Phenotyping of cancer-associated somatic mutations in the BCL2 transmembrane domain.

The BCL2 family of proteins controls cell death by modulating the permeabilization of the mitochondrial outer membrane through a fine-tuned equilibrium of interactions among anti- and pro-apoptotic members. The upregulation of anti-apoptotic BCL2 proteins represents an unfavorable prognostic factor in many tumor types due to their ability to shift the equilibrium toward cancer cell survival. Furthermore, cancer-associated somatic mutations in BCL2 genes interfere with the protein interaction network, thereby promoting cell survival. A range of studies have documented how these mutations affect the interactions between the cytosolic domains of BCL2 and evaluate the impact on cell death; however, as the BCL2 transmembrane interaction network remains poorly understood, somatic mutations affecting transmembrane regions have been classified as pathogenic-based solely on prediction algorithms. We comprehensively investigated cancer-associated somatic mutations affecting the transmembrane domain of BCL2 proteins and elucidated their effect on membrane insertion, hetero-interactions with the pro-apoptotic protein BAX, and modulation of cell death in cancer cells. Our findings reveal how specific mutations disrupt switchable interactions, alter the modulation of apoptosis, and contribute to cancer cell survival. These results provide experimental evidence to distinguish BCL2 transmembrane driver mutations from passenger mutations and provide new insight regarding selecting precision anti-tumor treatments.

Relación predictiva de los índices de adiposidad visceral y adiposidad disfuncional con el riesgo cardiovascular en población laboral / Predictive relationship of visceral adiposity index (VAI) and dysfunctiuonal adiposity index (DAI) with cardiovascular risk in the spanish working population

Objetivo: estimar la capacidad predictiva de los índices de adiposidad visceral (VAI) y disfuncional (DAI) en riesgo cardiovascular (RCV) de población laboral española. Métodos: análisis descriptivo en 418.343 trabajadores españoles de diferentes sectores durante la vigilancia de la salud en sus empresas. Se calculó el VAI y el DAI ajustándose a sus ecuaciones y el RCV mediante Registre Gironí del Cor (REGICOR), Systematic Coronary Risk Evaluation (SCORE) y estudio DORICA. Se estimó fortaleza asociativa mediante curvas de características operativas del receptor (ROC). El programa estadístico fue SPSS 27.0, considerando significación estadística p < 0,05. Resultados: los valores de RCV con los tres métodos son más elevados en hombres (p < 0,0001). El RCV más alto en mujeres se obtiene con REGICOR (1,58%) y en hombres con Score (11,28%). Con los métodos de valoración de RCV utilizados en ambos sexos, los valores medios de VAI y DAI van aumentando según lo hace el RCV. VAI y DAI son estimadores útiles de RCV en mujeres con DORICA AUC (área bajo la curva)-VAI 0,865 (intervalo de confianza [IC] 95%: 0,836-0,894) y AUC-DAI 0,859 (IC 95%: 0,829-0,888). En hombres, solo muestran moderada capacidad predictiva (valores AUC-VAI 0,774 (IC 95%: 0,768-0,780), AUC-DAI 0,762 (IC 95%: 0,756-0,768). La fortaleza asociativa es baja en ambos sexos con Score y REGICOR (AUC < 0,7). Conclusión: VAI y DAI aumentan sus valores medios según aumenta el RCV estimado con REGICOR, SCORE y DORICA. VAI y DAI tienen elevada capacidad predictiva con el RCV estimado con DORICA en mujeres y moderada fortaleza en hombres. (AU)

Aim: to estimate the predictive relationship of visceral adiposity (VAI) and dysfunctional adiposity (DAI) indices with cardiovascular risk (CVR) in the Spanish working population. Methods: descriptive analysis in 418,343 Spanish workers from different sectors during health monitoring in their companies. VAI and DAI were calculated according to their equations and cardiovascular risk was calculated using Regicor, Score and Dorica. Associative strength was estimated using ROC curves. The statistical programme used was SPSS 27.0, considering statistical significance P<0.05. Results: CVR values with the three methods are higher in men (P<0.0001). The highest CVR in women and men is obtained with Regicor (1.58%) and Score (11.28%), respectively. With the CVR evaluation methods used and in both sexes, the mean values of VAI and DAI increase as CVR increases. VAI and DAI are useful estimators of CVR in women with Dorica AUC -VAI 0.865 (95%CI 0.836-0.894) and AUC-DAI 0.859 (95%CI 0.829-0.888). In men they show only a moderate predictive relationship (AUC values for VAI 0.774 [95%CI 0.768-0.780] AUC DAI 0.762 [95%CI 0.756-0.768]). Strength of association is low in both sexes with Score and Regicor (AUC<0.7). Conclusion: VAI and DAI increase their mean values as estimated CVR increases with Regicor, Score and Dorica. VAI and DAI have a high predictive relationship with estimated CVR-Dorica in women and moderate strength in men. (AU)

Targeted Delivery of the Pan-Inflammasome Inhibitor MM01 as an Alternative Approach to Acute Lung Injury Therapy.

