. In addition, purified calpain protein was incubated with podocyte extracts, and a 45-kDa fragment of calcineurin was identified; this finding was confirmed in PAN-induced podocyte injury and calpain inhibition experiments. We conclude that calcineurin activity is abnormally increased during PAN-induced podocyte injury, whereas the expression of the full-length 60-kDa calcineurin protein is down-regulated due to over-activated calpain that cleaves calcineurin to form a 45-kDa fragment.

The nature and role of autoimmunity in cystic fibrosis lung immunopathology

Abstract Cystic Fibrosis (CF) arises due to the altered activity of the CF transmembrane regulator (CFTR) protein. While the lung disease in CF is associated with chronic infection by Pseudomonas aeruginosa (PA), identical CFTR mutations exhibit considerable clinical variability. To elucidate this, we evaluated the autoantibody response to bactericidal permeability-increasing protein (BPI) and anti-citrullinated proteins (ACPA) in CF patients (n=38) and compared it with the autoantibody profile in sera from Rheumatoid Arthritis (RA) patients (n=50), given the association of airway inflammation with ACPA induction in RA. The presence of ACPA was restricted to RA patients; no antibodies to BPI were seen. In contrast, 42% of CF patients had anti-BPI IgG, but no ACPA. Contrary to ACPA, the autoantibodies to BPI did not bind to post-translationally modified epitopes but mapped to the protein C-terminus (CT-BPI). Patients with high levels of anti-BPI IgG exhibited worse lung function (lower FEV1), the presence of anti-Cif antibodies (CFTR inhibitory factor), and the absence of the homozygous F508del CFTR mutation. Neutrophil incubation with PA led to the cleavage of BPI into a ~32kDa fragment corresponding to CT-BPI, in an elastase dependent process. In addition, anti-BPI IgG levels were higher in patients harboring the more pathogenic (mucoid) PA strain infection. These findings prompt the model that autoantibody responses reflect greater impairment of PA clearance, which is the hallmark of this chronic inflammatory disorder. Alternatively, given that no clear association between bacterial colonization and CF disease severity exists, the model that autoantibodies independently exacerbate CF lung pathology merits consideration.

Characterization of MUDENG, a novel anti-apoptotic protein.

MUDENG (Mu-2-related death-inducing gene, MuD) is revealed to be involved in cell death signaling. Astrocytes, the major glial cell type in the central nervous system, are a source of brain tumors. In this study, we examined MuD expression and function in human astroglioma cells. Stimulation of U251-MG cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resulted in a 40% decrease in cell viability and a 33% decrease in MuD protein levels, although not in MuD mRNA levels. To study the functional relevance of MuD expression, stable transfectants expressing high levels of MuD were generated. Stimulation of these transfectants with TRAIL resulted in enhanced cell survival (77% for stable and 46% for control transfectants). Depletion of MuD led to a marked reduction upon TRAIL stimulation in cell viability (22% in MuD-depleted cells and 54% in control cells). In addition, we observed that MuD depletion increased the susceptibility of the cells to TRAIL by enhancing the cleavage of caspase-3/-9 and BH3-interacting domain death agonist (Bid). A unique 25-kDa fragment of B-cell lymphoma 2 (Bcl-2) lacking BH4 was observed 60–180 min post TRAIL treatment in MuD-depleted cells, suggesting that Bcl-2 is converted from its anti-apoptotic form to the truncated pro-apoptotic form. Importantly, the TRAIL-mediated decrease in cell viability in MuD-depleted cells was abrogated upon Bid depletion, indicating that the role of MuD in apoptotic signaling takes place at the Bid and Bcl-2 junction. MuD localizes predominantly in the endoplasmic reticulum and partly in the mitochondria and its amounts are reduced 6 h post TRAIL stimulation, presumably via caspase-3-mediated MuD cleavage. Collectively, these results suggest that MuD, a novel signaling protein, not only possesses an anti-apoptotic function but may also constitute an important target for the design of ideal candidates for combinatorial treatment strategies for glioma cells.

