kDa Product Research Articles

Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry.

Human CLCA2 regulates store-operated calcium entry (SOCE) by interacting with Orai1 and STIM1. It is expressed as a 943aa type I transmembrane protein that is cleaved at amino acid 708 to produce a diffusible 100 kDa product. The N-terminal ectodomain contains a hydrolase-like subdomain with a conserved HEXXH zinc-binding motif that is proposed to cleave the precursor autoproteolytically. Here, we tested this hypothesis and its link to SOCE. We first studied the conditions for autocleavage in isolated membranes and then in a purified protein system. Cleavage was zinc-dependent and abolished by mutation of the E in the HEXXH motif to Q, E165Q. Cleavage efficiency increased with CLCA2 concentration, implying that it occurs in trans. Accordingly, the E165Q mutant was cleaved by co-transfected wildtype CLCA2. Moreover, CLCA2 precursors with different epitope tags co-immunoprecipitated. In a membrane-free system utilizing immunopurified protease and target, no cleavage occurred unless the target was first denatured, implying that membranes provide essential structural or conformational cues. Unexpectedly, cleavage caused a conformational shift: an N-terminal antibody that immunoprecipitated the precursor failed to precipitate the N-terminal product unless the product was first denatured with an ionic detergent. The E165Q mutation abolished the stimulation of SOCE caused by wildtype CLCA2, establishing that the metalloprotease activity is required for this regulatory function.

Geometric tumor embolic budding characterizes inflammatory breast cancer.

Inflammatory breast cancer (IBC) is characterized by numerous tumor emboli especially within dermal lymphatics. The explanation remains a mystery. This study combines experimental studies with two different IBC xenografts with image algorithmic studies utilizing human tissue microarrays (TMAs) of IBC vs non-IBC cases to support a novel hypothesis to explain IBC's sina qua non signature of florid lymphovascular emboli. In the human TMAs, compared to tumor features like nuclear grade (size), mitosis and Ki-67 immunoreactivity which show that IBC is only modestly more proliferative with larger nuclei than non-IBC, what really sets IBC apart is the markedly greater number of tumor emboli and distinctly smaller emboli whose numbers indicate geometric or exponential differences between IBC and non-IBC. In the experimental xenograft studies, Mary-X gives rise to tight spheroids in vitro which exhibit dynamic budding into smaller daughter spheroids whereas Karen-X exhibits only loose non-budding aggregates. Furthermore Mary-X emboli also bud dramatically into smaller daughter emboli in vivo. The mechanism that regulates this involves the generation of E-cad/NTF1, a calpain-mediated cleavage 100kDa product of 120kDa full length membrane E-cadherin. Inhibiting this calpain-mediated cleavage of E-cadherin by blocking either the calpain site of cleavage (SC) or the site of binding (SB) with specific decapeptides that both penetrate the cell membrane and mimic either the cleavage site or the binding site on E-cadherin, inhibits the generation of E-cad/NTF1 in a dose-dependent manner, reduces spheroid compactness and decreases budding. Since E-cad/NFT1 retains the p120ctn binding site but loses the α-and β-catenin sites, promoting its 360° distribution around the cell's membrane, the vacilating levels of this molecule trigger budding of both the spheroids as well as the emboli. Recurrent and geometric budding of parental emboli into daughter emboli then would account for the plethora of emboli seen in IBC.

Inducible expression of truncated NAC62 provides tolerance against Alternaria brassicicola and imparts developmental changes in Indian mustard

Indian mustard (Brassica juncea) faces significant yield loss due to the ‘Black Spot Disease,’ caused by a fungus Alternaria brassicicola. In plants, NAC transcription factors (NAC TFs) are known for their roles in development and stress tolerance. One such NAC TF, NAC 62, was induced during A. brassicicola challenge in Sinapis alba, a non-host resistant plant against this fungus. Sequence analyses of BjuNAC62 from B. juncea showed that it belonged to the membrane-bound class of transcription factors. Gene expression study revealed differential protein processing of NAC62 between B. juncea and S. alba on pathogen challenge. Furthermore, NAC62 processing to 25 kDa protein was found to be unique to the resistant plant during pathogenesis. Conditional expression of BjuNAC62ΔC, which lacks its transmembrane domain, in B. juncea showed improved tolerance to A. brassicicola. BjuNAC62ΔC processing to 25 kDa product was also observed in tolerant transgenic plants. Additionally, transgenic plants showed induced expression of genes associated with defense-related phytohormone signaling pathways on pathogen challenge. Again, altered phenotypes suggest a possible developmental effect of BjuNAC62∆C in transgenic plants. The overall results suggest that the processing of BjuNAC62 might be playing a crucial role in resistance response against Black Spot disease by modulating defense-associated genes.

