TMEM97 facilitates the activation of SOCE by downregulating the association of cholesterol to Orai1 in MDA-MB-231 cells.

The expression of TMEM97, a regulator of cholesterol transport, has been reported to be enhanced in some tumour cells. We have recently shown that TMEM97 is involved in the proliferation of the breast cancer cell line MDA-MB-231, probably through changes in store-operated calcium entry (SOCE). By using silencing and overexpression of TMEM97 in MDA-MB-231 cells (two manoeuvres that either reduce or increase the calcium influx, respectively), we show enhanced cholesterol uptake in these cells as compared to the non-tumoral breast cell line, MCF10A. The enhanced cholesterol uptake in MDA-MB-231 cells was inhibited by silencing TMEM97, while overexpression of this protein increased cholesterol uptake in MCF10A cells and, therefore, indicating that this protein plays a role in the enhanced cholesterol uptake in MDA-MB-231 cancer cell line. TMEM97 silencing and overexpression resulted in an increase and decrease in the association of cholesterol to the SOCE calcium channel Orai1, respectively. Interestingly, silencing of TMEM97 in MDA-MB-231 cells significantly reduced the co-localization of Orai1 with the SOCE regulatory protein STIM1. Finally, neither silencing nor overexpression of TMEM97 altered SOCE in MDA-MB-231 cells transfected with the cholesterol insensible mutant of Orai1(Y80E). Our results reveal a novel regulatory mechanism of SOCE that relies on TMEM97 activity that courses through the reduction of the cholesterol content in the plasma membrane, and subsequently, by impairing its interaction with Orai1.

Store-independent Orai1-mediated Ca2+ entry and cancer.

Ca2+ channels play an important role in the development of different types of cancer, and considerable progress has been made to understand the pathophysiological mechanisms underlying the role of Ca2+ influx in the development of different cancer hallmarks. Orai1 is among the most ubiquitous and multifunctional Ca2+ channels. Orai1 mediates the highly Ca2+-selective Ca2+ release-activated current (ICRAC) and participates in the less Ca2+-selective store-operated current (ISOC), along with STIM1 or STIM1 and TRPC1, respectively. Furthermore, Orai1 contributes to a variety of store-independent Ca2+ influx mechanisms, including the arachidonate-regulated Ca2+ current, together with Orai3 and the plasma membrane resident pool of STIM1, as well as the constitutive Ca2+ influx processes activated by the secretory pathway Ca2+-ATPase-2 (SPCA2) or supported by physical and functional interaction with the small conductance Ca2+-activated K+ channel 3 (SK3) or the voltage-dependent Kv10.1 channel. This review summarizes the current knowledge concerning the store-independent mechanisms of Ca2+ influx activation through Orai1 channels and their role in the development of different cancer features.

(-)­Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells.

Triple negative breast cancer is an aggressive type of cancer that does not respond to hormonal therapy and current therapeutic strategies are accompanied by side effects due to cytotoxic actions on normal tissues. Therefore, there is a need for the identification of anti-cancer compounds with negligible effects on non-tumoral cells. Here we show that (-)­oleocanthal (OLCT), a phenolic compound isolated from olive oil, selectively impairs MDA-MB-231 cell proliferation and viability without affecting the ability of non-tumoral MCF10A cells to proliferate or their viability. Similarly, OLCT selectively impairs the ability of MDA-MB-231 cells to migrate while the ability of MCF10A to migrate was unaffected. The effect of OLCT was not exclusive for triple negative breast cancer cells as we found that OLCT also attenuate cell viability and proliferation of MCF7 cells. Our results indicate that OLCT is unable to induce Ca2+ mobilization in non-tumoral cells. By contrast, OLCT induces Ca2+ entry in MCF7 and MDA-MB-231 cells, which is impaired by TRPC6 expression silencing. We have found that MDA-MB-231 and MCF7 cells overexpress the channel TRPC6 as compared to non-tumoral MCF10A and treatment with OLCT for 24-72 h downregulates TRPC6 expression in MDA-MB-231 cells. These findings indicate that OLCT impairs the ability of breast cancer cells to proliferate and migrate via downregulation of TRPC6 channel expression while having no effect on the biology of non-tumoral breast cells.

