Loss of INPP5K attenuates IP3-induced Ca2+ responses in the glioblastoma cell line U-251 MG cells.

INPP5K (inositol polyphosphate 5-phosphatase K) is an endoplasmic reticulum (ER)-resident enzyme that acts as a phosphoinositide (PI) 5-phosphatase, capable of dephosphorylating various PIs including PI 4,5-bisphosphate (PI(4,5)P2), a key phosphoinositide found in the plasma membrane. Given its ER localization and substrate specificity, INPP5K may play a role in ER-plasma membrane contact sites. Furthermore, PI(4,5)P2 serves as a substrate for phospholipase C, an enzyme activated downstream of extracellular agonists acting on Gq-coupled receptors or tyrosine-kinase receptors, leading to IP3 production and subsequent release of Ca2+ from the ER, the primary intracellular Ca2+ storage organelle. In this study, we investigated the impact of INPP5K on ER Ca2+ dynamics using a previously established INPP5K-knockdown U-251 MG glioblastoma cell model. We here describe that loss of INPP5K impairs agonist-induced, IP3 receptor (IP3R)-mediated Ca2+ mobilization in intact cells, while the ER Ca2+ content and store-operated Ca2+ influx remain unaffected. To further elucidate the underlying mechanisms, we examined Ca2+ release in permeabilized cells stimulated with exogenous IP3. Interestingly, the absence of INPP5K also disrupted IP3-induced Ca2+ release events. These results suggest that INPP5K may directly influence IP3R activity through mechanisms yet to be resolved. The findings from this study point towards role of INPP5K in modulating ER calcium dynamics, particularly in relation to IP3-mediated signaling pathways. However, further work is needed to establish the general nature of our findings and to unravel the exact molecular mechanisms underlying the interplay between INNP5K function and Ca2+ signaling.

Práticas Integrativas e Complementares em Saúde e Doenças Autoimunes: um mapa de evidências / Integrative and Complementary Health Practices and Autoimmune Diseases: a map of evidence

As doenças autoimunes (DAI) são causadas por alterações orgânicas relacionadas ao sistema imunológico e órgãos. Esses desequilíbrios podem causar danos à fisiologia de vários sistemas do organismo. Atualmente no Sistema Único de Saúde (SUS) existem 29 Práticas Integrativas Complementares (PICS), cujo objetivo é fornecer a população o acesso a um modo de cuidado que contempla o indivíduo em toda a sua complexidade, podendo assim promover a saúde dos portadores de doenças autoimunes. Objetivo: Construir um Mapa de Evidências por meio de um overview para identificar quais PICS têm sido usadas no tratamento das DAI selecionadas e analisar quais foram os desfechos em saúde dos pacientes em tratamento com PICS. Método: Foi realizada uma revisão de revisões (overview). Ocorreu busca de artigos científicos nas bases de dados PubMed, Scielo (Scientific Eletronic Library), Science Direct e Web of Science, utilizando os seguintes descritores: "Doenças Autoimunes", "Terapias Complementares". "Complementary Therapies" and "Artrite Reumatoide", "Lúpus Eritematoso Sistêmico"; "Doença de Crohn", "Diabetes Mellitus tipo 1", "esclerose múltipla", "psoríase", "vasculites" e o "vitiligo". Os critérios de inclusão foram: artigo original, disponível na íntegra, publicado até julho de 2022. Dois pesquisadores extraíram informações dos estudos selecionados através do software Rayyan. Conclusão: Este mapa de evidências possibilitou observar como as PICS podem auxiliar no tratamento das DAI, favorecendo o autocuidado e a independência dos portadores, melhorando sua qualidade de vida. Evidenciando dentre as PICS mais utilizadas a fitoterapia, acupuntura, yoga, massagem e hipnose. Espera-se com este mapa de evidências promover o uso das PICS para o tratamento das DAI para os usuários, profissionais de saúde e gestores.