Acute lung injury (ALI) is a severe pulmonary disorder responsible for high percentage of mortality and morbidity in intensive care unit patients. Current treatments are ineffective, so the development of efficient and specific therapies is an unmet medical need. The activation of NLPR3 inflammasome during ALI produces the release of proinflammatory factors and pyroptosis, a proinflammatory form of cell death that contributes to lung damage spreading. Herein, it is demonstrated that modulating inflammasome activation through inhibition of ASC oligomerization by the recently described MM01 compound can be an alternative pharmacotherapy against ALI. Besides, the added efficacy of using a drug delivery nanosystem designed to target the inflamed lungs is determined. The MM01 drug is incorporated into mesoporous silica nanoparticles capped with a peptide (TNFR-MM01-MSNs) to target tumor necrosis factor receptor-1 (TNFR-1) to proinflammatory macrophages. The prepared nanoparticles can deliver the cargo in a controlled manner after the preferential uptake by proinflammatory macrophages and exhibit anti-inflammatory activity. Finally, the therapeutic effect of MM01 free or nanoparticulated to inhibit inflammatory response and lung injury is successfully demonstrated in lipopolysaccharide-mouse model of ALI. The results suggest the potential of pan-inflammasome inhibitors as candidates for ALI therapy and the use of nanoparticles for targeted lung delivery.

[Predictive estimation of the Visceral Adiposity Index (VAI) and Dysfunctional Adiposity Index (DAI) for metabolic syndrome in a working population]. / Estimación predictiva en población laboral del Índice de adiposidad visceral (VAI) y adiposidad disfuncional (DAI) en síndrome metabólico.

OBJECTIVE: To evaluate metabolic syndrome using three methods proposed by  recognizedinternational institutions, and the visceral adiposity (VAI) and dysfunctional adiposity (DAI) indices for prediction and prevalence estimation in working populations. METHODS: Cross-sectional study in workers from different Spanish autonomous communities who underwent a health examination between January 2019 and September 2021 at four occupational risk prevention services. Metabolic syndrome was evaluated according to criteria from the National Cholesterol Education Program Adult Treatment Panel III (NCEP/ATP-III), the International Diabetes Federation (IDF) and  the Joint Interim Statement (JIS). VAI and DAI values were calculated using their specific formulas and their predictive capacity was measured using ROC curves. The SPSS 27.0 program was used, with statistical significance level set at p< 0.05. RESULTS: 418 343 workers were included, mostly men (58.8%), average age between 30 and 49 years (58.0%), social class III, mostly manual workers (75.9%) and nonsmokers (66.9%). The prevalence of metabolic syndrome differed  depending on the criteria used, being higher in men with IDF and JIS, and in women with ATPIII. For the three definitions of metabolic syndrome, the values of the area under the curve were > 0.8 (>80%). The highest VAI was obtained with the JIS, and the highest DAI with the ATPIII. The highest confidence index was for ATPIII and JIS. CONCLUSIONS: The VAI and ICD adiposity indices show high predictive capacity in metabolic syndrome with all three criteria used and can be useful for prevention activities in occupational health.

