The activity and surface presence of organic cation/carnitine transporter OCTN2 (SLC22A5) in breast cancer cells depends on AKT kinase.

l-carnitine is indispensable for transfer of fatty acids to mitochondria for the process of ß-oxidation, a process, whose significance in cancer has drawn attention in recent years. In humans majority of carnitine is delivered by diet and enters the cell due to activity of solute carriers (SLCs), mainly by ubiquitously expressed organic cation/carnitine transporter (OCTN2/SLC22A5). In control and cancer human breast epithelial cell lines the major fraction of OCTN2 is present as a not matured non-glycosylated form. Studies on overexpressed OCTN2 demonstrated an exclusive interaction with SEC24C, as the cargo-recognizing subunit of coatomer II in transporter exit from endoplasmic reticulum. Co-transfection with SEC24C dominant negative mutant completely abolished presence of the mature form of OCTN2, pointing to a possibility of trafficking regulation. SEC24C was previously shown to be phosphorylated by serine/threonine kinase AKT, known to be activated in cancer. Further studies on breast cell lines showed that inhibition of AKT with MK-2206 in control and cancer lines decreased level of OCTN2 mature form. Proximity ligation assay showed that phosphorylation of OCTN2 on threonine was significantly abolished by AKT inhibition with MK-2206. Carnitine transport was positively correlated with the level of OCTN2 phosphorylated by AKT on threonine moiety. The observed regulation of OCTN2 by AKT places this kinase in the center of metabolic control. This points to both proteins, AKT and OCTN2, as druggable targets, in particular in a combination therapy of breast cancer.

Involvement of CacyBP/SIP in differentiation and the immune response of HaCaT keratinocytes.

CacyBP/SIP is a multifunctional protein present in various cells and tissues. However, its expression and role in the epidermis has not been explored so far. In this work, using RT-qPCR, Western blot analysis and three-dimensional (3D) organotypic cultures of HaCaT keratinocytes we show that CacyBP/SIP is present in the epidermis. To investigate the possible role of CacyBP/SIP in keratinocytes we obtained CacyBP/SIP knockdown cells and studied the effect of CacyBP/SIP deficiency on their differentiation and response to viral infection. We found that CacyBP/SIP knockdown results in reduced expression of epidermal differentiation markers in both undifferentiated and differentiated HaCaT cells. Since epidermis is engaged in immune defense, the impact of CacyBP/SIP knockdown on this process was also analyzed. By applying RT-qPCR and Western blot it was found that poly(I:C), a synthetic analog of double-stranded RNA that mimics viral infection, stimulated the expression of genes involved in antiviral response, such as IFIT1, IFIT2 and OASL. Interestingly, following poly(I:C) stimulation, the level of expression of these genes was significantly lower in cells with CacyBP/SIP knockdown than control ones. Since the signaling pathway mediating cellular responses to viral infection involves, among others, the STAT1 transcription factor, we measured its activity using luciferase assay and found that it was lower in CacyBP/SIP knockdown HaCaT cells. Altogether, the presented results indicate that CacyBP/SIP promotes epidermal differentiation and might be involved in response of the skin cells to viral infection.

Ca2+- binding proteins of the S100 family in preeclampsia.

S100 proteins bind Ca2+ and regulate various signaling pathways inside and outside the cell. They are expressed in vertebrates and exhibit tissue and cell specific distribution. Of note, increased level of S100 proteins is observed in different pathologies such as cancers, nervous system diseases/neurodegeneration, inflammation or cardiovascular diseases. Certain S100 proteins can be found in serum and/or other body fluids, at an especially high level in pathological states. Thus, S100 proteins might serve as diagnostic markers in the clinic. Interestingly, expression of many S100 proteins is found to be associated with pregnancy, which suggests that alterations in their expression during pregnancy may regulate processes involved in embryo/fetus formation. In this review we summarize available literature data concerning the expression and possible function of S100 proteins in a disease of pregnant women known as preeclampsia or EPH-gestosis.

p-STAT3 is a PDC-E2 interacting partner in human cholangiocytes and hepatocytes with potential pathobiological implications.

The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases. This study investigated whether PDC-E2 interacts with STAT3 in human cholangiocytes (NHC) and hepatocytes (Hep-G2) under cholestatic conditions induced by glyco-chenodeoxycholic acid (GCDC). GCDC induced PDC-E2 expression in the cytoplasmic and nuclear fraction of NHC, whereas in Hep-G2 cells PDC-E2 expression was induced only in the cytoplasmic fraction. GCDC-treatment stimulated phosphorylation of STAT3 in the cytoplasmic fraction of NHC. siRNA-mediated gene silencing of PDC-E2 reduced the expression of pY-STAT3 in NHC but not in HepG2 cells. Immunoprecipitation and a proximity ligation assay clearly demonstrated that GCDC enhanced pY-STAT3 binding to PDC-E2 in the nuclear and cytoplasmic fraction of NHC cells. Staining with Mitotracker revealed mitochondrial co-localization of PDC-E2/pS-STAT3 complexes in NHC and Hep-G2 cells. In cirrhotic PBC livers the higher expression of both PDC-E2 and pY-STAT3 was observed. The immunoblot analysis demonstrated the occurrence of double bands of PDC-E2 protein in control livers, which was associated with a lower expression of pY-STAT3. Our data indicate the interaction between PDC-E2 and phosphorylated STAT3 under cholestatic conditions, which may play a role in the development of PBC.