Autoimmune diseases (AID) are caused by organic changes related to the immune system and organs. These imbalances can cause damage to the physiology of various body systems. Currently in the Unified Health System (SUS) there are 29 complementary therapies (PICS), whose objective is to provide the population with access to a way of care that contemplates the individual in all its complexity, thus being able to promote the health of people with diseases autoimmune. Objective: To build an Evidence Map through an overview to identify which PICS have been used in the treatment of selected IADs and to analyze the health outcomes of patients undergoing treatment with PICS. Method: A review of reviews was carried out. There was a search for scientific articles in the PubMed, Scielo (Scientific Electronic Library), Science Direct and Web of Science databases, using the following descriptors: "Autoimmune Diseases", "Complementary Therapies". "Complementary Therapies" and "Rheumatoid Arthritis", "Systemic Lupus Erythematosus"; "Crohn's disease", "Type 1 diabetes mellitus", "multiple sclerosis", "psoriasis", "vasculitis" and "vitiligo". The inclusion criteria were original article, available in full, published until July 2022. Two researchers extracted information from the selected studies using the Rayyan software. Conclusion: This evidence map made it possible to observe how PICS can help in the treatment of IAD, favoring self-care and independence of patients, improving their quality of life. Evidencing among the most used PICS: phytotherapy, acupuncture, yoga, massage, and hypnosis. This evidence map is expected to promote the use of PICS for the treatment of IAD for users, health professionals and managers.

A gestão terapêutica e a segurança do doente no Bloco Operatório / Therapeutic management and patient safety in the operating room

O presente relatório, descreve, analisa e reflete o processo de desenvolvimento e aquisição de competências especializadas, na área da pessoa em situação critica, adquiridas ao longo da Unidade Curricular - Estágio de Natureza Profissional (ENP), realizado no âmbito do Mestrado em Enfermagem Médico-Cirúrgica. Neste sentido, o ENP, decorreu no Bloco Operatório Central (BO) do Centro Hospitalar Póvoa de Varzim / Vila do Conde de 11 de janeiro a 11 de julho de 2021, tendo como principal objetivo o desenvolvimento de competências comuns e especificas em Enfermagem Médico-Cirúrgica, na área da prestação de cuidados à pessoa em situação crítica no BO. Para o desenvolvimento e aquisição destas competências, emergiram diversas atividades no contexto de estágio que foram realizadas com sustentação em bases teóricas e na evidência científica, de modo a garantir uma melhoria da qualidade dos cuidados e a excelência no exercício da profissão. Desta experiência, não é de mais descrever, a importância do papel do enfermeiro especialista em médico-cirúrgica no BO no cuidar ao doente crítico pela sua diferenciação na prestação de cuidados. No que diz respeito ao nosso desempenho neste estágio, destacamos o papel da formação profissional em enfermagem no desenvolvimento de competências, a aprendizagem e reflexão sobre as práticas no cuidar ao doente em situação crítica no BO e no conhecimento do agir em ambientes imprevisíveis e instáveis. No âmbito da investigação, recorremos a uma revisão integrativa da literatura, para dar resposta à problemática, a gestão terapêutica e a segurança do doente no Bloco Operatório. A pertinência do tema, decorre pela necessidade de cada vez mais se promover a cultura de segurança do doente, uma vez que é requisito essencial à qualidade dos cuidados de saúde e também pela minimização dos erros associados à terapêutica da enfermagem de anestesia, visto que estes podem desencadear um agravamento da situação do doente. Como principal resultado deste trabalho de investigação foram implementados novos procedimentos de enfermagem no Bloco Operatório. Estes, incidiram na rotulagem dos medicamentos de acordo com os medicamentos Look Alike e Sound Alike, medicamentos de Alto Risco e rotulagem com recurso a codificação por cores. A aprendizagem não é estanque, pelo que procuramos, enquanto futura Enfermeira Especialista, a melhoria contínua cuidados prestados com qualidade, eficácia e humanização dos mesmos.