Orai1 and Orai2 mediate store-operated calcium entry that regulates HL60 cell migration and FAK phosphorylation.

Store-operated Ca2+ entry (SOCE) is a major mechanism for the regulation of intracellular Ca2+ homeostasis and cellular function. Emerging evidence has revealed that altered expression and function of the molecular determinants of SOCE play a critical role in the development or maintenance of several cancer hallmarks, including enhanced proliferation and migration. Here we show that, in the acute myeloid leukemia cell line HL60, Orai2 is highly expressed at the transcript level, followed by the expression of Orai1. Using fluorescence Ca2+ imaging we found that Orai2 silencing significantly attenuated thapsigargin-induced SOCE, as well as knockdown of Orai1, while silencing the expression of both channels almost completely reduced SOCE, thus suggesting that SOCE in these cells is strongly dependent on Orai1 and Orai2. On the other hand, the expression of TRPC1, TRPC3 and TRPC6 is almost absent at the transcript and protein level. Bromodeoxyuridine cell proliferation assay revealed that Orai1 and Orai2 expression silencing significantly reduced HL60 cell proliferation. Furthermore, knockdown of Orai1 and Orai2 significantly attenuated the ability of HL60 to migrate in vitro as determined by transwell migration assay, probably due to the impairment of FAK tyrosine phosphorylation. These findings provide evidence for a role for Orai1 and Orai2, in SOCE and migration in the human HL60 promyeloblastic cell line. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility.

Macroautophagy (hereafter autophagy) is an evolutionarily highly conserved cellular process that participates in the maintenance of intracellular homeostasis through the degradation of most long-lived proteins and entire organelles. Autophagy participates in some reproductive events; however, there are not reports regarding the role of autophagy in the regulation of sperm physiology. Hence, the aim of this study was to investigate whether autophagy-related proteins are present and functionally active in human spermatozoa. Proteins related to autophagy/mitophagy process (LC3, Atg5, Atg16, Beclin 1, p62, m-TOR, AMPKα 1/2, and PINK1) were present in human spermatozoa. LC3 colocalized with p62 in the middle piece of the spermatozoa. Autophagy activation induced a significant increase in motility and a decrease in PINK1, TOM20 expression and caspase 3/7 activation. In contrast, autophagy inhibition resulted in decreased motility, viability, ATP and intracellular calcium concentration whereas PINK1, TOM20 expression, AMPK phosphorylation and caspase 3/7 activation were significantly increased. In conclusion our results show that autophagy related proteins and upstream regulators are present and functional in human spermatozoa. Modification of mitochondrial proteins expression after autophagy activation/inhibition may be indicating that a specialized form of autophagy named mitophagy may be regulating sperm function such as motility and viability and may be cooperating with apoptosis.

The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions.

Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of autophagy activation. The main objective of this study was to investigate whether LC3 is processed during cooling, freezing and the stressful conditions associated with these technologies. A secondary objective was to determine if LC3 processing can be modulated and if that may improve the quality of cryopreserved semen. LC3 processing was studied by Western blot with a specific antibody that recognized both LC3-I and LC3-II. Viability was assessed by flow cytometry. Modulation of LC3-I to LC3-II was studied with known autophagy activators (STF-62247 and rapamycin) or inhibitors (chloroquine and 3-MA) used in somatic cells. The results showed that conversion of LC3-I to LC3-II increased significantly during cooling at 4°C, freezing/thawing and each of the stressful conditions tested (UV radiation, oxidative stress, osmotic stress and changes in temperature). STF-62247 and rapamycin increased the LC3-II/LC3-I ratio and decreased the viability of equine sperm, whereas chloroquine and 3-MA inhibited LC3 processing and maintained the percentage of viable cells after 2 h of incubation at 37°C. Finally, refrigeration at 4°C for 96 h and freezing at -196°C in the presence of chloroquine and 3-MA resulted in higher percentages of viable cells. In conclusion, results showed that an 'autophagy-like' mechanism may be involved in the regulation of sperm viability during equine semen cryopreservation. Modulation of autophagy during these reproductive technologies may result in an improvement of semen quality and therefore in higher fertility rates.

Relationship between calcium mobilization and platelet α- and δ-granule secretion. A role for TRPC6 in thrombin-evoked δ-granule exocytosis.

