Nos últimos anos, um importante destaque tem sido dado aos linfócitos Th17 para o desenvolvimento e manutenção da inflamação associada à autoimunidade. A esclerose múltipla é uma doença autoimune desmielinizante do SNC, cuja patogênese está associada à resposta do padrão Th17. Evidências têm demonstrado que estas células são submetidas a uma reprogramação metabólica após serem ativadas, sendo essa adequação essencial para sua completa diferenciação e aquisição de funções efetoras. A enzima Piruvato Quinase M2 (PKM2) participa da etapa final da glicólise convertendo fosfoenolpiruvato em piruvato. Estudos recentes demonstraram que a fosforilação de PKM2 a torna capaz de translocar para o núcleo, onde adquire um papel no controle da expressão gênica. Nesse sentido, o objetivo deste estudo foi avaliar o envolvimento da PKM2 na diferenciação de linfócitos Th17, bem como sua participação no desenvolvimento da encefalomielite autoimune experimental (EAE), um modelo animal de esclerose múltipla. Observou-se que durante o processo de diferenciação, os linfócitos Th17 aumentam a expressão gênica de PKM2 bem como a sua forma fosforilada (Y105). De forma interessante, tanto a inibição farmacológica como a deleção gênica da PKM2 especificamente em linfócitos T promoveram uma redução da diferenciação e expansão da subpopulação Th17, que foi associada com diminuição na expressão de moléculas efetoras e fatores de transcrição chave para o estabelecimento do fenótipo Th17. Em um contexto de resposta autoimune, notou-se que PKM2 é superexpressa nos órgãos linfóides periféricos e sistema nervoso central de animais com EAE, sendo correlacionada com o infiltrado de células inflamatórias. Corroborando com os dados in vitro, a deficiência de PKM2 em linfócitos T promoveu redução dos sinais clínicos da EAE, acompanhada de baixa frequência de linfócitos Th17 e menor expressão de moléculas inflamatórias do perfil Th17. Adicionalmente, o tratamento farmacológico com o inibidor da PKM2 atenuou a progressão e gravidade da EAE. Portanto, esses achados implicam um importante papel para PKM2 em doenças autoimunes por regular o desenvolvimento e função de linfócitos Th17. / Over the past few years, an important highlight has been given to Th17 lymphocytes for the development and maintenance of autoimmunity-associated inflammation. Multiple sclerosis is a CNS demyelinating autoimmune disease that is associated to Th17-mediated response. Some evidences have demonstrated that those cells undergo metabolic reprogramming after being activated, which is essential for their complete differentiation and acquisition of effector functions. The enzyme Pyruvate kinase M2 (PKM2) participates at the final step of glycolysis by converting phosphoenolpyruvate into pyruvate. Recent studies have demonstrated that PKM2 phosphorylation allows its translocation into the nucleus, where it plays a role in controlling gene expression. Thus, the aim of this study was to evaluate the involvement of PKM2 in Th17 lymphocytes differentiation, as well as its role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. It was perceived that during differentiation process, Th17 lymphocytes increase PKM2 gene expression, and also its phosphorylated form (Y105). Interestingly, both pharmacological inhibition and T-lymphocyte-specific PKM2 gene deletion promoted a reduction in differentiation and expansion of Th17 subpopulation, being associated to diminished expression of effector molecules and key transcription factors for the establishment of Th17 phenotype. In the context of an autoimmune response, it was noticed that PKM2 is overexpressed in peripheral lymphoid organs and central nervous system of EAE-bearing mice, which was correlated with the inflammatory cell infiltration. Corroborating with in vitro data, the deficiency of PKM2 in T lymphocytes led to a reduction of EAE clinical score along with low Th17 frequency and diminished expression of Th17-related inflammatory molecules. Additionally, pharmacological treatment with the PKM2 inhibitor attenuated EAE progression and severity. Therefore, these findings imply an important role for PKM2 in autoimmune diseases by regulating the development and function of Th17 lymphocytes.