This report describes, analyzes and reflects the process of development and acquisition of specialized skills, in the area of the person in a critical situation, acquired throughout the Curricular Unit - Internship of a Professional Nature, carried out within the scope of the Master in Medical-Surgical Nursing. In this sense, the internship of a professional nature took place in the Operation Room of the Centro Hospitalar Póvoa de Varzim / Vila do Conde from January 11 to July 11, 2021, with the main objective of developing common and specific skills medical-surgical nursing, in the area of providing care to the person in critical condition in the Operation Room. For the development and acquisition of these skills, several activities emerged in the internship context that were carried out in a way based on theoretical bases and scientific evidence, in order to guarantee an improvement in the quality of care and excellence in the exercise of the profession. From this experience, it is not enough to describe the importance of the role of the medical-surgical nurse specialist in the Operation Room in caring for the critically ill patient due to their differentiation in the provision of care. With regard to our performance in this internship, we highlight the role of professional training in nursing in the development of skills, learning and reflection on practices in caring for the patient in a critical situation in the Operation Room and in the knowledge of acting in unpredictable and unstable environments. Within the scope of the investigation, we resorted to an integrative literature review, to respond to the problem, therapeutic management and patient safety in the Operation Room: contributions from anesthesia nursing. The relevance of the topic stems from the need to increasingly promote a patient safety culture, since it is an essential requirement for the quality of health care and due to the minimization of errors associated with anesthesia nursing therapy. As the main result of this investigation, new nursing procedures were implemented in the Operation Room. These were nothing more and nothing less than the labeling of medicines according to Look Alike and Sound Alike medicines, High Risk medicines and labeling using color coding. Learning is not watertight, so I seek, as a Specialist Nurse, the continuous improvement of care provided with quality, effectiveness and humanization.

APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin.

The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.

Consulta do Registo de Saúde Eletrónico ­ Análise ética de um caso clínico / Electronic Health Registry Query - Ethical Case Study / Consulta electrónica de registros de salud - Estudio de caso ético

Introducción: La bioética surgió de la necesidad de regular un código de conducta ética que evitara las prácticas médicas abusivas llevadas a cabo durante varios siglos. El Procedimiento Deliberativo (PD), aplicado a la medicina, ayuda a tomar decisiones éticas de forma objetiva (equilibrada, podenrada) y justa. Con la presentación de este caso clínico se pretende, como ejemplo, demostrar la importancia de aplicar el PD en la práctica clínica en medicina general y familiar (MGF). Caso clínico: Un hombre de 58 años que, después de un antígeno prostático específico alterado, fue derivado a una consulta de urología. Un mes después de la biopsia de próstata, asiste a la consulta de MGF con la confianza de que el resultado sería negativo. Sin su autorización, el médico de familia (MF) accedió al registro electrónico de salud y descubrió que el paciente tenía un adenocarcinoma de próstata, aunque no habia sido re-evaluado por Urologia. Se eligió el problema ético más importante y se definió el curso de acción óptimo que cumplía los criterios de legalidad, publicidad y temporalidad. Conclusión: La decisión del médico siempre debe beneficiar el paciente. El PD permite tomar decisiones estructuradas, por lo cual debe aplicarse en el contexto de la MGF frente a problemas éticos

Introduction: Bioethics emerged from the need to implement an ethical code of conduct that would avoid abusive clinical practice carried for centuries. The Deliberative Procedure (DP) applied to medicine helps in making ethical decisions in a well-reasoned and fair manner. This clinical case report is intended to exemplify the importance of applying the deliberative process in clinical practice in general practice/family medicine (GP/FM). Clinical case: Male, 58 years old referenced to urology office visit after positive Prostate-Specific Antigen (PSA) test. A month after prostatic biopsy he went to the medical consultation with his Family Doctor, believing the result was negative. His family doctor assessed his health electronic record without his authorization, and verified the result of the biopsy was a prostatic adenocarcinoma, without new urology appointment. It was elected the most important ethic problem and defined the action curse that accomplish the legality, publicity and temporary criteria. Conclusions: Whatever the physician decides, it should always be for the benefit of the wearer. DP allows structured decision making so it should be applied in the context of GP/FM in the face of ethical problems.

Introdução: A bioética surgiu da necessidade de regulamentar um código de conduta ética que evitasse as práticas médicas abusivas realizadas durante vários séculos. O Procedimento Deliberativo (PD) aplicado à medicina, ajuda na tomada de decisões éticas de forma ponderada e justa. Com a apresentação deste caso clínico pretende-se exemplificar a importância da aplicação do PD na prática clínica em Medicina Geral e Familiar (MGF). Apresentação do caso: Homem de 58 anos que no seguimento de um antígeno específico da próstata (PSA) alterado foi referenciado à consulta de urologia. Um mês após biópsia prostática recorre à consulta de MGF confiante de que o resultado seria negativo. Sem a sua autorização, o Médico de Família (MF) acedeu ao Registo de Saúde Eletrónico e verificou que o doente apresentava um adenocarcinoma da próstata, embora não tivesse ainda consulta de reavaliação de urologia. Foi eleito o problema ético mais importante e definido o curso de ação ótimo que cumpria os critérios de legalidade, publicidade e temporalidade. Conclusões: Qualquer que seja a decisão do médico, deve ser sempre em benefício da pessoa. O PD permite uma tomada de decisão estruturada pelo que deve ser aplicado no contexto de MGF perante a ocorrência de problemas éticos.