Changes in cytosolic Ca(2+) concentration ([Ca(2+)]c) regulate granule secretion in different cell types. Thrombin activates PAR1 and PAR4 receptors and promotes release of Ca(2+) from distinct intracellular stores, which, in turn, activates store-operated Ca(2+) entry (SOCE). A crucial step during platelet function is the release of physiological agonists stored in secretory granules to the extracellular compartment during activation. We aim to study the role of Ca(2+) mobilization from the extracellular compartment or from different intracellular stores in platelet granule secretion. By using flow cytometry, we have found that α- and δ-granules are secreted in thrombin-stimulated platelets in the absence of extracellular Ca(2+), and in a concentration-dependent manner. Our findings show that thrombin-stimulated granule secretion depends on Ca(2+) mobilization from intracellular stores. Analysis of the kinetics of granule secretion reveals that platelet stimulation with thrombin results in rapid release of α-granules which precedes the secretion of δ-granules. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 573-586 of TRPC6, inhibited thrombin-evoked δ-granule exocytosis. Our results indicate that the mechanisms underlying thrombin-induced α- and δ-granule secretion show differences in dependency on Ca(2+) mobilization.

Identification of apoptotic bodies in equine semen.

Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37 °C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin-V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer-assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.

Immunophilins are involved in the altered platelet aggregation observed in patients with type 2 diabetes mellitus.

UNLABELLED: Platelet hyperaggregability might contribute to vascular complications associated with type 2 diabetes mellitus (DM2).Experimental evidence supports a direct link between altered Ca(2+) entry and hyperaggregability in DM2 patients. OBJECTIVES: We aimed to investigate whether altered immunophilin expression and function are involved in the abnormal Ca(2+) entry observed in platelets from DM2 patients. RESULTS: Inhibition of immunophilins by tacrolimus (FK506) and sirolimus (rapamycin) reduced Ca(2+) entry in platelets from healthy donors and DM2 patients. Similarly, immunophilin inhibitors reduced platelet degranulation in both healthy and DM2 subjects. Nevertheless, α-granule secretion reduction was greater than that observed for dense granules in platelets from DM2 patients. However, no difference was observed in the inhibition of secretion in platelets from healthy subjects. Additionally, altered expression of FK506 binding protein-52 (FKBP52) and coupling to Ca(2+) channels were found in platelets from DM2 patients compared to healthy subjects. Finally, reduction in platelet function from healthy subjects and DM2 patients in the presence of immunophilin antagonists was observed, being this dysfunction more evident in platelets from DM2 patients. CONCLUSIONS: We suggest that, among others, FKBP52 expression and function are altered in platelets from DM2 patients, contributing to the altered Ca(2+) entry and hyperaggregability in these cells.

The membrane of the mammalian spermatozoa: much more than an inert envelope.

Sperm plasma membrane is a very important structure that functions to protect sperm against extracellular injuries and to respond to physiological challenges. It plays a crucial role during sperm capacitation, in sperm-egg interaction and, finally, in fertilization. Concerning sperm technology, possibly the most important factors causing damage in mammalian spermatozoa membranes are initiated by the osmotic stress generated by dehydration of the cells during freezing and thawing. These changes are rapidly derived to the plasma and organelle membranes that gradually experiment loss of membrane architecture, causing unbalanced production of reactive oxygen species and increased lipid peroxidation. Other procedures such as sperm sorting or liquid storage of sperm also induce harmful changes in the integrity of the membrane. The specific composition of lipids of the sperm membranes may provide clues for understanding the mechanisms behind the differences found in the response to stress in different species. In the present review, we deal with the composition, architecture and organization of the sperm plasma membrane, emphasizing the factors that can affect membrane integrity. The intracellular signalling pathways related with membrane reorganization during capacitation and acrosome reaction are also reviewed.

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation.