Nos últimos anos, um importante destaque tem sido dado aos linfócitos Th17 para o desenvolvimento e manutenção da inflamação associada à autoimunidade. A esclerose múltipla é uma doença autoimune desmielinizante do SNC, cuja patogênese está associada à resposta do padrão Th17. Evidências têm demonstrado que estas células são submetidas a uma reprogramação metabólica após serem ativadas, sendo essa adequação essencial para sua completa diferenciação e aquisição de funções efetoras. A enzima Piruvato Quinase M2 (PKM2) participa da etapa final da glicólise convertendo fosfoenolpiruvato em piruvato. Estudos recentes demonstraram que a fosforilação de PKM2 a torna capaz de translocar para o núcleo, onde adquire um papel no controle da expressão gênica. Nesse sentido, o objetivo deste estudo foi avaliar o envolvimento da PKM2 na diferenciação de linfócitos Th17, bem como sua participação no desenvolvimento da encefalomielite autoimune experimental (EAE), um modelo animal de esclerose múltipla. Observou-se que durante o processo de diferenciação, os linfócitos Th17 aumentam a expressão gênica de PKM2 bem como a sua forma fosforilada (Y105). De forma interessante, tanto a inibição farmacológica como a deleção gênica da PKM2 especificamente em linfócitos T promoveram uma redução da diferenciação e expansão da subpopulação Th17, que foi associada com diminuição na expressão de moléculas efetoras e fatores de transcrição chave para o estabelecimento do fenótipo Th17. Em um contexto de resposta autoimune, notou-se que PKM2 é superexpressa nos órgãos linfóides periféricos e sistema nervoso central de animais com EAE, sendo correlacionada com o infiltrado de células inflamatórias. Corroborando com os dados in vitro, a deficiência de PKM2 em linfócitos T promoveu redução dos sinais clínicos da EAE, acompanhada de baixa frequência de linfócitos Th17 e menor expressão de moléculas inflamatórias do perfil Th17. Adicionalmente, o tratamento farmacológico com o inibidor da PKM2 atenuou a progressão e gravidade da EAE. Portanto, esses achados implicam um importante papel para PKM2 em doenças autoimunes por regular o desenvolvimento e função de linfócitos Th17. / Over the past few years, an important highlight has been given to Th17 lymphocytes for the development and maintenance of autoimmunity-associated inflammation. Multiple sclerosis is a CNS demyelinating autoimmune disease that is associated to Th17-mediated response. Some evidences have demonstrated that those cells undergo metabolic reprogramming after being activated, which is essential for their complete differentiation and acquisition of effector functions. The enzyme Pyruvate kinase M2 (PKM2) participates at the final step of glycolysis by converting phosphoenolpyruvate into pyruvate. Recent studies have demonstrated that PKM2 phosphorylation allows its translocation into the nucleus, where it plays a role in controlling gene expression. Thus, the aim of this study was to evaluate the involvement of PKM2 in Th17 lymphocytes differentiation, as well as its role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. It was perceived that during differentiation process, Th17 lymphocytes increase PKM2 gene expression, and also its phosphorylated form (Y105). Interestingly, both pharmacological inhibition and T-lymphocyte-specific PKM2 gene deletion promoted a reduction in differentiation and expansion of Th17 subpopulation, being associated to diminished expression of effector molecules and key transcription factors for the establishment of Th17 phenotype. In the context of an autoimmune response, it was noticed that PKM2 is overexpressed in peripheral lymphoid organs and central nervous system of EAE-bearing mice, which was correlated with the inflammatory cell infiltration. Corroborating with in vitro data, the deficiency of PKM2 in T lymphocytes led to a reduction of EAE clinical score along with low Th17 frequency and diminished expression of Th17-related inflammatory molecules. Additionally, pharmacological treatment with the PKM2 inhibitor attenuated EAE progression and severity. Therefore, these findings imply an important role for PKM2 in autoimmune diseases by regulating the development and function of Th17 lymphocytes.