Introducción: Evaluar el síndrome metabólico utilizando tres métodos propuestos por instituciones internacionales de referencia, y los índices de adiposidad visceral (VAI) y adiposidad disfuncional (DAI), en la predicción y estimación de la prevalencia en población laboral. Métodos: Estudio transversal en trabajadores de distintas comunidades autónomas españolas a los que se les realizó un examen de salud entre enero 2019 y septiembre 2021. Se evaluó el síndrome metabólico con criterios del National Cholesterol Education Program Adult Treatment Panel III (NCEP/ATP-III), International Diabetes Federation (IDF) y Joint Interin Statement (JIS). Se calcularon los valores de VAI y DAI mediante sus fórmulas específicas y su capacidad predictiva mediante curvas ROC. Se utilizó el programa SPSS 27.0, considerando significación estadística p< 0,05. Resultados: Se incluyeron 418 343 trabajadores, la mayoría hombres (58,8%), de edad media entre 30 y 49 años (58,0%), clase social III, tipo de trabajo manual (75,9%) y no fumadores (66,9%). La prevalencia de síndrome metabólico muestra diferencias según el criterio utilizado, siendo superior en hombres con IDF y JIS, y en mujeres con ATPIII. Para las tres definiciones de síndrome metabólico, los valores del área bajo la curva fueron > 0,8 (>80%). El VAI más elevado se obtuvo con JIS, y el DAI más alto con ATPIII. El índice de mayor confianza fue para ATPIII y JIS. Conclusiones: Los índices de adiposidad VAI y DAI muestran una elevada capacidad predictiva del síndrome metabólico con los tres criterios utilizados y pueden ser de utilidad preventiva en salud laboral.

Estimación predictiva en población laboral del Índice de adiposidad visceral (VAI) y adiposidad disfuncional (DAI) en síndrome metabólico / Predictive estimation of the Visceral Adiposity Index (VAI) and Dysfunctional Adiposity Index (DAI) for metabolic syndrome in a working population

Introducción: Evaluar el síndrome metabólico utilizando tres métodos propuestos por ins-tituciones internacionales de referencia, y los índices de adiposidad visceral (VAI) y adiposi-dad disfuncional (DAI), en la predicción y estimación de la prevalencia en población laboral.Métodos: Estudio transversal en trabajadores de distintas comunidades autónomas espa-ñolas a los que se les realizó un examen de salud entre enero 2019 y septiembre 2021 por cuatro servicios de prevención de riesgos laborales. Se evaluó el síndrome metabólico con criterios del National Cholesterol Education Program Adult Treatment Panel III (NCEP/ATP-III), International Diabetes Federation (IDF) y Joint Interin Statement (JIS). Se calcularon los valores de VAI y DAI mediante sus fórmulas específicas y su capacidad predictiva mediante curvas ROC. Se utilizó el programa SPSS 27.0, considerando significación estadística p< 0,05.Resultados: Se incluyeron 418 343 trabajadores, la mayoría hombres (58,8%), de edad me-dia entre 30 y 49 años (58,0%), clase social III, tipo de trabajo manual (75,9%) y no fumado-res (66,9%). La prevalencia de síndrome metabólico muestra diferencias según el criterio utilizado, siendo superior en hombres con IDF y JIS, y en mujeres con ATPIII. Para las tres definiciones de síndrome metabólico, los valores del área bajo la curva fueron > 0,8 (>80%). El VAI más elevado se obtuvo con JIS, y el DAI más alto con ATPIII. El índice de mayor con-fianza fue para ATPIII y JIS.Conclusiones: Los índices de adiposidad VAI y DAI muestran una elevada capacidad pre-dictiva del síndrome metabólico con los tres criterios utilizados y pueden ser de utilidad preventiva en salud laboral (AU)

Objective: To evaluate metabolic syndrome using three methods proposed by recog-nizedinternational institutions, and the visceral adiposity (VAI) and dysfunctional adiposity (DAI) indices for prediction and prevalence estimation in working populations.Methods: Cross-sectional study in workers from different Spanish autonomous communi-ties who underwent a health examination between January 2019 and September 2021 at four occupational risk prevention services. Metabolic syndrome was evaluated according to criteria from the National Cholesterol Education Program Adult Treatment Panel III (NCEP/ATP-III), the International Diabetes Federation (IDF) and the Joint Interim Statement (JIS). VAI and DAI values were calculated using their specific formulas and their predictive ca-pacity was measured using ROC curves. The SPSS 27.0 program was used, with statistical significance level set at p< 0.05.Results: 418 343 workers were included, mostly men (58.8%), average age between 30 and 49 years (58.0%), social class III, mostly manual workers (75.9%) and nonsmokers (66.9%). The prevalence of metabolic syndrome differed depending on the criteria used, being high-er in men with IDF and JIS, and in women with ATPIII. For the three definitions of metabolic syndrome, the values of the area under the curve were > 0.8 (>80%). The highest VAI was obtained with the JIS, and the highest DAI with the ATPIII. The highest confidence index was for ATPIII and JIS. Conclusions: The VAI and ICD adiposity indices show high predictive capacity in metabolic syndrome with all three criteria used and can be useful for prevention activities in occupa-tional health (AU)

Nanodevices for the Efficient Codelivery of CRISPR-Cas9 Editing Machinery and an Entrapped Cargo: A Proposal for Dual Anti-Inflammatory Therapy.