Phosphoinositide 5-phosphatases SKIP and SHIP2 in ruffles, the endoplasmic reticulum and the nucleus: An update.

Phosphoinositides (PIs) are phosphorylated derivatives of phosphatidylinositol. They act as signaling molecules linked to essential cellular mechanisms in eukaryotic cells, such as cytoskeleton organization, mitosis, polarity, migration or invasion. PIs are phosphorylated and dephosphorylated by a large number of PI kinases and PI phosphatases acting at the 5-, 4- and 3- position of the inositol ring. PI 5-phosphatases i.e. OCRL, INPP5B, SHIP1/2, Synaptojanin 1/2, INPP5E, INPP5J, SKIP (INPP5K) are enzymes that dephosphorylate the 5-phosphate position of PIs. Several human genetic diseases such as the Lowe syndrome, some congenital muscular dystrophy and opsismodysplasia are due to mutations in PI phosphatases, resulting in loss-of-function. The PI phosphatases are also up or down regulated in several human cancers such as glioblastoma or breast cancer. Their cellular localization, that is dynamic and varies in response to stimuli, is an important issue to understand function. This is the case for two members of the PI 5-phosphatase SKIP and SHIP2. Both enzymes are in ruffles, plasma membranes, the endoplasmic reticulum, a situation that is unique for SKIP, and the nucleus. Following localization, PI 5-phosphatases act on specific cellular pools of PIs, which in turn interact with target proteins. Nuclear PIs have emerged as regulators of genome functions in different area of cell signaling. They often localize to nuclear speckles, as do several PI metabolizing kinases and phosphatases. We asked whether SKIP and SHIP2 could have an impact on nuclear PI(4,5)P2. In two glioblastoma cell models, lowering SKIP expression had an impact on nuclear PI(4,5)P2. In a model of SHIP2 deletion in MCF-7 cells, no change in nuclear PI(4,5)P2 was observed. Finally, we present evidence of an anti-tumoral role of SKIP in vivo, in xenografts using as model U87shSKIP cells.

Lipid phosphatases SKIP and SHIP2 regulate fibronectin-dependent cell migration in glioblastoma.

Cell migration is an important process that occurs during development and has also been linked to the motility of cancer cells. Cytoskeleton reorganization takes place in the migration process leading to lamellipodia formation. Understanding the molecular underpinnings of cell migration is particularly important in studies of glioblastoma, a highly invasive and aggressive cancer type. Two members of the phosphoinositide 5-phosphatase family, SKIP and SHIP2, have been associated with cell migration in glioblastoma; however, the precise role these enzymes play in the process-and whether they work in concert-remains unclear. Here, we compared phosphoinositide 5-phosphatases expression in glioblastoma primary cells and cell lines and showed that SHIP2 and SKIP expression greatly varies between different cell types, while OCRL, another phosphoinositide 5-phosphatase, is constitutively expressed. Upon adhesion of U-251 MG cells to fibronectin, SHIP2, SKIP, and PI(4,5)P2 colocalized in membrane ruffles. Upregulation of PI(4,5)P2 was observed in SKIP-depleted U-251 MG cells compared to control cells, but only when cells were adhered to fibronectin. Both PTEN-deficient (U-251) and PTEN-containing (LN229) glioblastoma cells showed a decrease in cell migration velocity in response to SKIP downregulation. Moreover, a SHIP2 catalytic inhibitor lowered cell migration velocity in the U-251 MG cell line. We conclude that integrin activation in U-251 cells leads to colocalization of both SKIP and SHIP2 in ruffles, where they act as potential drivers of cell migration. Depending on their expression levels in glioblastoma, phosphoinositide 5-phosphatases could cooperate and synergize in the regulation of cell migration and adhesion.

The impact of phosphoinositide 5-phosphatases on phosphoinositides in cell function and human disease.