L'oncoprotéine MDM2 est reconnue comme un régulateur négatif majeur du suppresseur de tumeur p53, mais plus de preuves indiquent que ses activités oncogéniques vont au-delà de p53. Ici, nous montrons que MDM2 est recruté à la chromatine indépendamment de p53 pour réguler un programme transcriptionnel complexe impliqué dans le métabolisme des acides aminés et l'homéostasie redox. L'identification des gènes cibles de MDM2 au niveau du génome entier met en évidence un rôle important pour les facteurs de transcription ATF3/4 dans le recrutement de MDM2 à la chromatine. Ce recrutement de MDM2 à la chromatine est un processus étroitement régulé qui se produit lors d'un stress oxydatif et lors d'une déprivation en serine/glycine et est modulé par la pyruvate kinase M2 (PKM2) qui est une enzyme métabolique. La déplétion de la protéine MDM2 endogène dans des cellules déficientes en p53 altère le métabolisme sérine/glycine, le rapport NAD+/NADH et le recyclage de la glutathion (GSH), important leurs état redox et leurs potentiel tumorigènique. Nos données illustrent une fonction précédemment insoupçonnée de MDM2 à la chromatine impliquée dans le métabolisme des cellules cancéreuses. / The mouse double minute 2 (MDM2) oncoprotein is recognized as a major negative regulator of the p53 tumor suppressor, but growing evidence indicates that its oncogenic activities extend beyond p53. Here, we show that MDM2 is recruited to chromatin independently of p53 to regulate a transcriptional program implicated in amino acid metabolism and redox homeostasis. Identification of MDM2 target genes at the whole-genome level highlights an important role for ATF3/4 transcription factors in tethering MDM2 to chromatin. MDM2 recruitment to chromatin is a tightly regulated process that occurs during oxidative stress and serine/glycine deprivation and is modulated by the pyruvate kinase M2 (PKM2) metabolic enzyme. Depletion of endogenous MDM2 in p53-deficient cells impairs serine/glycine metabolism, the NAD+/NADH ratio, and glutathione (GSH) recycling, impacting their redox state and tumorigenic potential. Collectively, our data illustrate a previously unsuspected function of chromatinbound MDM2 in cancer cell metabolism.

No description available.

Regulation of Glucose metabolism is known to play an important role in pathogenesis of many diseases. Primarily because deregulation of this metabolic pathway can lead to either apoptosis or extended life span of the cells involved. Viruses are parasitic in nature, they utilize the host cellular pathways to support their own progeny; hence it is expected that viruses would regulate the central glucose metabolism of infected host cells. Human immunodeficiency virus type 1 (HIV-1) causes acquired immune deficiency syndrome, and it uniquely infects both activated CD4+ T cells and terminally differentiated macrophages during the course of HIV-1 pathogenesis. While HIV-1 infection of CD4+ T cells induces G2 arrest and cell death within 2-3 days, HIV-1 infection of macrophages results in longer survival of infected cells and low constitutive viral production, generating viral reservoirs. Our studies show that HIV-1 infection lead to significant changes in the glycolytic pathway of infected cells by altering the enzymatic activity and protein expression of various glycolytic components. The data suggests that the two HIV-1 target cell types exhibit very different metabolic outcomes. During viral replication in monocyte/macrophage lineage cells we observe increase in glycolytic protein expression and the same proteins show no modulation in T-cell lines post viral replication. Similar differential regulation is observed in case of enzymatic activity of glycolytic enzymes as well. We also conducted proteomic studies in collaboration with the proteomics core. HIV-1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Vpr is known to cause cell cycle block in infected cell and bring about cell death. However, macrophages are resistant to cell death and are viral reservoir, even Vpr over expression does not cause apoptosis in these cell types. The goal of the study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. We observed that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. We then focused on infected monocyte macrophages to identify if glycolytic components such as HK and G6PD were regulated by HIV-1 infection/replication. We report that Hexokinase-1 (HK-1) enzyme expression increases post infection of PBMCs where as the enzymatic activity of HK decreases. Similar effect is seen with HIV-1 replication in latently infected monocyte cell lines U1. The G6PD enzyme activity and expression both increases in infected PBMCs and in U1 cells post induction of viral