The regulation of Sema4D exodomain shedding by protein kinase A in platelets

We have previously shown that Sema4D expressed on the platelet plasma membrane can be cleaved by the metalloprotease ADAM17, producing a 120-kDa exodomain fragment that retains biological activity and remnant fragments of 24–28 kDa that remain associated with the platelet membrane. This process is modulated by calmodulin. Here we investigated the potential role of protein kinase A (PKA) in these events. Using a pharmacological approach, we now show that inhibition of PKA by H89 is sufficient to induce Sema4D exodomain shedding, while activation of PKA inhibits agonist-initiated shedding. Studies on the regulatory mechanism show that the shedding induced by PKA inhibition is mediated by ADAM17, but, unlike agonist-induced shedding, does not involve the dissociation of calmodulin from the Sema4D cytoplasmic domain. In attempt to identify the cleavage sites for shedding, we found that ADAM17 mediates variable cleavages in the juxtamembrane region. Therefore, our data reveal a potential regulatory mechanism for the shedding of Sema4D in platelets.

Expression of xylosyltransferase-1 is modulated by fibronectin fragment in human articular chondrocytes

Purpose: Xylosyltransferase-1 (XT-1), encoded by xylt1 gene, is an essential anabolic enzyme to catalyze the initial and rate-determining step in glycosaminoglycan chain synthesis. The effect of fibronectin fragments (FN-fs), generated by proteolytic cleavage of FN and known as damage-associated molecular pattern (DAMP) molecules, on cartilage metabolism was poorly characterized. In this study we examined 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f)-mediated XT-1 expression mechanism and its signaling pathway, determining the role of 29-kDa FN-f in cartilage matrix synthesis. Methods: Human articular chondrocytes were enzymatically isolated from articular cartilage and cultured in monolayer. In 29-kDa FN-f-stimulated chondrocytes, the relative levels of mRNA and protein for XT-1 were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. In order to investigate the effects of 29-kDa FN-f on XT-1, human chondrocytes were transfected with small interfering RNAs (siRNAs) targeting TLR-2. Results: The level of aggrecan and XT-1 in human osteoarthritis cartilage was significantly decreased compared to normal cartilage. XT-1 expression in cultured primary articluar chondrocytes showed a periodic oscillation in both mRNA and protein level. 29-kDa FN-f significantly suppressed the mRNA and protein levels of XT-1 at 14 h and 24 h, respectively. Inhibition of mitogen activated protein kinase and nuclear factor-κB signaling pathway restored 29-kDa FN-f-inhibited XT-1 expression. Knockdown of toll like receptor-2 (TLR-2) using small interference RNA revealed that the decrease of XT-1 expression by 29-kDa FN-f is mediated by TLR-2 signaling pathway. In addition, Sp3, a repressor of XT-1 promoter, was up-regulated by 29-kDa FN-f. Knockdown and overexpression experiments revealed that XT-1 expression was modulated by 29-kDa FN-f-stimulated Sp3 in primary articular chondrocytes. XT- expression was promoted by AP-1 inhibitor and suppressed by AP-1 activator. Both c-jun and c-fos were activated by 29-kDa FN-f. Conclusions: These results demonstrated that 29-kDa FN-f plays a detrimental role in the regulation of cartilage extracellular matrix formation including XT-1 expression.

Hyaluronan and hyaluronidase, which is better for embryo development?

Our aim was to examine size-specific effects of Hyaluronan (HA) on preimplantation embryo development. We investigated the effects of Hyalovet (HA, 500–750 kDa; the size produced by HA synthase-3, which is abundant in the oviduct), or HA treated with Hyaluronidase-2 (Hyal2; also expressed in the oviduct that breaks down HA into 20 kDa fragments). In experiment 1 (in vivo), oviducts of synchronized and superovulated ewes (n = 20) were surgically exposed on Day 2 post-mating, ligated, and infused with either Hyalovet, Hyalovet + Hyal2, Hyal2, or PBS (control). Ewes were killed 5 days later for recovery of embryos and oviductal epithelial cells (OEC). Blastocyst rates were significantly higher in Hyal2 and Hyalovet + Hyal2 oviducts. Hyaluronidase-2 infusion resulted in higher blastocyst cell numbers and hatching rates. This was associated with increased HSP70 expression in OEC. In contrast, Hyalovet resulted in the lowest development to blastocyst stage and lowest hatching rates, and decreased IGF2 and IGFBP2 expression in OEC. IGF1 and IL1α expression were not affected. In experiment 2, to rule out indirect effects of oviductal factors, ovine embryos were produced and cultured with the same treatments in vitro from Day 2 to 8. Hyaluronidase-2, but not Hyalovet, enhanced blastocyst formation and reduced inner cell mass apoptosis. Hyalovet inhibited hatching. In conclusion, the presence of large-size HA (500–750 kDa) in the vicinity of developing embryos appears to disturb the oviductal environment and embryo development in vivo and in vitro. In contrast, we show evidence that breakdown of HA into smaller fragments is required to maximize embryo development and blastocyst quality.