Discovery and Characterization of an Aberrant Small Form of Glycoprotein I of Herpes Simplex Virus Type I in Cell Culture.

ABSTRACTThe 380-to-393-amino-acid glycoprotein I (gI) encoded by herpes simplex virus 1 (HSV-1) is a critical mediator for viral cell-to-cell spread and syncytium formation. Here we report a previously unrecognized aberrant form of gI in HSV-1-infected cells. Production of this molecule is independent of cell type and viral strains. It had an unexpected gel migration size of approximately 23 kDa, was packaged into viral particles, and could be coimmunoprecipitated by antibodies to both N and C termini of gI. Deep sequencing failed to detect alternative RNA splicing, and the in vitro transcribed full-length mRNA gave rise to the 23 kDa protein in transfected cells. Combined mass spectrometry and antibody probing analyses detected peptide information across different regions of gI, suggesting the possibility of a full-length gI but with abnormal migration behavior. In line with this notion, the HA insertion mutagenesis revealed a stable fold in the gI extracellular region aa.38-196 resistant to denaturing conditions, whereas small deletions within this region failed the antibodies to detect the fast, but not the slow-moving species of gI. It is also intriguing that the structure could be perturbed to some extent by a gBsyn mutation, leading to exposure or shielding of the gI epitopes. Thus, the HSV-1 gI apparently adopts a very stable fold in its natural form, rendering it an unusual biophysical property. Our findings provide novel insight into the biological properties of HSV gI and have important implications in understanding the viral spread and pathogenesis.IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but its behavior during infection has remained poorly defined. Along with the classic 66 kDa product, here we report a previously unrecognized, approximately 23 kDa form of gI. Biochemical and genetics analyses revealed that this molecule represents the full-length form of gI but adopts a stable fold in its extracellular domain that is resistant to denatured conditions, thus contributing to the aberrant migration rate. Our results revealed a novel property of HSV-1 gI and have important implications in understanding viral pathogenesis.

Use of Trichoderma koningiopsis chitinase to enhance the insecticidal activity of Beauveria bassiana against Diatraea saccharalis.

Trichoderma is a well-known soil-borne fungus, highly efficient producer of extracellular enzymes including chitinases. The aim of this study was to recover a chitinase from fermentation waste after harvesting Trichoderma koningiopsis Th003 conidia and assess its potential as an enhancer of Beauveria bassiana insecticidal activity against Diatraea saccharalis. T. koningiopsis was produced by solid fermentation, conidia were harvested, and a crude extract (CE) was recovered by washing the residual substrate (rice:wheat bran). The partially purified chitinase (PPC) (75 kDa product) with N-acetyl-β-glucosaminidase activity was obtained by chromatography to 29.3-fold with optimal activity at pH 5 and 55°C. Both the CE and the PPC were mixed with B. bassiana Bv062 conidia and assessed in a bioassay against D. saccharalis larvae. The CE and PPC from T. koningiopsis Th003 did not affect the germination or viability of B. bassiana conidia and enhanced its insecticidal activity when used at 0.06 U/ml enzymatic activity with a 24.5% reduction in B. bassiana lethal time (LT90 ). This study demonstrated the potential of chitinases produced by T. koningiopsis in solid fermentation to be recovered from the waste substrate and used as an additive to enhance B. bassiana, adding value to the main waste from the Trichoderma biopesticide/biofertilizer industries.