In this article, we report one of the few examples of nanoparticles capable of simultaneously delivering CRISPR-Cas9 gene-editing machinery and releasing drugs for one-shot treatments. Considering the complexity of inflammation in diseases, the synergistic effect of nanoparticles for gene-editing/drug therapy is evaluated in an in vitro inflammatory model as proof of concept. Mesoporous silica nanoparticles (MSNs), able to deliver the CRISPR/Cas9 machinery to edit gasdermin D (GSDMD), a key protein involved in inflammatory cell death, and the anti-inflammatory drug VX-765 (GSDMD45CRISPR-VX-MSNs), were prepared. Nanoparticles allow high cargo loading and CRISPR-Cas9 plasmid protection and, thus, achieve the controlled codelivery of CRISPR-Cas9 and the drug in cells. Nanoparticles exhibit GSDMD gene editing by downregulating inflammatory cell death and achieving a combined effect on decreasing the inflammatory response by the codelivery of VX-765. Taken together, our results show the potential of MSNs as a versatile platform by allowing multiple combinations for gene editing and drug therapy to prepare advanced nanodevices to meet possible biomedical needs.

Identification of NLRP3PYD Homo-Oligomerization Inhibitors with Anti-Inflammatory Activity.

Inflammasomes are multiprotein complexes that represent critical elements of the inflammatory response. The dysregulation of the best-characterized complex, the NLRP3 inflammasome, has been linked to the pathogenesis of diseases such as multiple sclerosis, type 2 diabetes mellitus, Alzheimer's disease, and cancer. While there exist molecular inhibitors specific for the various components of inflammasome complexes, no currently reported inhibitors specifically target NLRP3PYD homo-oligomerization. In the present study, we describe the identification of QM380 and QM381 as NLRP3PYD homo-oligomerization inhibitors after screening small molecules from the MyriaScreen library using a split-luciferase complementation assay. Our results demonstrate that these NLRP3PYD inhibitors interfere with ASC speck formation, inhibit pro-inflammatory cytokine IL1-ß release, and decrease pyroptotic cell death. We employed spectroscopic techniques and computational docking analyses with QM380 and QM381 and the PYD domain to confirm the experimental results and predict possible mechanisms underlying the inhibition of NLRP3PYD homo-interactions.

Understanding MCL1: from cellular function and regulation to pharmacological inhibition.

Myeloid cell leukemia-1 (MCL1), an antiapoptotic member of the BCL2 family characterized by a short half-life, functions as a rapid sensor that regulates cell death and other relevant processes that include cell cycle progression and mitochondrial homeostasis. In cancer, MCL1 overexpression contributes to cell survival and resistance to diverse chemotherapeutic agents; for this reason, several MCL1 inhibitors are currently under preclinical and clinical development for cancer treatment. However, the nonapoptotic functions of MCL1 may influence their therapeutic potential. Overall, the complexity of MCL1 regulation and function represent challenges to the clinical application of MCL1 inhibitors. We now summarize the current knowledge regarding MCL1 structure, regulation, and function that could impact the clinical success of MCL1 inhibitors.

Identification of an ASC oligomerization inhibitor for the treatment of inflammatory diseases.

The ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) protein is an scaffold component of different inflammasomes, intracellular multiprotein platforms of the innate immune system that are activated in response to pathogens or intracellular damage. The formation of ASC specks, initiated by different inflammasome receptors, promotes the recruitment and activation of procaspase-1, thereby triggering pyroptotic inflammatory cell death and pro-inflammatory cytokine release. Here we describe MM01 as the first-in-class small-molecule inhibitor of ASC that interferes with ASC speck formation. MM01 inhibition of ASC oligomerization prevents activation of procaspase-1 in vitro and inhibits the activation of different ASC-dependent inflammasomes in cell lines and primary cultures. Furthermore, MM01 inhibits inflammation in vivo in a mouse model of inflammasome-induced peritonitis. Overall, we highlight MM01 as a novel broad-spectrum inflammasome inhibitor for the potential treatment of multifactorial diseases involving the dysregulation of multiple inflammasomes.

Targeted-lung delivery of dexamethasone using gated mesoporous silica nanoparticles. A new therapeutic approach for acute lung injury treatment.