The effect of elapsed time on cardiac troponin-T (cTnT) degradation and its dependency on the cause of death

PurposeThe aim of the present study is to evaluate the effect of elapsed time on cardiac troponin-T degradation and its dependency on the cause of death. MethodsThe cases included in this study were divided into six groups depending upon the cause of death without any prior history of disease that died in the hospital and their exact time of death was known. The analysis involves extraction of the protein, separation by denaturing gel electrophoresis and visualization by Western blot. ResultsWestern blot data shows the rate of degradation of cTnT into lower molecular weight fragments with respect to time. In cases of control group the greatest amount of protein breakdown was observed within the first 64 h while in MI cases within first 6 h, the original band of cTnT (42 kDa) decreased markedly into seven major fragments, with 25 kDa & 20 kDa fragments being the most prominent. In burn group, at 41.40 h blot shows maximum fragmentation. In electrocution group the greatest amount of protein breakdown was observed within the first 50 Hrs. Within asphyxia cases, the original band of cTnT (42 kDa) decreased markedly into many major and minor fragments which continues up to 210 Hrs while the original band of cTnT (42 kDa) in poisoning cases decreased markedly into many major & minor fragments up to 140 h but after it blot shows only intact protein of very less intensity with few minor fragments. ConclusionIt can be observed that in case of death due to MI, the intact cTnI fragmented at a much faster rate than in burn, electrocution, control, poisoning and asphyxia group. Thus, the rate of fragmentation of intact cTnT into lower molecular weight fragments depends upon the cause of death.

Distinct oxidative cleavage and modification of bovine [Cu– Zn]-SOD by an ascorbic acid/Cu(II) system: Identification of novel copper binding site on SOD molecule

We investigated the combined effect of ascorbate and copper [Asc/Cu(II)] on the integrity of bovine [Cu–Zn]-superoxide dismutase (bSOD1) as a model system to study the metal catalyzed oxidation (MCO) and fragmentation of proteins. We found Asc/Cu(II) mediates specific cleavage of bSOD1 and generates 12.5 and 3.2kDa fragments in addition to oxidation/carbonylation of the protein. The effect of other tested transition metals, a metal chelator, and hydrogen peroxide on the cleavage and oxidation indicated that binding of copper to a previously unknown site on SOD1 is responsible for the Asc/Cu(II) specific cleavage and oxidation. We utilized tandem mass spectrometry to identify the specific cleavage sites of Asc/Cu(II)-treated bSOD1. Analyses of tryptic- and AspN-peptides have demonstrated the cleavage to occur at Gly31 with peptide bond breakage with Thr30 and Ser32 through diamide and α-amidation pathways, respectively. The three-dimensional structure of bSOD1 reveals the imidazole ring of His19 localized within 5Å from the α-carbon of Gly31 providing a structural basis that copper ion, most likely coordinated by His19, catalyzes the specific cleavage reaction.

Clinical impacts of mesothelin expression in gastrointestinal carcinomas.

Mesothelin, C-ERC/mesothelin is a 40-kDa cell surface glycoprotein that is normally present on normal mesothelial cells lining the pleura, peritoneum, and pericardium. Moreover, mesothelin has been shown to be overexpressed in several human cancers, including virtually all mesothelioma and pancreatic cancer, approximately 70% of ovarian cancer and extra bile duct cancer, and 50% of lung adenocarcinomas and gastric cancer. The full-length human mesothelin gene encodes the primary product, a 71-kDa precursor protein. The 71-kDa mesothelin precursor is cleaved into two products, 40-kDa C-terminal fragment that remains membrane-bound via glycosylphosphatidylinositol anchor, and a 31-kDa N-terminal fragment, megakaryocyte potentiating factor, which is secreted into the blood. The biological functions of mesothelin remain largely unknown. However, results of recent studies have suggested that the mesothelin may play a role of cell proliferation and migration. In pancreatic cancer, mesothelin expression was immunohistochemically observed in all cases, but absent in normal pancreas and in chronic pancreatitis. Furthermore, the expression of mesothelin was correlated with an poorer patient outcome in several human cancers. The limited mesothelin expression in normal tissues and high expression in many cancers makes it an attractive candidate for cancer therapy. The present review discusses the expression and function of mesothelin in cancer cells and the utility of mesothelin as a target of cancer therapy.