Regulation of S100A10 Gene Expression.

S100A10, a member of the S100 family of Ca2+-binding proteins, is a widely distributed protein involved in many cellular and extracellular processes. The best recognized role of S100A10 is the regulation, via interaction with annexin A2, of plasminogen conversion to plasmin. Plasmin, together with other proteases, induces degradation of the extracellular matrix (ECM), which is an important step in tumor progression. Additionally, S100A10 interacts with 5-hydroxytryptamine 1B (5-HT1B) receptor, which influences neurotransmitter binding and, through that, depressive symptoms. Taking this into account, it is evident that S100A10 expression in the cell should be under strict control. In this work, we summarize available literature data concerning the physiological stimuli and transcription factors that influence S100A10 expression. We also present our original results showing for the first time regulation of S100A10 expression by grainyhead-like 2 transcription factor (GRHL2). By applying in silico analysis, we have found two highly conserved GRHL2 binding sites in the 1st intron of the gene encoding S100A10 protein. Using chromatin immunoprecipitation (ChIP) and luciferase assays, we have shown that GRHL2 directly binds to these sites and that this DNA region can affect transcription of S100A10.

Ca2+-dependent binding of S100A6 to cofilin-1 regulates actin filament polymerization-depolymerization dynamics.

S100A6 is a Ca2+-binding protein belonging to the S100 family. Many reports indicate that S100A6 is involved in actin filament organization, however the mechanism of S100A6 action in this process is not fully understood. By screening S100A6 binding partners in NIH3T3 mouse fibroblasts, we have found that S100A6 binds cofilin-1, a protein required for the dynamics of actin polymerization and depolymerization. By applying various biochemical and cell biology assays, we have shown that S100A6 bound to cofilin-1 in a Ca2+-dependent manner and increased cofilin-1 affinity for F-actin. Microscopic analysis indicated that S100A6 significantly decreased severing of the actin filaments induced by cofilin-1. Moreover, in the presence of cofilin-1, S100A6 stabilized the filaments by inhibiting their depolymerization. When S100A6 was present at sub-stoichiometric concentrations in relation to actin, polymerization of G-actin accelerated by cofilin-1 was increased. At higher S100A6:actin ratios the polymerization rate was decreased. Altogether, these results show that S100A6 regulates actin filament dynamics by controlling activity of cofilin-1 and suggest that this regulation is Ca2+ -dependent.

Binding of S100A6 to actin and the actin-tropomyosin complex.

S100A6 is a low molecular weight Ca2+-binding protein belonging to the S100 family. Many reports indicate that in the cell S100A6 has an influence on the organization of actin filaments, but so far no direct interaction between S100A6 and actin has been shown. In the present study we investigated binding of S100A6 to actin and the actin-tropomyosin complex. The analyses were performed on G- and F-actin and two tropomyosin isoforms-Tpm1.6 and Tpm1.8. Using purified proteins and a variety of biochemical approaches we have shown that, in a Ca2+-bound form, S100A6 directly interacts with G- and F-actin and with tropomyosin, preferentially with isoform Tpm1.8. S100A6 and tropomyosin bind to the same population of filaments and the presence of tropomyosin on the microfilament facilitates the binding of S100A6. By applying proximity ligation assay we have found that in NIH3T3 fibroblasts S100A6 forms complexes both with actin and with tropomyosin. These results indicate that S100A6, through direct interactions with actin and tropomyosin, might regulate the organization and functional properties of microfilaments.

CacyBP/SIP in the rat spinal cord in norm and after transection - Influence on the phosphorylation state of ERK1/2 and p38 kinases.