BACKGROUND: All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP(3)Rs)-binding (CIRB) site involved in the regulation of TRPC channel function. OBJECTIVES: To assess the basis of CaM/IP(3)Rs binding to the CIRB site of TRPC6 and its role in platelet physiology. METHODS: Protein association was detected by co-immunoprecipitation and Western blotting, Ca(2+) mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry. RESULTS: Co-immunoprecipitation of TRPC6 with CaM or the IP(3)Rs at different cytosolic free Ca(2+) concentrations ([Ca(2+)](c)) indicates that the association between these proteins is finely regulated by cytosolic Ca(2+) via association of CaM and displacement of the IP(3)Rs at high [Ca(2+)](c). Thrombin-stimulated association of TRPC6 with CaM or the IP(3)Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca(2+)-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP(3)Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca(2+) entry in the presence of low or high, respectively, extracellular Ca(2+) concentrations, as well as altered thrombin-evoked platelet aggregation. CONCLUSIONS: Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca(2+) entry and aggregation through its interaction with CaM and IP(3)Rs.

Homocysteine, intracellular signaling and thrombotic disorders.

Homocysteine, a sulphur-containing amino acid derived from methionine, has been presented as an independent risk factor for cardiovascular disorders, including atherosclerosis and thrombogenesis. The mechanisms underlying homocysteine-induced effects have been intensively investigated over the last two decades. Homocysteine can induce oxidative stress promoting oxidant injury to vascular and blood cells. Hyperhomocysteinemia often results in intracellular Ca2+ mobilization, endoplasmic reticulum (ER) stress, with the subsequent development of apoptotic events, chronic inflammation leading to endothelial dysfunction and remodeling of the extracellular matrix. Homocysteine has also been reported to induce modulation of gene expression through alteration of the methylation status. The effects of elevated concentrations of circulating homocysteine on the vascular wall, platelet function and coagulation factors promote the development of a pro-coagulant state. The pathophysiological significance of homocysteine in the development of vascular disorders through the induction of endothelial dysfunction and abnormal platelet activity and blood coagulation is discussed in this review.

STIM1, Orai1 and hTRPC1 are important for thrombin- and ADP-induced aggregation in human platelets.

Ca(2+) entry, particularly store-operated Ca(2+) entry (SOCE), has been reported to be crucial for a variety of cellular functions. SOCE is a mechanism regulated by the Ca(2+) content of the stores, where the intraluminal Ca(2+) sensor STromal Interaction Molecule 1 (STIM1) has been reported to communicate the filling state of the intracellular Ca(2+) stores to the store-operated Ca(2+)-permeable channels in the plasma membrane, likely involving Orai1 and TRPC proteins, such as TRPC1. Here we have investigated the role of Orai1, STIM1 and TRPC1 in platelet aggregation, an event that occurs during the process of thrombosis and hemostasis. Electrotransjection of cells with anti-STIM1 (25-139) antibody, directed towards the Ca(2+)-binding motif, significantly reduced thrombin-induced aggregation and prevented ADP-evoked response. Extracellular application of the anti-STIM1 antibody, in order to block the function of plasma membrane-located STIM1, reduced thrombin- and ADP-stimulated platelet aggregation to a lesser extent. Introduction of an anti-Orai1 (288-301) antibody, which binds the STIM1-binding site located in the Orai1 C-terminus, or extracellular application of anti-hTRPC1 (557-571) antibody to impair hTRPC1 channel function, significantly reduced thrombin- and ADP-induced platelet aggregation. These findings suggest a role of STIM1, Orai1 and hTRPC1 in thrombin- and ADP-induced platelet aggregation probably through the regulation of Ca(2+) entry, which might become targets for the development of therapeutic strategies to treat platelet hyperactivity and thrombosis disorders.

N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine induces apoptosis through the activation of caspases-3 and -8 in human platelets. A role for endoplasmic reticulum stress.