Caspase cleavage of iASPP potentiates its ability to inhibit p53 and NF-κB.

An intriguing biological question relating to cell signaling is how the inflammatory mediator NF-kB and the tumour suppressor protein p53 can be induced by similar triggers, like DNA damage or infection, yet have seemingly opposing or sometimes cooperative biological functions. For example, the NF-κB subunit RelA/p65 has been shown to inhibit apoptosis, whereas p53 induces apoptosis. One potential explanation may be their co-regulation by common cellular factors: inhibitor of Apoptosis Stimulating p53 Protein (iASPP) is one such common regulator of both RelA/p65 and p53. Here we show that iASPP is a novel substrate of caspases in response to apoptotic stimuli. Caspase cleaves the N-terminal region of iASPP at SSLD294 resulting in a prominent 80kDa fragment of iASPP. This caspase cleavage site is conserved in various species from zebrafish to Homo sapiens. The 80kDa fragment of iASPP translocates from the cytoplasm to the nucleus via the RaDAR nuclear import pathway, independent of p53. The 80kDa iASPP fragment can bind and inhibit p53 or RelA/p65 more efficiently than full-length iASPP. Overall, these data reveal a potential novel regulation of p53 and RelA/p65 activities in response to apoptotic stimuli.

Filanesib Primarily Initiates the Apoptotic Program By Activating Bax through a Calpain-Dependent Mechanism

Introduction:Filanesib (ARRY-520) is a highly selective inhibitor of kinesin spindle protein (KSP), a microtubule motor protein active in proliferating cells, which plays an essential role in assembly and maintaining of the bipolar spindle. In cells arrested by KSP inhibition, Mcl-1 is rapidly depleted resulting in cell death, and consequently cells that are dependent on this pro-survival protein, such as myeloma cells, are particularly sensitive to filanesib. We investigated the mechanisms underlying the antimyeloma effect of this agent, focusing on other Bcl-2 family members.Methods: In vitro action of filanesib, alone and in combination with calpain inhibitor PD150606, was evaluated in multiple myeloma (MM) cell lines by MTT assay, Annexin V staining and cell cycle profile analysis by flow cytometry. MM cells were transiently transfected with non- targeting control short interfering RNA (NT-siRNA), Bax siRNA ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. Expression levels of different proteins were analyzed by Western-Blot.Results:We previously showed that all 11 MM cell lines tested were sensitive to filanesib and that sensitivity to this agent correlated with Bax levels. For these experiments, we focused on 3 cell lines with different Bax expression and sensitivity to filanesib: OPM-2 and MM1S (sensitive and high Bax levels) and U266 (less sensitive and low Bax levels). Treatment of MM1S with this agent triggered the translocation to the mitochondria of several proapoptotic Bcl-2 family members such as Noxa, Bim and Bax with several downstream effects. The mitochondrial translocation and activation of Noxa is key in the degradation of Mcl-1 by mediating the translocation of this protein from the cytosol to the mitochondria promoting its degradation. Regarding Bim, filanesib also induced the early (12-24 hours) expression of several proapoptotic isoforms of Bim that also translocated to the mytochondria. As previously reported, Bax is the top determinant of sensitivity to filanesib. In the present study, remarkably, once translocated into the mitochondria, Bax was also cleaved into the very potent proapoptotic 18 kDa fragment. All these events triggered the mitochondrial release of cytochrome C (caspase dependent apoptosis) and AIF (caspase independent apoptosis).In order to confirm the role of Bax in filanesib-induced apoptosis, we knocked-down Bax in MM1S by using small interfering RNA. This approach clearly decreased the sensitivity of these cells to filanesib, as treatment with 10 nM for 24 hours induced only 26% apoptosis in the siRNA-Bax cells as compared with 50% in the non-targeted cells (as compared with 58% vs 61% for bortezomib).Furthermore, treatment with filanesib also induced cleavage of effector caspases (3 and 7) in all cell lines studied (OPM2, MM1S, U266). PARP was also cleaved in these cells, but it was previous to caspase activation in the most sensitive cell lines (OPM2, MM1S) suggesting a caspase-independent mechanism of apoptosis. This was confirmed by pre-treatment with the pan caspase-inhibitor Z-VAD-FMK, which did not rescue OPM2 and MM1S cells from apoptosis.Interestingly, one potential mechanism that could link both effects is the activity of calpain, a cysteine protease involved in caspase-independent apoptosis. This protein is a well-known caspase-independent way of processing PARP into the 60 kDa fragment, and has also been described as being responsible for Bax cleavage into the 18-kDa fragment. Consistent with this hypothesis, pre-treatment with the calpain inhibitor PD150606 clearly reduced the activity of filanesib in these cells (35 % to 70 % of survival) as assessed by MTT.Finally, consistent with the previous hypothesis, the less sensitive U266 cell line contained undetectable Bax protein suggesting that filanesib was not able to trigger caspase-independent apoptosis. However, a secondary caspase dependent apoptosis mechanism was confirmed as the pan-caspase inhibitor ZVAD-FMK was able to almost completely abrogate the activity of filanesib.Conclusions: Our results show that filanesib primarily initiates apoptosis by activating Bax in a caspase-independent manner, probably via calpain, a powerful accelerator of the apoptotic process. In addition, Noxa and BIM appear to be crucial for modulating Mcl-1 proteasomal degradation and Bax activation.This work was funded in part by the company Array BioPharma. DisclosuresTunquist:Array BioPharma: Employment. Mateos:Takeda: Consultancy; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Ocio:Mundipharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding; MSD: Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Array BioPharma: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Pharmamar: Consultancy, Research Funding; Janssen: Honoraria.