Analysis of the immunomodulatory activity of excreted and secreted products from Haemonchus placei transition infective larvae (xL3)

The excretory/secretory (E/S) products released by infective transitory larvae (xL3) of Haemonchus placei have an important biological function in stimulating immune mechanisms during the invasive process. Our objective was to analyse the modulatory activity of 15 and 70 kDa E/S products from H. placei xL3. Both E/S products were collected from xL3in vitro cultures at 24 and 72 h. Proteins were confirmed by SDS-PAGE, and the corresponding spots were elicited by gel isoelectrofocusing (IEF) and characterised by mass spectrometry. Additionally, flow cytometry of CD4+/γδ+ T cells and immune gene expression were performed by proliferation assays using each E/S product to stimulate lymphocyte and peripheral blood mononuclear cells (PBMCs) from non-infected calves. The IEF results displayed two spots of 7.0 and 5.7 pI for the 15 and 70 kDa products, respectively. Additionally, 29 and 17 peptides from the 15 and 70 kDa E/S products, respectively, were identified with the hypothetical neurotransmitter and enzymatic functions necessary for larval development. The relative expression displayed upregulation of IL4, 5, 6, 8, 10, 13, IFNγ, and FCεR1A genes (from 2.0- to 17.6-fold, p < 0.05) stimulated by the 15 and 70 kDa proteins, indicating specific genes against haemonchosis. Although the percentage of median florescence intensity (MFI%) of CD4+/γδ+ T cells did not change for both E/S products compared to the negative control and concanavalin-A stimulated cells as the positive control (p > 0.05), the 15-kDa protein reduced the levels of both T cells, and the 70-kDa proteins increased the γδ+ cells slightly. Additionally, there was increased PBMCs proliferation by the 70 kDa proteins (p < 0.05), denoting the biological role of other immune cells. The 15 and 70 kDa protein E/S products from H. placei xL3 showed modulation of the immune response, and although more studies are required, they indicate important functions in the host/parasite interaction.

Abstract A20: 17-Aminogeldanamycin inhibits cytoprotective UPR pathways and cooperates with inhibitors of the MAPK signaling cascade in apoptosis induction

Abstract Geldanamycin is an inhibitor of Heat Shock Protein 90 (HSP90). Attenuation of HSP90 activity results in accumulation of unfolded proteins, induction of endoplasmic reticulum (ER) stress, and activation of unfolded protein response (UPR). HSP90 is a chaperone protein participating in appropriate folding of more than 100 proteins, including IRE1α and BRAFV600E. In melanoma cells, BRAFV600E was correlated with chronically induced ER stress and involved in UPR-induced resistance to apoptosis. As geldanamycin is highly hepatotoxic, geldanamycin derivatives exerting similar proapoptotic properties but higher selectivity towards cancer cells are needed. In the present study, 17-aminogeldanamycin exerted higher proapoptotic activity than geldanamycin in patient-derived melanoma cell populations as demonstrated by using fluorescence time-lapse imaging (IncuCyte ZOOM). We have also shown that 17-aminogeldanamycin inhibits ERK1/2 activity and cell proliferation,as well as induces apoptosis transiently, enhancing expression of HSP70 and GRP78. GRP78 regulates activity of the UPR pathway, and we have demonstrated that 17-aminogeldanamycin inhibits IRE1α-dependent pathway of UPR responsible for degradation of unfolded proteins and reduces levels of 36 kDa product of ATF6 cleavage in selected melanoma cell populations. Moreover, we have shown that combination of 17-aminogeldanamycin and trametinib induces apoptosis earlier and more efficiently. Our study suggests that cooperation of 17-aminogeldanamycin and trametinib might result from concurrent inhibition of the MAPK and protective UPR pathways but can be also associated with the basal UPR activity, which is diverse among drug-naïve melanoma cell populations. Further studies are necessary to fully explore antimelanoma capabilities of 17-aminogeldanamycin and its combination with targeted therapy. This work was financially supported by Grant 2014/15/B/NZ7/00947 from National Science Centre (Poland), and 502-03/1-156-01/502-14-318 from Medical University of Lodz. Citation Format: Aleksandra H. Mielczarek-Lewandowska, Mariusz L. Hartman, Malgorzata M. Sztiller-Sikorska, Marta Osrodek, Anna E. Gajos-Michniewicz, Malgorzata E. Czyz. 17-Aminogeldanamycin inhibits cytoprotective UPR pathways and cooperates with inhibitors of the MAPK signaling cascade in apoptosis induction [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A20.

Myocardial MMP-2 contributes to SERCA2a proteolysis during cardiac ischaemia-reperfusion injury.