Phosphoinositides (PIs) are recognized as major signaling molecules in many different functions of eukaryotic cells. PIs can be dephosphorylated by multiple phosphatase activities at the 5-, 4-, and 3- positions. Human PI 5-phosphatases belong to a family of 10 members. Except for inositol polyphosphate 5-phosphatase A, they all catalyze the dephosphorylation of PI(4,5)P2 and/or PI(3,4,5)P3 at the 5- position. PI 5-phosphatases thus directly control the levels of PI(3,4,5)P3 and participate in the fine-tuning regulatory mechanisms of PI(3,4)P2 and PI(4,5)P2 Second messenger functions have been demonstrated for PI(3,4)P2 in invadopodium maturation and lamellipodia formation. PI 5-phosphatases can use several substrates on isolated enzymes, and it has been challenging to establish their real substrate in vivo. PI(4,5)P2 has multiple functions in signaling, including interacting with scaffold proteins, ion channels, and cytoskeleton proteins. PI 5-phosphatase isoenzymes have been individually implicated in human diseases, such as the oculocerebrorenal syndrome of Lowe, through mechanisms that include lipid control. Oncogenic and tumor-suppressive functions of PI 5-phosphatases have also been reported in different cell contexts. The mechanisms responsible for genetic diseases and for oncogenic or tumor-suppressive functions are not fully understood. The regulation of PI 5-phosphatases is thus crucial in understanding cell functions.

Inhibition of SHIP2 activity inhibits cell migration and could prevent metastasis in breast cancer cells.

Metastasis of breast cancer cells to distant organs is responsible for ∼50% of breast cancer-related deaths in women worldwide. SHIP2 (also known as INPPL1) is a phosphoinositide 5-phosphatase for phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2]. Here we show, through depletion of SHIP2 in triple negative MDA-MB-231 cells and the use of SHIP2 inhibitors, that cell migration appears to be positively controlled by SHIP2. The effect of SHIP2 on migration, as observed in MDA-MB-231 cells, appears to be mediated by PI(3,4)P2. Adhesion on fibronectin is always increased in SHIP2-depleted cells. Apoptosis measured in MDA-MB-231 cells is also increased in SHIP2-depleted cells as compared to control cells. In xenograft mice, SHIP2-depleted MDA-MB-231 cells form significantly smaller tumors than those formed by control cells and less metastasis is detected in lung sections. Our data reveal a general role for SHIP2 in the control of cell migration in breast cancer cells and a second messenger role for PI(3,4)P2 in the migration mechanism. In MDA-MB-231 cells, SHIP2 has a function in apoptosis in cells incubated in vitro and in mouse tumor-derived cells, which could account for its role on tumor growth determined in vivo.

Phosphoinositide 5-phosphatase activities control cell motility in glioblastoma: Two phosphoinositides PI(4,5)P2 and PI(3,4)P2 are involved.

Inositol polyphosphate 5-phosphatases or phosphoinositide 5-phosphatases (PI 5-phosphatases) are enzymes that can act on soluble inositol phosphates and/or phosphoinositides (PIs). Several PI 5-phosphatases have been linked to human genetic diseases, in particular the Lowe protein or OCRL which is mutated in the Lowe syndrome. There are 10 different members of this family and 9 of them can use PIs as substrate. One of these substrates, PI(3,4,5)P3 binds to specific PH domains and recruits as effectors specific proteins to signaling complexes. Protein kinase B is one target protein and activation of the kinase will have a major impact on cell proliferation, survival and cell metabolism. Two other PIs, PI(4,5)P2 and PI(3,4)P2, are produced or used as substrates of PI 5-phosphatases (OCRL, INPP5B, SHIP1/2, SYNJ1/2, INPP5K, INPP5J, INPP5E). The inositol lipids may influence many aspects of cytoskeletal organization, lamellipodia formation and F-actin polymerization. PI 5-phosphatases have been reported to control cell migration, adhesion, polarity and cell invasion particularly in cancer cells. In glioblastoma, reducing SHIP2 expression can positively or negatively affect the speed of cell migration depending on the glioblastoma cell type. The two PI 5-phosphatases SHIP2 or SKIP could be localized at the plasma membrane and can reduce either PI(3,4,5)P3 or PI(4,5)P2 abundance. In the glioblastoma 1321 N1 cells, SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration.

The direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysis.