Acute lung injury (ALI) is a critical inflammatory syndrome, characterized by increased diffuse inflammation and severe lung damage, which represents a clinical concern due to the high morbidity and mortality in critical patients. In last years, there has been a need to develop more effective treatments for ALI, and targeted drug delivery to inflamed lungs has become an attractive research field. Here, we present a nanodevice based on mesoporous silica nanoparticles loaded with dexamethasone (a glucocorticoid extensively used for ALI treatment) and capped with a peptide that targets the TNFR1 receptor expressed in pro-inflammatory macrophages (TNFR-Dex-MSNs) and avoids cargo leakage. TNFR-Dex-MSNs nanoparticles are preferentially internalized by pro-inflammatory macrophages, which overexpressed the TNFR1 receptor, with the subsequent cargo release upon the enzymatic hydrolysis of the capping peptide in lysosomes. Moreover, TNFR-Dex-MSNs are able to reduce the levels of TNF-α and IL-1ß cytokines in activated pro-inflammatory M1 macrophages. The anti-inflammatory effect of TNFR-Dex-MSNs is also tested in an in vivo ALI mice model. The administered nanodevice (intravenously by tail vein injection) accumulated in the injured lungs and the controlled dexamethasone release reduces markedly the inflammatory response (TNF-α IL-6 and IL-1ß levels). The attenuation in lung damage, after treatment with TNFR-Dex-MSNs, is also confirmed by histopathological studies. Besides, the targeted-lung dexamethasone delivery results in a decrease of dexamethasone derived side-effects, suggesting that targeted nanoparticles can be used for therapy in ALI and could help to overcome the clinical limitations of current treatments.

BOK-MCL1 transmembrane interactions: a challenging target for cancer therapy.

Myeloid cell leukemia 1 (MCL1) gene amplification occurs in a wide range of human cancers and protein overexpression associates with malignant cell growth and evasion of apoptosis. We recently reported that disrupting the interaction between the transmembrane domains of MCL1 and BCL-2 related ovarian killer (BOK) induces cell death, thereby suggesting a new target site for anti-tumorigenic strategies.

Mcl-1 and Bok transmembrane domains: Unexpected players in the modulation of apoptosis.

The Bcl-2 protein family comprises both pro- and antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family members can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate preferentially to the endoplasmic reticulum (ER), heterooligomerization between the TMDs of Mcl-1 and Bok predominantly takes place at the mitochondrial membrane. Strikingly, the coexpression of Mcl-1 and Bok TMDs produces an increase in ER mitochondrial-associated membranes, suggesting an active role of Mcl-1 in the induced mitochondrial targeting of Bok. Finally, the introduction of Mcl-1 TMD somatic mutations detected in cancer patients alters the TMD interaction pattern to provide the Mcl-1 protein with enhanced antiapoptotic activity, thereby highlighting the clinical relevance of Mcl-1 TMD interactions.

Preclinical antitumor efficacy of senescence-inducing chemotherapy combined with a nanoSenolytic.

The induction of senescence produces a stable cell cycle arrest in cancer cells, thereby inhibiting tumor growth; however, the incomplete immune cell-mediated clearance of senescent cells may favor tumor relapse, limiting the long-term anti-tumorigenic effect of such drugs. A combination of senescence induction and the elimination of senescent cells may, therefore, represent an efficient means to inhibit tumor relapse. In this study, we explored the antitumor efficacy of a combinatory senogenic and targeted senolytic therapy in an immunocompetent orthotopic mouse model of the aggressive triple negative breast cancer subtype. Following palbociclib-induced senogenesis and senolysis by treatment with nano-encapsulated senolytic agent navitoclax, we observed inhibited tumor growth, reduced metastases, and a reduction in the systemic toxicity of navitoclax. We believe that this combination treatment approach may have relevance to other senescence-inducing chemotherapeutic drugs and additional tumor types. SIGNIFICANCE: While the application of senescence inducers represents a successful treatment strategy in breast cancer patients, some patients still relapse, perhaps due to the subsequent accumulation of senescent cells in the body that can promote tumor recurrence. We now demonstrate that a combination treatment of a senescence inducer and a senolytic nanoparticle selectively eliminates senescent cells, delays tumor growth, and reduces metastases in a mouse model of aggressive breast cancer. Collectively, our results support targeted senolysis as a new therapeutic opportunity to improve outcomes in breast cancer patients.

Identification and validation of uterine stimulant methylergometrine as a potential inhibitor of caspase-1 activation.