replication with PMA. We also found that HK-1 translocate to the mitochondria of U1 cells post induction of HIV-1. It is known that the product of HK activity, Glucose 6-phosphate (G6P) releases HKI from the outer leaflet of mitochondria. Hence we conclude that the viral infection decreases HK activity to have less G6P produced in cell and increases G6PD enzyme activity ensuring the remaining G6P is quickly used up, supporting the adherence of outer mitochondrial membrane bound HK1. This sequence of cellular events ensures longer survival of infected cells supporting the viral progeny to propagate in the cell. We further show that suppressing the Pentose phosphate pathway (PPP) by blocking G6PD activity is not only detrimental to the survival of the infected cells it also suppresses viral replication and promoter level transactivation of the viral LTR. Next we sought to identify if glycolytic enzyme PKM2, that is also known to play a nonmetabolic dual role as a protein kinase regulating gene transcription has any effect on the transcription of HIV-LTR. Our study demonstrates upregulation of pyruvate kinase isoform M2 (PKM2) expression in whole cell extracts and nuclear extracts of HIV-1JRFL infected PBMCs and during reactivation of HIV-1 in chronically infected U1 cells. We then focused on understanding the potential role of PKM2 on HIV-1 LTR transactivation. Our studies demonstrate that over expression of PKM2 leads to transactivation of the HIV-1 LTR reporter construct. Using various deletions constructs of HIV-1 LTR, we mapped the region spanning between -120 bp to -80 bp to be essential for PKM2 mediated transactivation. This region contains the NFKB DNA binding site and mutation of NFKB binding site attenuated PKM2 mediated transactivation of HIV-LTR. Chromatin immune-precipitation (ChIP) analysis confirmed interaction of PKM2 with HIV-1 LTR. Our studies suggest that PKM2 is a transcriptional co-activator of HIV-1 LTR. Hence it opens up another possible target to curb HIV-1 replication at transcriptional level. This study sheds light on the regulation of glycolytic pathway of host cells by HIV-1 infection and its consequences for the virus, opening up new avenues to target viral replication and identify glycolytic markers of HIV-1 pathogenesis. / Biology

A psoríase é uma doença inflamatória crônica com uma elevada incidência, que afeta a pele. A patogênese da psoríase caracteriza-se pela participação de inúmeras células, incluindo os queratinócitos que são as principais células efetoras da citocina IL-17, críticas para a doença, que produzidas pelas células T. Evidências crescentes sugerem o importante papel da piruvato quinase M2 (PKM2) na regulação da resposta inflamatória, mas o mecanismo subjacente permanece obscuro. Nesse sentido, no presente estudo investigamos o papel da PKM2 no desenvolvimento da psoríase. Observamos o aumento de PKM2 em biópsia humana, em modelo de psoríase induzida por imiquimode e em modelos espontâneos K14-IL-17Aind e DC-IL-17Aind. Em adição, esse aumento observado na enzima foi predominante nos queratinócitos e isso foi associado a marcadores de ativação de queratinócitos. Utilizando o inibidor de PKM2, Shikonin (SKN), como abordagem farmacológica, observamos que o tratamento com esse composto foi capaz de reverter a psoríase experimental e a reduzir marcadores associados a doença como: K17, LCN2, TNF-?, KC, S100A8, S100A9, IL-6 e IL-17A. Associado a isso, observamos a redução na frequência de células T (?? e ??) produtoras de IL-17 e do número de neutrófilos na pele em modelo de imiquimode após inibição da PKM2. O SKN, também, reduziu o número de neutrófilos no modelo DC-IL-17Aind. Em nosso próximo passo, observamos que queratinócitos HACAT estimulados com IL-17A apresentou um aumento da expressão de PKM2 e que a sua inibição foi associada a redução da ativação de queratinócitos e de mediadores inflamatórios como a IL-8. Além disso, a deleção da PKM2, utilizando a tecnologia CRISPR/Cas9, reduziu a expressão do receptor de IL-17. Por fim, o desenvolvimento da psoríase por imiquimode foi atenuada em animais deficientes para PKM2 em queratinócitos (K14-PKM2fl/+), no qual foi observado a redução de neutrófilos na pele e, além disso, evidenciamos a redução da expressão de IL-17A nesses animais. O conjunto de resultados apresentados nesse trabalho demonstram que a PKM2 apresenta um papel crítico no desenvolvimento da psoríase e que a ativação do receptor de IL-17 promove um aumento da PKM2 em queratinócitos e esta contribui para ativação de mediadores que é responsável diretamente para o desenvolvimento da psoríase. Esses resultados, ainda, sugerem a PKM2 como um biomarcador para diagnóstico da psoríase e consequentemente, um potencial alvo