Matrix metalloproteinase-2 (MMP-2) is a zinc-dependent protease which contributes to cardiac contractile dysfunction when activated during myocardial ischaemia-reperfusion (IR) injury. MMP-2 is localized to several subcellular sites inside cardiac myocytes; however, its role in the sarcoplasmic reticulum (SR) is unknown. The Ca2+ ATPase SERCA2a, which pumps cytosolic Ca2+ into the SR to facilitate muscle relaxation, is degraded in cardiac IR injury; however, the protease responsible for this is unclear. We hypothesized that MMP-2 contributes to cardiac contractile dysfunction by proteolyzing SERCA2a, thereby impairing its activity in IR injury. Isolated rat hearts were subjected to IR injury in the presence or absence of the selective MMP inhibitor ARP-100, or perfused aerobically as a control. Inhibition of MMP activity with ARP-100 significantly improved the recovery of cardiac mechanical function and prevented the increase of a 70 kDa SERCA2a degradation fragment following IR injury, although 110 kDa SERCA2a and phospholamban levels appeared unchanged. Electrophoresis of IR heart samples followed by LC-MS/MS confirmed the presence of a SERCA2a fragment of ∼70 kDa. MMP-2 activity co-purified with SR-enriched microsomes prepared from the isolated rat hearts. Endogenous SERCA2a in SR-enriched microsomes was proteolyzed to ∼70 kDa products when incubated in vitro with exogenous MMP-2. MMP-2 also cleaved purified porcine SERCA2a in vitro. SERCA activity in SR-enriched microsomes was decreased by IR injury; however, this was not prevented with ARP-100. This study shows that MMP-2 activity is found in SR-enriched microsomes from heart muscle and that SERCA2a is proteolyzed by MMP-2. The cardioprotective actions of MMP inhibition in myocardial IR injury may include the prevention of SERCA2a degradation.

Stimuli-responsive polymer-doxorubicin conjugate: Antitumor mechanism and potential as nano-prodrug

Polymer-drug conjugates has significantly improved the anti-tumor efficacy of chemotherapeutic drugs and alleviated their side effects. N-(1,3-dihydroxypropan-2-yl) methacrylamide (DHPMA) copolymer was synthesized via RAFT polymerization and polymer-doxorubicin (DOX) (diblock pDHPMA-DOX) were formed by conjugation, resulting in a self-aggregation-induced nanoprodrug with a favorable size of 21 nm and great stability. The nanoprodrug with a molecular weight (MW) of 95 kDa released drugs in response to tumor microenvironmental pH variations and they were enzymatically hydrolyzed into low MW segments (45 kDa). The nanoprodrug was transported through the endolysosomal pathway, released the drug into the cytoplasm and some was localized in the mitochondria, resulting in disruption of the cellular actin cytoskeleton. Cellular apoptosis was also associated with reduction in the mitochondrial potential caused by the nanoprodrug. Notably, the nanoprodrug had a significantly prolonged blood circulation time with an elimination half time of 9.8 h, displayed high accumulation within tumors, and improved the in vivo therapeutic efficacy against 4T1 xenograft tumors compared to free DOX. The tumor xenograft immunohistochemistry study clearly indicated tumor inhibition was through the inhibition of cell proliferation and antiangiogenic effects. Our studies demonstrated that the diblock pDHPMA-DOX nanoprodrug with a controlled molecular structure is promising to alleviate adverse effects of free DOX and have a great potential as an efficient anticancer agent. Statement of SignificanceIn this work, we prepared a biodegradable diblock DHPMA polymer-doxorubicin conjugate via one-pot of RAFT polymerization and conjugate chemistry. The conjugate-based nanoprodrug was internalized by endocytosis to intracellularly release DOX and further induce disruption of mitochondrial functions, actin cytoskeleton alterations and cellular apoptosis. The nanoprodrug with a high molecular weight (MW) (95 kDa) showed a long blood circulation time and achieved high accumulation into tumors. The nanoprodrug was degraded into low MW (∼45 kDa) products below the renal threshold, which ensured its biosafety. Additionally, the multi-stimuli-responsive nanoprodrug demonstrated an enhanced antitumor efficacy against 4T1 breast tumors and alleviated side effects, showing a great potential as an efficient and safe anticancer agent.