Fibronectin fragment-induced expression of matrix metalloproteinases is mediated by MyD88-dependent TLR-2 signaling pathway in human chondrocytes.

IntroductionFibronectin fragments (FN-fs) are increased in the cartilage of patients with osteoarthritis (OA) and have a potent chondrolytic effect. However, little is known about the cellular receptors and signaling mechanisms that are mediated by FN-fs. We investigated whether the 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) regulates cartilage catabolism via the Toll-like receptor (TLR)-2 signaling pathway in human chondrocytes.MethodsSmall interfering RNA was used to knock down TLR-2 and myeloid differentiation factor 88 (MyD88). TLR-2 was overexpressed in chondrocytes transfected with a TLR-2 expression plasmid. The expression levels of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 were analyzed using quantitative real-time reverse transcription polymerase chain reactions, immunoblotting, or enzyme-linked immunosorbent assay. The effect of TLR-2 on 29-kDa FN-f-mediated signaling pathways was investigated by immunoblotting.ResultsTLR-2, TLR-3, TLR-4, and TLR-5 mRNA were significantly overexpressed in OA cartilage compared with normal cartilage, whereas no significant difference of TLR-1 mRNA expression was found. 29-kDa FN-f significantly increased TLR-2 expression in human chondrocytes in a dose- and time-dependent manner. Knockdown of TLR-2 or MyD88, the latter a downstream adaptor of TLR-2, significantly inhibited 29-kDa FN-f-induced MMP production at the mRNA and protein levels. Conversely, TLR-2 overexpression led to enhanced MMP production by 29-kDa FN-f. In addition, TLR-2 knockdown apparently inhibited 29-kDa FN-f-mediated activation of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, and p38, but not of c-Jun N-terminal kinase or extracellular signal-regulated kinase. Exposure to synovial fluid (SF) from affected joints of patients with OA elevated MMP-1, MMP-3, and MMP-13 expression markedly in primary chondrocytes without reducing cell viability. However, TLR-2 knockdown in chondrocytes significantly suppressed SF-induced MMP induction.ConclusionsOur data demonstrate that the MyD88-dependent TLR-2 signaling pathway may be responsible for 29-kDa FN-f-mediated cartilage catabolic responses. Our results will enhance understanding of cartilage catabolic mechanisms driven by cartilage degradation products, including FN-f. The modulation of TLR-2 signaling activated by damage-associated molecular patterns, including 29-kDa FN-f, is a potential therapeutic strategy for the prevention of cartilage degradation in OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0833-9) contains supplementary material, which is available to authorized users.