Inflammasomes are intracellular multiprotein complexes of the innate immune system. Upon an inflammatory insult, such as infection or intracellular damage, a nucleotide-binding oligomerization domain-like receptor (NLR) sensor protein and the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain) are assembled to activate protease procaspase-1. This protease processes pro-IL-1ß and pro-IL-18 cytokines, which are released to induce the inflammatory response. De-regulation of inflammasome contributes to the progression of several diseases, such as Alzheimer's disease, diabetes, cancer, inflammatory and autoimmune disorders. We herein describe the identification of methylergometrine (MEM), a drug currently used as a smooth muscle constrictor during postpartum hemorrhage, as an inhibitor of the inflammasome complex in ASC-mediated procaspase-1 activation screening. MEM inhibits the activation of the nucleotide-binding oligomerization domain-like receptor protein 1 (NLRP1) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasomes in cellular models upon different pro-inflammatory stimuli. Our results suggest that MEM has the potential to reposition in the treatment of inflammatory diseases with the advantages of established safety and clinical data.

Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes.

The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation.

Apaf1 inhibition promotes cell recovery from apoptosis.

The protein apoptotic protease activating factor 1 (Apaf1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. We have developed a vital method that allows fluorescence-activated cell sorting of cells at different stages of the apoptotic pathway and demonstrated that upon pharmacological inhibition of Apaf1, cells recover from doxorubicin- or hypoxia-induced early apoptosis to normal healthy cell. Inhibiting Apaf1 not only prevents procaspase-9 activation but delays massive mitochondrial damage allowing cell recovery.

Apaf-1 inhibitors protect from unwanted cell death in in vivo models of kidney ischemia and chemotherapy induced ototoxicity.

BACKGROUND: Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need. METHODOLOGY AND PRINCIPAL FINDINGS: The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia. CONCLUSIONS: Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases.

Dynamics and dispensability of variant-specific histone H1 Lys-26/Ser-27 and Thr-165 post-translational modifications.

In mammals, the linker histone H1, involved in DNA packaging into chromatin, is represented by a family of variants. H1 tails undergo post-translational modifications (PTMs) that can be detected by mass spectrometry. We developed antibodies to analyze several of these as yet unexplored PTMs including the combination of H1.4 K26 acetylation or trimethylation and S27 phosphorylation. H1.2-T165 phosphorylation was detected at S and G2/M phases of the cell cycle and was dispensable for chromatin binding and cell proliferation; while the H1.4-K26 residue was essential for proper cell cycle progression. We conclude that histone H1 PTMs are dynamic over the cell cycle and that the recognition of modified lysines may be affected by phosphorylation of adjacent residues.

Role of CDK5/cyclin complexes in ischemia-induced death and survival of renal tubular cells.

Ischemia reperfusion processes induce damage in renal tubules and compromise the viability of kidney transplants. Understanding the molecular events responsible for tubule damage and recovery would help to develop new strategies for organ preservation. CDK5 has been traditionally considered a neuronal kinase with dual roles in cell death and survival. Here, we demonstrate that CDK5 and their regulators p35/p25 and cyclin I are also expressed in renal tubular cells. We show that treatment with CDK inhibitors promotes the formation of pro-survival CDK5/cyclin I complexes and enhances cell survival upon an ischemia reperfusion pro-apoptotic insult. These findings support the benefit of treating with CDK inhibitors for renal preservation, assisting renal tubule protection.

Mapping of six somatic linker histone H1 variants in human breast cancer cells uncovers specific features of H1.2.

Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Despite being key structural components of chromatin, it is not known whether the different variants have specific roles in the regulation of nuclear processes or are differentially distributed throughout the genome. Using variant-specific antibodies to H1 and hemagglutinin (HA)-tagged recombinant H1 variants expressed in breast cancer cells, we have investigated the distribution of six H1 variants in promoters and genome-wide. H1 is depleted at promoters depending on its transcriptional status and differs between variants. Notably, H1.2 is less abundant than other variants at the transcription start sites of inactive genes, and promoters enriched in H1.2 are different from those enriched in other variants and tend to be repressed. Additionally, H1.2 is enriched at chromosomal domains characterized by low guanine-cytosine (GC) content and is associated with lamina-associated domains. Meanwhile, other variants are associated with higher GC content, CpG islands and gene-rich domains. For instance, H1.0 and H1X are enriched at gene-rich chromosomes, whereas H1.2 is depleted. In short, histone H1 is not uniformly distributed along the genome and there are differences between variants, H1.2 being the one showing the most specific pattern and strongest correlation with low gene expression.