INTRODUCTION: CacyBP/SIP is a multifunctional protein present in various mammalian tissues, among them in brain. Recently, it has been shown that CacyBP/SIP exhibits phosphatase activity towards ERK1/2 and p38 kinases. OBJECTIVES: The aim of our study was to analyze the localization and level of CacyBP/SIP and its substrates, phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated p38 (p-p38) kinases, in an intact and transected rat spinal cord. METHODS: To achieve our goals we have performed Western blot/densitometric analysis and double immunofluorescence staining using rat spinal cord tissue, intact and after total transection at different time points. RESULTS: We have observed a decrease in the level of CacyBP/SIP and an increase in the level of p-ERK1/2 and of p-p38 in fragments of the spinal cord excised 1 and 3 months after transection. Moreover, immunofluorescence staining has shown that CacyBP/SIP, p-ERK1/2 or p-p38 co-localized with a neuronal marker, NeuN, and with an oligodendrocyte marker, Olig2. CONCLUSION: The inverse correlation between CacyBP/SIP and p-ERK1/2 or p-p38 levels suggests that CacyBP/SIP may dephosphorylate p-ERK1/2 and p-p38 kinases and be involved in neural plasticity following spinal cord injury.

The HtrA3 protease promotes drug-induced death of lung cancer cells by cleavage of the X-linked inhibitor of apoptosis protein (XIAP).

HtrA3 is a proapoptotic protease shown to promote drug-induced cytotoxicity in lung cancer cells and proposed to have an antitumor effect. However, at the molecular level, the role of HtrA3 in cell death induction is poorly understood. There are two HtrA3 isoforms, a long and a short one, termed HtrA3L and HtrA3S. By performing pull down assays, co-immunoprecipitation and ELISA, we showed that HtrA3 formed complexes with the X-linked inhibitor of apoptosis protein (XIAP). The recombinant HtrA3 variants ΔN-HtrA3L and -S, lacking the N-terminal regions that are not essential for protease activity, cleaved XIAP with a comparable efficiency, though ΔN-HtrA3S was more active in the presence of cellular extract, suggesting the existence of an activating factor. Immunofluorescence and proximity ligation assays indicated that HtrA3 partially co-localized with XIAP. Exogenous ΔN-HtrA3L/S promoted apoptotic death of lung cancer cells treated with etoposide and caused a significant decrease of cellular XIAP levels, in a way dependent on HtrA3 proteolytic activity. These results collectively indicate that both HtrA3 isoforms stimulate drug-induced apoptotic death of lung cancer cells via XIAP cleavage and thus help to understand the molecular mechanism of HtrA3 function in apoptosis and in cancer cell death caused by chemotherapy.

Tubulin-dependent secretion of S100A6 and cellular signaling pathways activated by S100A6-integrin ß1 interaction.

S100A6 is a calcium binding protein expressed mainly in fibroblasts and epithelial cells. Interestingly, S100A6 is also present in extracellular fluids. Recently we have shown that S100A6 is secreted by WJMS cells and binds to integrin ß1 (Jurewicz et al., 2014). In this work we describe for the first time the mechanism of S100A6 secretion and signaling pathways activated by the S100A6-integrin ß1 complex. We show that colchicine suppressed the release of S100A6 into the cell medium, which indicates that the protein might be secreted via a tubulin-dependent pathway. By applying double immunogold labeling and immunofluorescence staining we have shown that S100A6 associates with microtubules in WJMS cells. Furthermore, results obtained from immunoprecipitation and proximity ligation assay (PLA), and from in vitro assays, reveal that S100A6 is able to form complexes with α and ß tubulin in these cells, and that the S100A6-tubulin interaction is direct. We have also found that the S100A6 protein, due to binding to integrin ß1, activates integrin-linked kinase (ILK), focal adhesion kinase (FAK) and p21-activated kinase (PAK). Our results suggest that binding of S100A6 to integrin ß1 affects cell adhesion/proliferation due to activation of ILK and FAK signaling pathways.

[Preeclampsia - a disease of pregnant women]. / Preeklampsja ­ choroba kobiet w ciazy.

Preeclampsia, also known as EPH-gestosis, is a pregnancy-specific syndrome. It affects 3-5% of pregnant women and is characterized by edemas, high blood pressure and proteinuria. Moreover, in women with preeclampsia dysfunction of many organs, such as kidney and liver, is diagnosed, while in the case of fetus growth restriction is observed. Preeclampsia, when left untreated, can lead to death. In low-income countries, this disorder is one of the main causes of maternal and child mortality. Preeclampsia predisposes women in later life to cardiovascular diseases. So far, in acute cases of preeclampsia stabilization of the mother and fetus and finally termination of pregnancy at a time optimal for both sides can only be considered. In this work, available literature data concerning the causes of preeclampsia, its symptoms, techniques for diagnosis, methods for prevention and new approaches to treatment were collected.

Stress-Dependent Changes in the CacyBP/SIP Interacting Protein S100A6 in the Mouse Brain.