Biochemical classification of tauopathies by immunoblot, protein sequence and mass spectrometric analyses of sarkosyl-insoluble and trypsin-resistant tau.

Intracellular filamentous tau pathology is the defining feature of tauopathies, which form a subset of neurodegenerative diseases. We have analyzed pathological tau in Alzheimer’s disease, and in frontotemporal lobar degeneration associated with tauopathy to include cases with Pick bodies, corticobasal degeneration, progressive supranuclear palsy, and ones due to intronic mutations in MAPT. We found that the C-terminal band pattern of the pathological tau species is distinct for each disease. Immunoblot analysis of trypsin-resistant tau indicated that the different band patterns of the 7–18 kDa fragments in these diseases likely reflect different conformations of tau molecular species. Protein sequence and mass spectrometric analyses revealed the carboxyl-terminal region (residues 243–406) of tau comprises the protease-resistant core units of the tau aggregates, and the sequence lengths and precise regions involved are different among the diseases. These unique assembled tau cores may be used to classify and diagnose disease strains. Based on these results, we propose a new clinicopathological classification of tauopathies based on the biochemical properties of tau.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1503-3) contains supplementary material, which is available to authorized users.

Suppressive effect of ATP on the denaturation of sarcoplasmic reticulum (Ca2+)-ATPase from southern bluefin tuna Thunnus maccoyii and its biochemical properties

Sarcoplasmic reticulum (SR) was prepared from ordinary white muscle of southern bluefin tuna. The chymotryptic digestibility, pH and temperature dependent Ca2+-ATPase activity of SR, as well as the thermal and freezing stability with different additives including ATP were examined. A major component of SR Ca2+-ATPase, which was about 100 kDa by SDS-PAGE analysis, was digested to 60 kDa and 38 kDa fragments by α-chymotrypsin. The SR Ca2+-ATPase activity was 0.3 μmol Pi/min/mg at 25 °C. There was no break point in the Arrhenius plot of temperature-dependent Ca2+-ATPase activity. The activation energy of Ca2+-ATPase was 35.44 kJ/mol. The pH dependence of SR Ca2+-ATPase activity of southern bluefin tuna showed a clear peak with a maximum activity at pH 6.5, and SR Ca2+-ATPase was half inactivated when heated at 45 °C for 4 min. SR Ca2+-ATPase was almost inactivated by pH 5.5 acid treatment, for 2 h at 0 °C. Denaturation by acid treatment was strongly suppressed by the addition of 2 mM ATP. ATP also showed a strong suppressive effect against denaturation of SR Ca2+-ATPase by freezing and heating. These results indicate that ATP reduces denaturation of SR Ca2+-ATPase and that enhancement of ATP levels during freezing has the potential to improve meat quality.

Zinc pyrithione inhibits caspase-3 activity, promotes ErbB1-ErbB2 heterodimerization and suppresses ErbB2 downregulation in cardiomyocytes subjected to ischemia/reperfusion

Heart tissue becomes zinc-depleted and the capacity to mobilize labile zinc is diminished, indicating zinc dyshomeostasis during ischemia/reperfusion (I/R). Apparently, zinc pyrithione restores the basal zinc levels during I/R and prevents apoptosis by activating phosphatidyl inositol-3-kinase/Akt and targeting mitochondrial permeability transition. Receptor tyrosine kinases of the ErbB family (ErbB1 to ErbB4) are cell surface proteins that can regulate cell growth, proliferation and survival. Previous studies have shown that zinc pyrithione-induced activation of PI3kinase/Akt requires ErbB2 expression. On the other hand, while I/R decreases ErbB2 levels causing cardiomyocyte dysfunction and cell death, zinc pyrithione restores ErbB2 levels and maintains cardiomyocyte function. H9c2 cells expressed all the four ErbBs, although the expression of ErbB1 and ErbB2 were higher compared to ErbB3 and ErbB4. Hypoxia/Reoxygenation (H/R) had opposing effects on the mRNA expression of ErbB1 and ErbB2. ErbB2 mRNA levels were enhanced, but corresponding ErbB2 protein levels decreased after reoxygenation. H/R induced the degradation of ErbB2 in caspase-3 dependent manner, with the formation of a 25kDa fragment. This fragment could be detected after H/R only upon treatment of the cells with a proteasomal inhibitor, ALLN, suggesting that caspase-mediated cleavage of 185kDa ErbB2 results in C-terminal cleavage and formation of 25kDa fragment, which is further degraded by proteasome. Heterodimerization and phosphorylation of ErbB2/ErbB1 which decreased upon reoxygenation, was promoted by zinc pyrithione. Zinc pyrithione effectively suppressed the caspase activation, decreased the proteolytic cleavage of ErbB2, enhanced the phosphorylation and activation of ErbB1-ErbB2 complexes and improved the cell survival after hypoxia/reoxygenation.

