Neutrophils activate alveolar macrophages by producing caspase-6-mediated cleavage of IL-1 receptor-associated kinase-M

Alveolar macrophages (AMs) are exposed to respirable microbial particles. Similar to phagocytes in the gastrointestinal tract, AMs can suppress inflammation after exposure to nonpathogenic organisms. IL-1R-associated kinase-M (IRAK-M) is one inhibitor of innate immunity, normally suppressing pulmonary inflammation. During pneumonia, polymorphonuclear neutrophils (PMNs) are recruited by chemotactic factors released by AMs to produce an intense inflammation. We report that intact IRAK-M is strongly expressed in resting human AMs but is cleaved in patients with pneumonia via PMN-mediated induction of caspase-6 (CASP-6) activity. PMN contact is necessary and PMN membranes are sufficient for CASP-6 induction in macrophages. PMNs fail to induce TNF-α fully in macrophages expressing CASP-6 cleavage-resistant IRAK-M. Without CASP-6 expression, PMN stimulation fails to cleave IRAK-M, degrade IκBα, or induce TNF-α. CASP-6(-/-) mice subjected to cecal ligation and puncture have impaired TNF-α production in the lung and decreased mortality. LPS did not induce or require CASP-6 activity demonstrating that TLR2/4 signaling is independent from the CASP-6 regulated pathway. These data define a central role for CASP-6 in PMN-driven macrophage activation and identify IRAK-M as an important target for CASP-6. PMNs de-repress AMs via CASP-6-mediated IRAK-M cleavage. This regulatory system will blunt lung inflammation unless PMNs infiltrate the alveolar spaces.     

Proapoptotic histone H1.2 induces CASP-3 and -7 activation by forming a protein complex with CYT c, APAF-1 and CASP-9

Cytochrome c (CYT c) is a protein that employs the caspase recruitment domain (CARD)-containing proteins APAF-1 and CASP-9 to activate effectors CASP-3 and -7. By using affinity labeling techniques and mass spectrometry analysis, we show that histone H1.2 is a regulator of caspases upon UV irradiation. We demonstrated that histone H1.2 forms a protein complex with APAF-1, CASP-9 and CYT c upon UV irradiation. In cell-free systems, we show that histone H1.2 triggers activation of CASP-3 and -7 via APAF-1 and CASP-9. We therefore conclude that upon DNA damage histone H1.2 acts as a positive regulator of apoptosome formation.     

Quantitative biochemical characterization and biotechnological production of caspase modulator,XIAP: Therapeutic implications for apoptosis-associated diseases

Background

Regulating apoptosis is a common and essential therapeutic strategy for cancer and neurodegenerative disorders. Based on basic studies of apoptotic mechanisms, various researches have attempted to overcome the pathogenesis of such diseases by activating or inhibiting apoptosis. Generally, the biochemical characteristics of the target molecules should be evaluated along with understanding of their mechanisms of action during drug development. Among apoptotic regulators, XIAP serves as a potent negative regulator to block apoptosis through the inhibition of caspase (CASP)-9 and -3/7. Although XIAP is an attractive target with such apoptotic-modulating property, biochemical and biophysical studies of XIAP are still challenging.

N–H bond cleavage of ammonia on graphene-like B36 borophene: DFT studies

The prediction of domain/linker residues in protein sequences is a crucial task in the functional classification of proteins, homology-based protein structure prediction, and high-throughput structural genomics. In this work, a novel consensus-based machine-learning technique was applied for residue-level prediction of the domain/linker annotations in protein sequences using ordered/disordered regions along protein chains and a set of physicochemical properties. Six different classifiers—decision tree, Gaussian naïve Bayes, linear discriminant analysis, support vector machine, random forest, and multilayer perceptron—were exhaustively explored for the residue-level prediction of domain/linker regions. The protein sequences from the curated CATH database were used for training and cross-validation experiments. Test results obtained by applying the developed PDP-CON tool to the mutually exclusive, independent proteins of the CASP-8, CASP-9, and CASP-10 databases are reported. An n-star quality consensus approach was used to combine the results yielded by different classifiers. The average PDP-CON accuracy and F-measure values for the CASP targets were found to be 0.86 and 0.91, respectively. The dataset, source code, and all supplementary materials for this work are available at https://cmaterju.org/cmaterbioinfo/ for noncommercial use.