The CacyBP/SIP target S100A6 is widely present in the nervous system, and its up-regulation is associated with certain neurodegenerative diseases. Here, we examined the involvement of S100A6 protein in stress responses in mice. Using Western blotting, we observed a marked change in brainstem structures, whereby stressed mice showed approximately one-third the protein level produced in the control group. A decreased level of S100A6 protein in stressed animals was also detected in the olfactory bulb and the cerebellum and stress-related structures such as the hippocampus and the hypothalamus. Additionally, using immunohistochemistry, high levels of S100A6 expression were observed in astrocytes localized in the border zones of all brain ventricles, tanycytes of the ventro-lateral walls of the hypothalamus, including the arcuate nucleus (ARH) and low levels of this protein were in neurons of the olfactory bulb, the hippocampus, the thalamus, the cerebral cortex, the brainstem and the cerebellum. Although S100A6-expressing cells in all these brain structures did not change their phenotype in response to stress, the intensity of immunofluorescent labeling in all studied structures was lower in stressed mice than in control animals. For example, in the ARH, where extremely strong immunostaining was observed, the number of immunolabeled fibers was decreased by approximately half in the stressed group compared with the controls. Although these results are descriptive and do not give clue about functional role of S100A6 in stress, they indicate that the level of S100A6 decreases in several brain structures in response to chronic mild stress, suggesting that this protein may modify stress responses.

Translation elongation factor eEF1A1 is a novel partner of a multifunctional protein Sgt1.

Mammalian translation elongation factor eEF1A is involved in ribosomal polypeptide synthesis. Also, the protein fulfills many additional duties in an eukaryotic cell. Here, we identified a novel partner of the eEF1A1 isoform, namely Sgt1, a protein that possesses co-chaperon properties and participates in antiviral defense processes. By applying different methods, we demonstrated the interaction between eEF1A1 and Sgt1 using both purified proteins and cell lysates. We also found that the D2 and D3 domains of eEF1A1 and the TPR domain of Sgt1 are involved in complex formation. Modeling of the Sgt1-eEF1A1 complex suggested both shape and charge complementarities of the eEF1A1-Sgt1 interface stabilized by a number of salt bridges. As long as such interaction mode is typical more for protein-nucleic acid interaction we suggested a possibility that Sgt1 competes with viral RNA for binding to eEF1A and obtained in vitro evidence to this effect.

S100A6 is secreted from Wharton's jelly mesenchymal stem cells and interacts with integrin ß1.

S100A6 is a calcium binding protein belonging to the S100 family. In this work we examined the function of extracellular S100A6. Using mesenchymal stem cells isolated from Wharton's jelly of the umbilical cord (WJMS cells) we have shown that S100A6 is secreted by these cells, and when added to the medium, increases their adhesion and inhibits proliferation. The search for a potential target/receptor of S100A6 in the membrane fraction of WJMS cells allowed us to identify some proteins, among them integrin ß1, which interacts with S100A6 in a calcium dependent manner. The interaction between S100A6 and integrin ß1, was then confirmed by ELISA using purified proteins. Applying specific antibodies against integrin ß1 reversed the effect on cell adhesion and proliferation observed in the presence of S100A6 which indicates that S100A6 exerts its function due to interaction with integrin ß1. Since the data show the influence of extracellular S100A6 on cells isolated from Wharton's jelly, our results might help to establish molecular mechanisms leading to some pathologies characteristic for this tissue.

Status epilepticus induces long lasting increase in S100A6 expression in astrocytes.

In the present work we examined expression and localization of the S100A6 protein in rat brain in a model of epilepsy induced by Status Epilepticus evoked by amygdala stimulation. We demonstrate, through the use of the reverse transcriptase-polymerase chain reaction technique, that mRNA level of S100A6 was increased in cortex while, as found by immunoblotting, the level of the S100A6 protein was significantly higher in the cortex and in the CA1 area of the hippocampus at day 14 after stimulation. Immunohistochemical studies performed on rat brain slices indicated that S100A6 immunoreactivity was elevated in GFAP-positive astrocytes in the hippocampus and cortex starting from day 1, and further increased at day 4 and 14 after stimulation. Interestingly, in a subpopulation of astrocytes, up-regulation of S100A6 was associated with an increased level of ß-catenin, a protein involved in regulation of S100A6 expression. Altogether, our data show a widespread and prolonged up-regulation of S100A6 in the epileptic brain and indicate that an increase in S100A6 immunoreactivity is related to astrogliosis.

CacyBP/SIP as a novel modulator of the thin filament.

The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin-tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.

Identification and localization of S100A6 in human umbilical cord.

S100A6, a calcium-binding protein also known as calcyclin, was detected in human umbilical cord by immunoblotting. Immunohistochemical studies showed an intensive reaction for S100A6 in the walls of vessels and Wharton's jelly. In the latter, S100A6 was found not only in the myofibroblasts but also in the ECM (extracellular matrix) surrounding these cells. Affinity chromatography of S100A6 resin indicated that Wharton's jelly contains some proteins that could bind to S100A6. Thus these novel results show the presence of S100A6 in umbilical cord and suggest the involvement of this protein in intra- and extra-cellular signalling pathways in this tissue.