Platelet-Rich Plasma Attenuates 30-kDa Fibronectin Fragment–Induced Chemokine and Matrix Metalloproteinase Expression by Meniscocytes and Articular Chondrocytes

Background: Proteolytic fragments of fibronectin have catabolic effects on cartilage and menisci. Platelet-rich plasma (PRP) is increasingly being used to treat a range of joint conditions, but it is unknown whether PRP influences fibronectin fragment (FN-f) procatabolic activity. Hypotheses: The procatabolic activity of FN-f on meniscocytes and articular chondrocytes is attenuated by cotreatment with PRP. Study Design: Controlled laboratory study. Methods: Human meniscocytes were treated with FN-f (30 kDa) with or without PRP coincubation, and gene expression was analyzed by complementary DNA microarray analysis. Validation of altered expression of known and novel chemokine and protease genes was undertaken by real-time polymerase chain reaction (RT-PCR) in articular chondrocytes and meniscocytes. Chemokine release was assayed by enzyme-linked immunosorbent assay, and intracellular pathway signaling was evaluated by Western immunoblotting. Results: Microarray analysis and RT-PCR showed increased expression of matrix metalloproteinase (MMP)1, MMP2, MMP3, MMP9, MMP13, interleukin (IL)–6, IL-8 (CXCL8), CCL5, CCL20, and CXCL10 chemokines in meniscocytes after treatment with FN-f. Upregulation of these genes was significantly attenuated by PRP. Similar results were seen with articular chondrocytes, although no changes in MMP2 or MMP9 levels were identified. PRP-induced suppression of gene expression was associated with activation of Akt and p44/p42. Conclusion: PRP treatment attenuates the 30-kDa FN-f–induced expression of a range of proinflammatory chemokines and MMPs, including IL-8, IL-6, CCL20, CCL5, CXCL10, MMP1, MMP3, and MMP13, by both meniscocytes and articular chondrocytes. Clinical Relevance: These observations provide support for the use and further trials of PRP in management of cartilage and meniscal injuries.

Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme

The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised.

Formation of 55-kDa fragments under impaired coordination bonds and hydrophobic interactions in peripheral light-harvesting complexes isolated from photosynthetic purple bacteria

Size exclusion chromatography was used to assess the relative size of intact and diphenylamine-treated (DPA, with suppressed carotenoid synthesis) peripheral light-harvesting complexes (LH2 complexes) of the sulfurbacterium Allochromatium minutissimum. Both LH2 complexes were nonamers and had the same elution volume V(e), coinciding with that for the LH2 complex of Rhodoblastus acidophilus (strain 10050). Their molecular mass was 150 kDa. Bot pheophytinization of bacteriochlorophyll (BChl) at low pH and treatment with the detergent LDAO, affecting the hydrophobic interactions between the neighboring protomers, result in the fragmentation of the ring of the isolated LH2 complexes and formation of 55-kDa fragments with molecular masses corresponding to one-third of the initial value. Fragmentation caused by both pheophytinization and detergent treatment was much more rapid in DPA-treated LH2 complexes than in the intact ones. The 55-kDa fragments formed at low pH values contained monomeric bacteriopheophytin, while the fragments of a similar molecular mass formed at pH 8.0 in the presence of the detergent contained monomeric BChl. The observed fragmentation was hypothesized to reflect the inherent C3 symmetry of the LH2 complexes, with the preliminarily assembled trimers used as building blocks.