BACKGROUND: Apoptosis or programmed cell death involves a number of biochemical events, including the activation of caspases, which lead to specific cell morphology changes and ultimately cell death. Traditionally, two apoptotic pathways have been described: the cell-surface death receptor-dependent extrinsic pathway and the mitochondria-dependent intrinsic pathway. Alternatively, apoptosis has been reported to be induced by endoplasmic reticulum (ER) stress, which is mainly induced by a reduction in intraluminal free Ca(2+) concentration ([Ca(2+)](ER)). OBJECTIVES: The present study aimed to investigate the development of apoptotic events after ER stress induced by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), an ER Ca(2+) chelator, in human platelets. METHODS: Changes in cytosolic free Ca(2+) concentration, caspase activity and phosphatidylserine externalization were determined by fluorimetric techniques. RESULTS: Our results indicate that TPEN reduces the amount of free Ca(2+) releasable by the Ca(2+)-mobilizing agonist thrombin. TPEN induced activation of caspase-3, -8 and -9 and subsequent phosphatidylserine externalization. The ability of TPEN to induce phosphatidylserine externalization was smaller than that of thrombin. In addition, TPEN was able to induce phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2 alpha). TPEN-mediated caspase-3 activation requires functional caspase-8, but is independent of H(2)O(2) generation. Activation of caspase-3 and -8 by TPEN was prevented by salubrinal, an agent that prevents ER stress-induced apoptosis. CONCLUSION: These findings provide experimental evidence for the existence of ER stress-mediated apoptosis in human platelets, a process that might limit platelet life span upon prolonged stimulation with agonists.

Identification of protein tyrosine phosphatases and dual-specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation.

Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in boar and stallion sperm lysates, we unequivocally identified two PTPs (PTPRB and PTPN11) and two DSPs (DUSP3 and DUSP4). In dog sperm lysates, only PTPN11, DUSP3, and DUSP4 were detected. In all these species, we did not detect the specific signal with anti-PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, or PTPRZ antibodies. Positive matches were further investigated by indirect immunofluorescence and confocal microscopy. Results showed that PTPRB was associated with the plasma membrane in the head and tail of boar and stallion spermatozoa. In agreement with Western blotting results, PTPRB antibodies did not show immunoreactivity in dog sperm analyzed by immunofluorescence. In the three species, DUSP4 was mainly found in the tail of spermatozoa, with little or no immunoreactivity in the head. PTPN11 was mainly located in the postacrosomal region in the head, whereas DUSP3 immunoreactivity was extended within the acrosome. PTPN11 and DUSP3 showed immunoreactivity in the tail that was restricted to the midpiece. Finally, we incubated boar, stallion, and dog spermatozoa with pervanadate and sodium orthovanadate, two PTP inhibitors, and analyzed overall protein tyrosine phosphorylation and assessed sperm motility. Sodium orthovanadate and pervanadate showed concentration-dependent inhibition of sperm motility that was rapid and reversible. Pervanadate also increased tyrosine phosphorylation of different proteins in capacitated and noncapacitated spermatozoa. Results showed that the phosphatases PTPN11, DUSP4, and DUSP3 are present in boar, stallion, and dog spermatozoa. PTPRB is also present in boar and stallion spermatozoa but was not detected in dog. The subcellular distribution of the identified phosphatases is diverse, suggesting that they likely have specific roles in sperm. Finally, PTP activity has a positive role in the regulation of motility and is involved in protein tyrosine phosphorylation in mammalian sperm.

Effect of homocysteine on calcium mobilization and platelet function in type 2 diabetes mellitus.

Type 2 diabetes mellitus induces a characteristic platelet hyperactivity that might be due to several factors including oxidative stress and abnormal intracellular Ca(2+) homeostasis. Hyperhomocysteinaemia is considered a risk factor in the development of thrombosis although its effect on platelet function and the mechanisms involved are still poorly understood. Here we show that homocysteine induce a concentration-dependent increase in endogenous production of reactive oxygen species (ROS), which was significantly greater in platelets from diabetic patients than in controls. Platelet treatment with homocysteine resulted in Ca2+ release from the dense tubular system and the acidic stores. Ca2+ mobilization-induced by homocysteine consisted in two components, an initial slow increase in intracellular free Ca (+) concentration ([Ca2+]i) and a rapid and marked increase in [Ca2+]i, th second leading to the activation of platelet aggregation. As well as ROS generation, Ca2+ mobilization and platelet aggregation were significantly greater in platelets from diabetic donors than in controls, which indicate that platelets from diabetic donors are more sensitive to homocysteine. These findings, together with the hyperhomocysteinaemia reported in diabetic patients, strongly suggest that homocysteine might be considered a risk factor in the development of cardiovascular complications associated to type 2 diabetes mellitus.