Impact of Escherichia coli K12 and O18:K1 on human platelets: Differential effects on platelet activation, RNAs and proteins

Blood platelets can interact with bacteria, possibly leading to platelet activation, cytokine and microparticle release and immune signalling. Besides, bacteria can also affect the platelet RNA content. We investigated the impact of non-pathogenic K12 and pathogenic O18:K1 Escherichia (E.) coli strains on platelet activation, RNA expression patterns, and selected proteins. Depending on bacteria concentration, contact of platelets with E. coli K12 lead to an increase of P-selectin (24–51.3%), CD63 (15.9–24.3%), PAC-1 (3.8–14.9%) and bound fibrinogen (22.4–39%) on the surface. E. coli O18:K1 did not affect these markers. Sequencing analysis of total RNA showed that E. coli K12 caused a significant concentration change of 103 spliced mRNAs, of which 74 decreased. For the RNAs of HMBS (logFC = +5.73), ATP2C1 (logFC = −3.13) and LRCH4 (logFC = −4.07) changes were detectable by thromboSeq and Tuxedo pipelines. By Western blot we observed the conversion of HMBS protein from a 47 kDA to 40 kDa product by E. coli K12, O18:K1 and by purified lipopolysaccharide. While ATP2C1 protein was released from platelets, E. coli either reduced the secretion or broke down the released protein making it undetectable by antibodies. Our results demonstrate that different E. coli strains influence activation, RNA and protein levels differently which may affect platelet-bacteria crosstalk.

Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques.

Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.

In vitro hydrolysis of Bacillus thuringiensis Cry1Ac toxin by gut proteases of Nilaparvata lugens (Stål) and binding assays of Cry1Ac toxin with brush border membrane of N. lugens midgut

ABSTRACTBacillus thuringiensis (Bt) and transgenic crops carrying cry genes are widely used in the management of lepidopteran and coleopteran pests. However, almost none of the Cry toxins have insecticidal properties against sap-sucking insects, such as planthoppers, leafhoppers and aphids. To understand the low insecticidal activity of Cry1Ac toxin on sap-sucking insects, we investigated two critical steps in the Bt-intoxication cascade: the proteolytic processing of Cry1Ac toxin by gut proteases, and the binding of Cry1Ac to brush border membrane vesicles (BBMV) of Nilaparvata lugens. Proteolytic processing of Cry1Ac protoxin by N. lugens gut proteases resulted in an ∼65 kDa product, similar to the expected size of the trypsin-activated Cry1Ac toxin. In addition, activation of cysteine proteases in N. lugens gut increased the efficiency of proteolytic activities in the processing of Cry1Ac. However, feeding N. lugens nymphs with either Cry1Ac protoxin or trypsin-activated Cry1Ac toxin resulted in low mortalities. The LC50 of Cry1Ac protoxin and trypsin-activated Cry1Ac was 198.92 and 450.18 μg/mL, respectively. In vitro binding analysis of BBMV with the pre-activated Cry1Ac showed that Cry1Ac toxin could specifically bind to the BBMV. However, binding competition with 500-fold molar excess GalNAc (N-acetyl-d-galactosamine) suggested that the binding was not mediated by GalNAc-like glycoproteins. These results indicate that Cry1Ac toxin could be successfully processed by the treatment of N. lugens gut proteases. However, the binding of Cry1Ac toxin to the midgut brush border membrane was not mediated by GalNAc-like glycoprotein. This may be responsible for the low susceptibility of N. lugens to Cry1Ac.

Enzymatic sources and physio-pathological functions of soluble (pro)renin receptor.

(Pro)renin receptor (PRR) belongs to type I transmembrane receptor family and binds both prorenin and renin, representing a potential regulator of the activity of the renin-angiotensin system. Soluble form of PRR (sPRR) is generated by intracellular protease-mediated cleavage of full-length PRR. The purpose of this review is to highlight recent advances in understanding the mechanisms of action and production of sPRR. It has recently been demonstrated that site-1-protease (S1P) plays a dominant role in the generation of sPRR. New evidence is also emerging to support a biological function of sPRR in the physiological regulation of fluid homeostasis as well as pathogenesis of chronic kidney disease. sPRR is a 28 kDa product of PRR cleavage via S1P-mediated protease activity. Not only does sPRR regulate renal tubular water transport, but it also mediates pathogenic responses to renal cellular injury. sPRR is likely involved in a wide range of physio-pathological processes.

The Vip3Ag4 Insecticidal Protoxin from Bacillus thuringiensis Adopts A Tetrameric Configuration That Is Maintained on Proteolysis.