Hydrogenases are highly active enzymes for hydrogen production and oxidation. [NiFeSe] hydrogenases, in which selenocysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H2 production. In contrast to [NiFe] hydrogenases, they display reduced H2 inhibition and are rapidly reactivated after contact with oxygen. Here we report an expression system for production of recombinant [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough and study of a selenocysteine-to-cysteine variant (Sec489Cys) in which, for the first time, a [NiFeSe] hydrogenase was converted to a [NiFe] type. This modification led to severely reduced Ni incorporation, revealing the direct involvement of this residue in the maturation process. The Ni-depleted protein could be partly reconstituted to generate an enzyme showing much lower activity and inactive states characteristic of [NiFe] hydrogenases. The Ni-Sec489Cys variant shows that selenium has a crucial role in protection against oxidative damage and the high catalytic activities of the [NiFeSe] hydrogenases.

The SHIP2 interactor Myo1c is required for cell migration in 1321 N1 glioblastoma cells.

The phosphoinositide 5-phosphatases consist of several enzymes that have been shown to modulate cell migration and invasion. SHIP2, one family member, is known to interact with growth factor receptors and cytoskeletal proteins. In a human model of glioblastoma 1321 N1 cells, we recently identified Myo1c as a new interactor of SHIP2. This was shown in a complex of proteins also containing filamin A. We show here that SHIP2 localization at lamellipodia and ruffles is impaired in Myo1c depleted cells. In the absence of Myo1c, N1 cells tend to associate to form clusters. Cell migration is very much reduced in Myo1c depleted cells, concomitantly with a decrease in FAK Tyr397 phosphorylation, focal adhesion length and PI(4,5)P2 immunostaining. In N1 cells, Myo1c is thus important for lamellipodia formation to assemble a protein complex containing SHIP2 to facilitate cell migration.

New Functions of the Inositol Polyphosphate 5-Phosphatases in Cancer.

Inositol polyphosphate 5-phosphatases act on inositol phosphates and phosphoinositides as substrates. They are 10 different isoenzymes and several splice variants in the human genome that are involved in a series of human pathologies such as the Lowe syndrome, the Joubert and MORM syndromes, breast cancer, glioblastoma, gastric cancer and several other type of cancers. Inositol 5-phosphatases can be amplified in human cancer cells, whereas the 3- and 4- phosphatase tumor suppressor PTEN and INPP4B, repectively are often repressed or deleted. The inositol 5-phosphatases are critically involved in a complex network of higly regulated phosphoinositides, affecting the lipid content of PI(3, 4, 5)P3, PI(4, 5)P2 and PI(3, 4)P2. This has an impact on the normal behavior of many intracellular target proteins e.g. protein kinase B (PKB/Akt) or actin binding proteins and final biological responses. The production of PI(3, 4P)2 by dephosphorylation of the substrate PI(3, 4, 5)P3 is particularly important as it produces a new signal messenger in the control of cell migration, invasion and endocytosis. New inhibitors/activators of inositol 5- phosphatases have recently been identified for the possible control of their activity in several human pathologies such as inflamation and cancer.

The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris†Hildenborough.

Flavin-based electron bifurcation (FBEB) is an important mechanism for the energy metabolism of anaerobes. A new family of NADH dehydrogenases, the flavin oxidoreductase (FlxABCD, previously called FloxABCD), was proposed to perform FBEB in sulphate-reducing organisms coupled with heterodisulfide reductase (HdrABC). We found that the hdrABC-flxABCD gene cluster is widespread among anaerobic bacteria, pointing to a general and important role in their bioenergetics. In this work, we studied FlxABCD of Desulfovibrio vulgaris Hildenborough. The hdr-flx genes are part of the same transcriptional unit and are increased in transcription during growth in ethanol-sulfate, and to a less extent during pyruvate fermentation. Two mutant strains were generated: one where expression of the hdr-flx genes was interrupted and another lacking the flxA gene. Both strains were unable to grow with ethanol-sulfate, whereas growth was restored in a flxA-complemented strain. The mutant strains also produced very reduced amounts of ethanol compared with the wild type during pyruvate fermentation. Our results show that in D. vulgaris, the FlxABCD-HdrABC proteins are essential for NADH oxidation during growth on ethanol, probably involving a FBEB mechanism that leads to reduction of ferredoxin and the small protein DsrC, while in fermentation they operate in reverse, reducing NAD(+) for ethanol production.

Unifying concepts in anaerobic respiration: insights from dissimilatory sulfur metabolism.