Protein complex immunological separation assay (ProCISA): a technique for investigating single protein properties

Analysis of the posttranslational modification of proteins, such as phosphorylation,might yield misleading results due to the presence of other proteins with similar electrophoreticproperties that coimmunoprecipitate with the target protein. The aim ofthe present work was to develop a reliable, easy and economical technique to completelyisolate a protein from its complex. Here we present a new assay developed tofully isolate proteins from macromolecular complexes that consists of an initialSDS/PAGE (under reducing conditions), which isolates the target protein, followedby transfer of the proteins to a buffer, from which the target protein is recaptured byconventional immunoprecipitation. This technique, that we have termed “ProteinComplex Immunological Separation Assay” (ProCISA), successfully separated proteinsof different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes.We show that ProCISA allows the investigation of the tyrosine phosphorylationstate of isolated proteins. This technique could also be used to study other posttranslationalmodifications without risk of misleading results resulting from contaminationwith other proteins of similar electrophoretic mobility which complex with theprotein of interest (AU)

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Thrombin induces activation and translocation of Bid, Bax and Bak to the mitochondria in human platelets.

BACKGROUND: Thrombin is a physiological platelet agonist that activates apoptotic events, including cytochrome c release and phosphatidylserine exposure; however, the mechanisms underlying these events remain unclear. OBJECTIVES: The present study is aimed to investigate whether thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak. METHODS: Changes in the mitochondrial membrane potential were registered using the dye JC-1; Bid, Bax and Bak translocation to the mitochondria was detected by immunoprecipitation and Western blotting in samples from mitochondrial and cytosolic fractions. RESULTS: Treatment of platelets with thrombin or ADP induces activation and mitochondrial association of active Bid, Bax and Bak. Translocation of Bid and Bax to the mitochondria was reduced by cytochalasin D, latrunculin A or jasplakinolide. Platelet exposure to exogenous H(2)O(2) (10 microm) results in activation of Bid and Bax, which was found to be similar to the effect of thrombin. Thrombin evokes mitochondrial membrane depolarization, which is attenuated by catalase. CONCLUSION: Our results indicate that thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak, which is likely to be one of the apoptotic events in human platelets.

Protein complex immunological separation assay (ProCISA): a technique for investigating single protein properties.

Analysis of the posttranslational modification of proteins, such as phosphorylation, might yield misleading results due to the presence of other proteins with similar electrophoretic properties that coimmunoprecipitate with the target protein. The aim of the present work was to develop a reliable, easy and economical technique to completely isolate a protein from its complex. Here we present a new assay developed to fully isolate proteins from macromolecular complexes that consists of an initial SDS/PAGE (under reducing conditions), which isolates the target protein, followed by transfer of the proteins to a buffer, from which the target protein is recaptured by conventional immunoprecipitation. This technique, that we have termed "Protein Complex Immunological Separation Assay" (ProCISA), successfully separated proteins of different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes. We show that ProCISA allows the investigation of the tyrosine phosphorylation state of isolated proteins. This technique could also be used to study other posttranslational modifications without risk of misleading results resulting from contamination with other proteins of similar electrophoretic mobility which complex with the protein of interest.

Effect of homocysteine on calcium mobilization and platelet function in type 2 diabetes mellitus.

Type 2 diabetes mellitus induces a characteristic platelet hyperactivity that might be due to several factors including oxidative stress and abnormal intracellular Ca2+ homeostasis. Hyperhomocysteinaemia is considered a risk factor in the development of thrombosis although its effect on platelet function and the mechanisms involved are still poorly understood. Here we show that homocysteine (Hcy) induce a concentration-dependent increase in endogenous production of reactive oxygen species (ROS), which was significantly greater in platelets from diabetic patients than in controls. Platelet treatment with Hcy resulted in Ca2+ release from the dense tubular system and the acidic stores. Ca2+ mobilisation-induced by Hcy consisted in two components, an initial slow increase in intracellular free Ca2+ concentration ([Ca2+]i) and a rapid and marked increase in [Ca2+]i, the second leading to the activation of platelet aggregation. As well as ROS generation, Ca2+ mobilization and platelet aggregation were significantly greater in platelets from diabetic donors than in controls, which indicate that platelets from diabetic donors are more sensitive to Hcy. These findings, together with the hyperhomocysteinaemia reported in diabetic patients, strongly suggest that Hcy might be considered a risk factor in the development of cardiovascular complications associated to type 2 diabetes mellitus.