terapêutico para tratamento dessa doença e outras doenças inflamatórias. / Psoriasis is a chronic inflammatory skin disease with high incidence in the global population. The pathogenesis of psoriasis is characterized by involvement of many cells, including keratinocytes that are targets for IL-17-producing T cells. Evidences suggests a critical role of pyruvate kinase M2 (PKM2) in inflammatory response, but the underlying mechanism remains unclear. In this context, here we investigated the role of PKM2 in the development of psoriasis. We observed overexpression of PKM2 in psoriatic human skin, imiquimod-induced psoriasis and spontaneous K14-IL-17Aind and DC-IL-17Aind models. In addition, the overexpression of this enzyme was observed in keratinocytes associated with keratinocytes activation markers. Using the PKM2 inhibitor, Shikonin (SKN), as a pharmacological approach, we observed that the treatment with this compound was able to reduce experimental psoriasis and disease-associated markers such as K17, LCN2, TNF-?, KC, S100A8, S100A9, IL-6 and IL-17A. Moreover, we observed reduction of frequency of IL-17-producing T cells (?? and ??) and the number of neutrophils in the skin after imiquimod application plus inhibition of PKM2. SKN, also, reduced the number of neutrophils in the DC-IL-17Aind model. In our next step, we observed overexpression of PKM2 in human keratinocytes HACAT stimulated with IL-17A and that its inhibition was associated with less keratinocytes activation and inflammatory mediators such as IL-8. In addition, deletion of PKM2, using CRISPR/Cas9 technology, reduced IL-17 receptor expression. Finally, the development of imiquimod-induced psoriasis was attenuated in PKM2-deficient mice in keratinocytes (K14-PKM2f/+), with reduction in the number of neutrophils in the skin. In addition, we evidenced the reduction of IL-17A expression these animals. Taken together, these results demonstrate that PKM2 plays a critical role in the development of psoriasis and that IL-17 receptor activation promotes an increase of PKM2 in keratinocytes and this contributes to the release of mediators that is directly responsible for development of psoriasis. These results, suggest PKM2 as a biomarker for the diagnosis of psoriasis and consequently a potential therapeutic target for the treatment of this disease and other inflammatory diseases.

No description available.

Schweinfurthins are a family of natural products with significant anti-cancer activities. They were originally identified in the National Cancer Institute (NCI) human 60 cancer cell line screening. The growth inhibition profile of schweinfurthins is distinct from other clinically used anti-cancer agents, indicating that they have a novel mechanism of action or have a previously unrecognized protein target. Previous studies showed that schweinfurthins affect multiple cellular processes in cancer cells. For example, schweinfurthins can alter cytoskeleton organization, induce ER stress and apoptosis, and inhibit the mevalonate pathway. The mevalonate pathway is responsible for the production of isoprenoids and cholesterol, which have been shown to play regulatory roles in the Hedgehog (Hh) signaling pathway. In this study, we found that the Hh signaling pathway in NIH-3T3 and SF-295 cells was inhibited by schweinfurthins. The supplementation of mevalonate and cholesterol partially restored Hh signaling, indicating that schweinfurthins inhibit Hh signaling partially by down-regulating the products from the mevalonate pathway. Interestingly, schweinfurthins in combination with cyclopamine, an inhibitor of the Hh singaling pathway, synergistically decreased cell viability. In order to better understand the underlying mechanism of the anti-cancer action of schweinfurthins, we attempted to identify the protein target of schweifnurthins. Affinity chromatography was performed to pull down the protein target. We found that schweinfurhtins bound to the M2 isoform of pyruvate kinase (PKM2) and inhibit its pyruvate kinase activity. Knockdown of PKM2 by siRNA increased the sensitivity of SF-295 cells to schweinfurthins. The inhibition of PKM2 by schweinfurthins led to a reduction in the rate of glycolysis in cancer cells. Fructose 1,6-bisphosphate (FBP), an activator of PKM2, could alleviate schweinfurthin-mediated inhibition on PKM2 and glycolysis. Notably, FBP could also partially reverse the reduction of cell viability in the presence of schweinfurthins. Taken together, these studies revealed the mechanism by which schweinfurthins inhibit Hh signaling. In addition, we uncovered PKM2 as a schwienfurthin target and highlighted the importance of glycolysis suppression as a mechanism of the anti-cancer action of schweinfurthins.