Characterization of CPAF Critical Residues and Secretion during Chlamydia trachomatis Infection

CPAF (chlamydial protease-like activity factor), a Chlamydia serine protease, is activated via proximity-induced intermolecular dimerization that triggers processing and removal of an inhibitory peptide occupying the CPAF substrate-binding groove. An active CPAF is a homodimer of two identical intramolecular heterodimers, each consisting of 29-kDa N-terminal and 35-kDa C-terminal fragments. However, critical residues for CPAF intermolecular dimerization, catalytic activity, and processing were defined in cell-free systems. Complementation of a CPAF-deficient chlamydial organism with a plasmid-encoded CPAF has enabled us to characterize CPAF during infection. The transformants expressing CPAF mutated at intermolecular dimerization, catalytic, or cleavage residues still produced active CPAF, although at a lower efficiency, indicating that CPAF can tolerate more mutations inside Chlamydia-infected cells than in cell-free systems. Only by simultaneously mutating both intermolecular dimerization and catalytic residues was CPAF activation completely blocked during infection, both indicating the importance of the critical residues identified in the cell-free systems and exploring the limit of CPAF's tolerance for mutations in the intracellular environment. We further found that active CPAF was always detected in the host cell cytoplasm while nonactive CPAF was restricted to within the chlamydial inclusions, regardless of how the infected cell samples were treated. Thus, CPAF translocation into the host cell cytoplasm correlates with CPAF enzymatic activity and is not altered by sample treatment conditions. These observations have provided new evidence for CPAF activation and translocation, which should encourage continued investigation of CPAF in chlamydial pathogenesis.

Dynamic Changes in Equatorial Segment Protein 1 (SPESP1) Glycosylation During Mouse Spermiogenesis.

ESP1/SPESP1 is a testis-specific, postmeiotic gene expressed in round spermatids that encodes equatorial segment protein 1, an intra-acrosomal protein found in the acrosomal matrix and on the luminal surface of the inner and outer acrosomal membranes within the equatorial segment domain of mature spermatozoa. A comparison of testicular protein extracts with caput, corpus, and caudal epididymal sperm proteins revealed striking differences in the apparent masses of SPESP1 isoforms. The predominant isoforms of SPESP1 in the testis were 77 and 67 kDa, with 47-kDa forms present to a minor degree. In contrast, SPESP1 isoforms of 47 and 43 kDa were found in caput, corpus, and caudal sperm, indicating that SPESP1 undergoes noticeable mass changes during spermiogenesis and/or subsequent transport to the epididymis. On two-dimensional (2D) SDS-PAGE, testicular SPESP1 isoforms resolved as a train of pI values from 4.9 to 5.2. Immunoprecipitated 77-kDa SPESP1 from testis reacted with the glycoprofile stain after one-dimensional and 2D gel electrophoresis, indicating that the 77-kDa testicular isoform was highly glycosylated. One charge variant of the 67-kDa isoform was also glycoprofile positive after 2D gel resolution. The 47- and 43-kDa isoforms of SPESP1 from epididymal sperm did not stain with glycoprofile, suggesting an absence of, or few, glycoprofile-sensitive glycoconjugates in epididymal SPESP1. Treatment of testicular extracts with a variety of glycosidases resulted in mass shifts in immunoreactive SPESP1, indicating that testicular SPESP1 was glycosylated and that terminal sialic acid, N- and O-glycans were present. A mixture of deglycosidase enzymes (including PNGase-F, neuraminidase, beta1-4 galactosidase, endo-alpha-N-acetylgalactosaminidase, and beta N-acetyl-glucosaminidase) completely eliminated the 77- and 67-kDa SPESP1 bands and resulted in the appearance of 75-, 60-, 55-, 50-, 47-, and 43-kDa forms, confirming that both the 77- and 67-kDa testicular forms of SPESP1 contain complex carbohydrate residues. Treatment of caudal epididymal sperm with PNGase-F enzymes showed a faint deglycosylated band at 30 kDa, but neuraminidase did not result in any molecular shift, indicating that epididymal sperm SPESP1 did not contain sialic acid/N-acetylglucosamine residues. These findings are consistent with the hypothesis that SPSPESP1 undergoes significant glycosylation in the testis and that the majority of these glycoconjugates are removed by the time sperm reach the caput epididymis. Studies of the fate of SPESP1 after the acrosome reaction localized SPESP1 to the equatorial segment region in both noncapacitated and capacitated, acrosome-reacted sperm. During capacitation, SPESP1 underwent proteolysis, resulting in a 27-kDa fragment. Zona-free oocytes incubated with recSPESP1 protein showed complementary binding sites on the microvillar oolemmal domain. Both recSPESP1 and anti-recSPESP1 antibody inhibited in vitro fertilization.