The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins, but the molecular details of their activity are not understood. As a first step in the structural characterisation of these proteins, we have analysed their secondary structure and resolved the surface topology of a tetrameric complex of the Vip3Ag4 protein by transmission electron microscopy. Sites sensitive to proteolysis by trypsin are identified and the trypsin-cleaved protein appears to retain a similar structure as an octomeric complex comprising four copies each of the ~65 kDa and ~21 kDa products of proteolysis. This processed form of the toxin may represent the active toxin. The quality and monodispersity of the protein produced in this study make Vip3Ag4 a candidate for more detailed structural analysis using cryo-electron microscopy.

Proteolytic inactivation of anti-angiogenic vasohibin-1 by cancer cells

Vasohibin-1 (VASH1) is an angiogenesis inhibitor synthesized by endothelial cells (ECs) under conditions associated with physiological and pathological angiogenesis including cancers. VASH1, which is a 44-kDa protein, is processed after its translation and secretion, and a 29 kDa product cleaved both N-terminal and C-terminal end loses its anti-angiogenic activity. Here, we tested whether cancer cells modulate the processing of VASH1. When mouse EC line MS1 stably overexpressing the human VASH1 gene (MS1-hVASH1) and various cancer cell lines were co-cultured, there was an increased processing of hVASH1 protein in the culture media. This augmented processing was abrogated by a general cysteine protease inhibitor, E-64, and also by a specific calpain inhibitor, MDL28170. Recombinant hVASH1 protein was degraded by µ-calpain in vitro, which degradation was blocked by calpeptin. Conditioned media from co-cultures had little effect on the migration of human umbilical vein endothelial cells, but exhibited an inhibitory effect on their migration when collected in the presence of MDL28170; and this inhibitory effect was blocked by neutralizing anti-hVASH1 mAb. These results indicate that cancer cells proteolytically inactivate VASH1 protein secreted by ECs in the tumour microenvironment.

Microvesicles shed from fibroblasts act as metalloproteinase carriers in a 3-D collagen matrix.

This study shows that fibroblasts migrating into a collagen matrix release numerous microvesicles into the surrounding medium. By spreading in regions of the matrix far distant from cells of origin, microvesicles carry metalloproteinase 9 (MMP-9) to act upon the collagen fibrils. As a result, the collagen matrix is gradually transformed from a laminar to a fibrillar type of architecture. As shown by western blots and gelatin zymography, MMP-9 is secreted as a 92 kDa precursor and activated upon release of 82 kDa product into the culture medium. Activation is more efficient under three-dimensional than in two-dimensional culturing conditions. While MMP-9 labeling is associated with intraluminal vesicles clustered inside the microvesicles, the microvesicle’s integrin β1 marker is bound to the outer membrane. The intraluminal vesicles are recruited from the cortical cytoplasm and eventually released following uploading inside the microvesicle. Here, we propose that fusion of the intraluminal vesicles with the outer microvesicle’s membrane could work as a mechanism controlling the extent to which MMP-9 is first activated and then released extracellularly.

Grapevine leafroll associated virus 4 strain 9: complete genome and quantitative analysis of virus-derived small interfering RNA populations.

The complete genome sequence of one isolate of Grapevine leafroll-associated virus 4 strain 9 (GLRaV-4 strain 9) was determined using a library constructed from the sequencing of the small RNA population. The RNA genome consists of 13,858 nucleotides and contains seven open reading frames. A 5’ non coding end (218 nt) is followed by ORF1 encoding the 261.5 kDa methyltransferase/helicase domains then, through a +1 ribosomal frameshift, by ORF1b encoding a 58.28 kDa product corresponding to the replicase RdRp. The ORFs that follow encode the 5.32 kDa P5 protein, the 58.12 kDa HSP70h protein, the 60.18 kDa protein, the 29.9 kDa coat protein and finally the 23.2 kDa p23 protein followed by a 127 nt non coding 3’ end. The analysis of virus-derived siRNA populations aligning to the GLRaV-4 strain 9 genome showed a major representation of 21-nt reads, suggesting the involvement of DCL4 in virus silencing. The profile of the vsiRNAs density along the viral genome differed clearly from that of GLRaV-3, the representative member of subgroup I of the genus Ampelovirus, that was present in the same plant.

In vitro culture of Parascaris equorum larvae and initial investigation of parasite excretory-secretory products.