Cancer cells take more glucose to provide energy and phosphoryl intermediates for cancer progression. Meanwhile, energy-provider function of mitochondria in cancer cells is disrupted. This phenomenon is so-called Warburg effect, which is discovered over eighty years ago. The detail mechanisms for Warburg effect are not well defined. How glycolytic enzymes contribute to cancer progression is not well known. PKM2 is a glycolytic enzyme dominantly localized in the cytosol, catalyzing the production of ATP from PEP. In this study, we discovered that there were more nuclear PKM2 expressed in highly proliferative cancer cells. The nuclear PKM2 levels are correlated with cell proliferation rates. According to our microarry analyses, MEK5 gene was upregulated in PKM2 overexpression cells. Our studies showed that PKM2 regulated MEK5 gene transcription to promote cell proliferation. Moreover, nuclear PKM2 phosphorylated Stat3 at Y705 site using PEP as a phosphoryl group donor to regulate MEK5 gene transcription. Our study also showed that double phosphorylated p68 RNA helicase at Y593/595 interacted with PKM2 at its FBP binding site. Under the stimulation of growth factors, p68 interacted with PKM2 to promote the conversion from tetrameraic to dimeric form so as to regulate its protein kinase activity. Overexpression PKM2 in less aggressive cancer cells induced the formation of multinuclei by regulating Cdc14A gene transcription. Overall, this study presents a step forward in understanding the Warburg effect.

P68 RNA helicase (p68), as a DEAD family protein, is a typical RNA helicase protein. P68 functions in many other biological processes, which include the regulations of the gene transcription, cell proliferation and cell differentiation. In our group, Y593 phosphorylated p68 was found to have a function in the epithelial mesynchymal transition, which is an important process for cancer metastasis. In the present study, we found that p68 is a nucleocytoplasmic shuttling protein. The protein carries two functional nuclear exporting signal sequences and two nuclear localization signal sequences. Calmodulin, a calcium sensor protein, is well known to play roles in cell migration by regulating the activities of its target proteins at the leading edge. Calmodulin interacts with p68 at the IQ motif of p68. However, the biological function of this interaction is not known. In this study, we found that the p68/calmodulin protein complex functions as a microtubule motor in migrating cells. The shuttling function of p68 along with the motor function of p68/calmodulin causes the leading edge distribution of calmodulin in migrating cells. Disruption the interaction between p68 and calmodulin inhibits cancer cell metastasis in an established mouse model. On the other hand, Y593-Y595 double phosphorylated p68 were found to interact with PKM2, an important tumor isoform of pyruvate kinase. The shuttling function of p68 is reasoned to promote the dimer formation of PKM2 and transport the PKM2 to the cell nucleus. The nuclear PKM2 was found to function as a protein kinase to promote cell proliferation. In specific, the nuclear PKM2 phosphorylates and activates Stat3, an important transcription factor functions in cell proliferation. Overall, p68 is found to have functions in both cell migration and cell proliferation, and these two functions depend on the nucleocytoplasmic shuttling activity and the post-translational modification of p68.