Behind the versatile nature of prokaryotic energy metabolism is a set of redox proteins having a highly modular character. It has become increasingly recognized that a limited number of redox modules or building blocks appear grouped in different arrangements, giving rise to different proteins and functionalities. This modularity most likely reveals a common and ancient origin for these redox modules, and is obviously reflected in similar energy conservation mechanisms. The dissimilation of sulfur compounds was probably one of the earliest biological strategies used by primitive organisms to obtain energy. Here, we review some of the redox proteins involved in dissimilatory sulfur metabolism, focusing on sulfate reducing organisms, and highlight links between these proteins and others involved in different processes of anaerobic respiration. Noteworthy are links to the complex iron-sulfur molybdoenzyme family, and heterodisulfide reductases of methanogenic archaea. We discuss how chemiosmotic and electron bifurcation/confurcation may be involved in energy conservation during sulfate reduction, and how introduction of an additional module, multiheme cytochromes c, opens an alternative bioenergetic strategy that seems to increase metabolic versatility. Finally, we highlight new families of heterodisulfide reductase-related proteins from non-methanogenic organisms, which indicate a widespread distribution for these protein modules and may indicate a more general involvement of thiol/disulfide conversions in energy metabolism. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

The Membrane QmoABC Complex Interacts Directly with the Dissimilatory Adenosine 5'-Phosphosulfate Reductase in Sulfate Reducing Bacteria.

The adenosine 5'-phosphosulfate reductase (AprAB) is the enzyme responsible for the reduction of adenosine 5'-phosphosulfate (APS) to sulfite in the biological process of dissimilatory sulfate reduction, which is carried out by a ubiquitous group of sulfate reducing prokaryotes. The electron donor for AprAB has not been clearly identified, but was proposed to be the QmoABC membrane complex, since an aprBA-qmoABC gene cluster is found in many sulfate reducing and sulfur-oxidizing bacteria. The QmoABC complex is essential for sulfate reduction, but electron transfer between QmoABC and AprAB has not been reported. In this work we provide the first direct evidence that QmoABC and AprAB interact in Desulfovibrio spp., using co-immunoprecipitation, cross-linking Far-Western blot, tag-affinity purification, and surface plasmon resonance studies. This showed that the QmoABC-AprAB complex has a strong steady-state affinity (K(D) = 90 ± 3 nM), but has a transient character due to a fast dissociation rate. Far-Western blot identified QmoA as the Qmo subunit most involved in the interaction. Nevertheless, electron transfer from menaquinol analogs to APS through anaerobically purified QmoABC and AprAB could not be detected. We propose that this reaction requires the involvement of a third partner to allow electron flow driven by a reverse electron bifurcation process, i.e., electron confurcation. This process is deemed essential to allow coupling of APS reduction to chemiosmotic energy conservation.

A comparative genomic analysis of energy metabolism in sulfate reducing bacteria and archaea.

The number of sequenced genomes of sulfate reducing organisms (SRO) has increased significantly in the recent years, providing an opportunity for a broader perspective into their energy metabolism. In this work we carried out a comparative survey of energy metabolism genes found in 25 available genomes of SRO. This analysis revealed a higher diversity of possible energy conserving pathways than classically considered to be present in these organisms, and permitted the identification of new proteins not known to be present in this group. The Deltaproteobacteria (and Thermodesulfovibrio yellowstonii) are characterized by a large number of cytochromes c and cytochrome c-associated membrane redox complexes, indicating that periplasmic electron transfer pathways are important in these bacteria. The Archaea and Clostridia groups contain practically no cytochromes c or associated membrane complexes. However, despite the absence of a periplasmic space, a few extracytoplasmic membrane redox proteins were detected in the Gram-positive bacteria. Several ion-translocating complexes were detected in SRO including H(+)-pyrophosphatases, complex I homologs, Rnf, and Ech/Coo hydrogenases. Furthermore, we found evidence that cytoplasmic electron bifurcating mechanisms, recently described for other anaerobes, are also likely to play an important role in energy metabolism of SRO. A number of cytoplasmic [NiFe] and [FeFe] hydrogenases, formate dehydrogenases, and heterodisulfide reductase-related proteins are likely candidates to be involved in energy coupling through electron bifurcation, from diverse electron donors such as H(2), formate, pyruvate, NAD(P)H, ß-oxidation, and others. In conclusion, this analysis indicates that energy metabolism of SRO is far more versatile than previously considered, and that both chemiosmotic and flavin-based electron bifurcating mechanisms provide alternative strategies for energy conservation.