Currently, diagnosis of Parascaris equorum infection in equids is limited to patent infections. The goals of this study were to culture P. equorum larvae in vitro and identify excretory-secretory (ES) products for prepatent diagnostic testing. Parascaris equorum L2/L3 larvae were hatched and cultured for up to 3 weeks for ES product collection. Fifth stage (L5) P. equorum were also cultured for ES product collection. Examination of ES fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver stain revealed L2/L3 products ranging from 12-94 kDa and L5 products ranging from 12-189 kDa. Western blot analyses were conducted using polyclonal antibodies produced against P. equorum or Baylisascaris procyonis L2/L3 ES products, sera from rabbits inoculated with B. procyonis or Toxocara canis eggs, and sera from animals naturally infected with P. equorum or T. canis. Western blot results indicated parasite antigens migrating at 19 and 34 kDa may be useful for specifically detecting P. equorum infections.

Comparison of protein degradation, protein oxidation, and μ-calpain activation between pale, soft, and exudative and red, firm, and nonexudative pork during postmortem aging.

The objective of this study was to investigate the differences in protein modifications between pale, soft, and exudative (PSE) and red, firm, and nonexudative (RFN) pork during postmortem (PM) aging. Longissimus dorsi (LD) including 8 PSE and 8 RFN muscles were individually removed from 16 carcasses. These 16 LD muscles were vacuum packaged at 24 h after slaughter and stored at 4°C for 1, 3, and 5 d. The centrifugation loss, drip loss, color, protein solubility, protein oxidation, protein degradation including desmin, troponin T, and integrin, and μ-calpain activation were determined. The pH of PSE samples was significantly lower than that of RFN samples at both 1 and 24 h PM (P < 0.05). The L* values of PSE pork were significantly greater than that of RFN pork at different time point during PM storage (P < 0.01). The centrifugation loss of PSE samples at d 1 was extremely greater than samples from RFN pork (P < 0.01). The cumulative drip loss for d 0 to 1, d 0 to 3, and d 0 to 5 in PSE pork were significantly greater than that from RFN pork (P < 0.05). The carbonyl content of myofibrillar proteins was not significantly different between PSE and RFN pork samples (P > 0.05). In addition, PSE pork presented a lower solubility of sarcoplasmic protein, myofibrillar protein, and total protein than RFN pork except the solubility of myofibrillar protein at d 1 (P < 0.05). The intensity of intact desmin and troponin T 2 in PSE pork at d 3 and 5 were significantly greater than that in RFN pork (P < 0.05), whereas no significant difference was detected at d 1. The intensity of intact troponin T 1 in PSE pork at d 5 was greater than that in RFN pork (P < 0.05). However, more degradation products of integrin were detected in PSE pork compared to that of RFN pork at d 1 (P < 0.05). Red, firm, and nonexudative pork presented lower intensity of intact 80 kDa calpain and greater intensity of autolyzed 76 kDa product compared to PSE pork (P < 0.01). The results indicate that the degree of μ-calpain activation, the extent of protein degradation including desmin and integrin, and the level of protein solubility in PSE pork could contribute to its low water holding capacity during PM storage.

Understanding structural and functional aspects of PII snake venom metalloproteinases: Characterization of BlatH1, a hemorrhagic dimeric enzyme from the venom of Bothriechis lateralis

A new homodimeric PII metalloproteinase, named BlatH1, was purified from the venom of the Central American arboreal viperid snake Bothriechis lateralis by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. BlatH1 is a glycoprotein of 84 kDa. The mature protein contains a metalloproteinase domain, with the characteristic zinc-binding motif (HEXXHXXGXXH) followed by the sequence CIM at the Met-turn. In the disintegrin domain, the tripeptide sequence TDN substitutes the characteristic RGD motif found in many disintegrins. BlatH1 hydrolyzed azocasein, gelatin and fibrinogen, and exerts a potent local and systemic hemorrhagic activity in mice. The hemorrhagic activity of BlatH1 is not inhibited by the plasma proteinase inhibitor α2-macroglobulin, although the SVMP is able to cleave this plasma inhibitor, generating a 90 kDa product. BlatH1 inhibits ADP- and collagen-induced human platelet aggregation (IC50 = 0.3 μM and 0.7 μM for ADP and collagen, respectively). This activity is abrogated when the enzyme is preincubated with the metalloproteinase inhibitor Batimastat, implying that it depends on proteolysis. In agreement, a synthetic peptide containing the sequence TDN of the disintegrin domain is unable to inhibit platelet aggregation. BlatH1 is a valuable tool to understand the structural determinants of toxicity in PII SVMPs.