     

Caspase-7 ablation modulates UPR,reprograms TRAF2-JNK apoptosis and protects T17M rhodopsin mice from severe retinal degeneration

The UPR is activated in the mouse retina expressing misfolded T17M rhodopsin (RHO) during autosomal dominant retinitis pigmentosa (ADRP) progression. Therefore, the goal of this study is to validate the UPR-induced caspase-7 as a new therapeutic target that modulates the UPR, reduces the level of apoptosis and protects the ADRP retina from retinal degeneration and light-induced damage. Mice were analyzed using ERG, SD-OCT and histology to determine the role of caspase-7 ablation. The results of these experiments demonstrate the significant preservation of photoreceptors and their function in T17M RHO CASP-7 retinas from P30 to P90 compared with control mice. These mice were also protected from the light-induced decline in the ERG responses and apoptosis. The RNA and protein analyses of T17M RHO+Csp7-siRNA, Tn+Csp7-siRNA 661W cells and T17M RHO CASP-7 retinas revealed that caspase-7 ablation reprograms the UPR and reduces JNK-induced apoptosis. This reduction is believed to occur through the downregulation of the mTOR and Hif1a proteins. In addition, decline in activated PARP1 was detected in T17M RHO CASP-7 retina. Altogether, our findings indicate that the targeting of caspase-7 in T17M RHO mice could be a feasible therapeutic strategy for advanced stages of ADRP.     

Quality of Life amongst Older Brazilians: A Cross-Cultural Validation of the CASP-19 into Brazilian-Portuguese

Introduction

As population ageing becomes a global phenomenon the need to understand the quality of life of older people around the world has become increasingly salient. The CASP-19 is a well established measure of quality of later life. The scale is composed of 19 items which map onto the four domains of control (C), Autonomy (A), Self-Realisation (S) and Pleasure (P). It has already been translated to 12 languages and has been used in a number of national and international studies. However use of the scale outside of Europe has been very limited. The objective of this study was to translate and evaluate the use of the CASP-19 amongst older Brazilians.

Methods

The CASP-19 was translated from English to Portuguese, back-translated and submitted to an analysis of equivalence by a committee of judges. The scale was then administered to a sample of community dwelling older people in Recife, Brazil (n = 87), and tested for psychometric properties. The Control and Pleasure domains exhibited good internal consistency. By removing one item from each of the Autonomy and Self Realisation domains their internal consistency was improved.

Results

The mean age of the sample was 75.6±0.7 years, subjects were mainly female (52.9%), white (52.9%), who lived without a partner (54%), and had a monthly income varying from USD 340.00 to USD 850.00. Translation and cross-cultural adaptation permitted good understanding and applicability of final version. Psychometric analyses revealed that the removal of two items improved the internal consistency of the Autonomy and Pleasure domains. Confirmatory factor analyses suggest that a 16 item, four factor, model best fits the data.

Conclusion

In this small exploratory study the CASP-19 Brazil demonstrated good psychometric properties. It was easy to use for both participants and researchers. Hopefully future studies in Brazil will employ the scale so that more direct cross national comparisons can be made with older people in Europe and the US.     

AS602801 sensitizes glioma cells to temozolomide and vincristine by blocking gap junction communication between glioma cells and astrocytes

Previous studies showed that the chemotherapeutic effect of temozolomide (TMZ) and vincristine (VCR) against glioma might be blunted by the co-culture with astrocytes, and connexin-43 (CX43) was thought to play a vital role in the communication between glioma cells and astrocytes. In this study, we aimed to investigate the combined chemotherapeutic effect of AS602801 and TMZ/ VCR in glioma cells both. Dye transfer assay was used to evaluate the gap junction activity between U251 cells and astrocytes. Western blot and immunohistochemistry were carried out to analyse the expression of p-JNK, CX43 and CASP-3 proteins treated under different conditions. AS602801 significantly suppressed the gap junction activity between U251 cells and astrocytes. The expression of p-JNK and CX43 was remarkably inhibited by AS602801. TMZ/VCR-induced apoptosis of glioma cells was effectively enhanced by AS602801 treatment. Accordingly, the inhibitory role of TMZ/VCR in the expression of p-JNK, CX43 and CASP-3 in glioma cells was notably restored by AS602801. Furthermore, in a glioma cell xenograft, AS602801 showed an apparent capability to enhance TMZ/VCR-induced tumour cell apoptosis through altering the expression of p-JNK, CX43 and CASP-3. The findings of this study demonstrated that the co-culture of glioma cells with astrocytes blunted the tumour killing effect of TMZ and VCR. AS602801 down-regulated CX43 expression by inhibiting JNK. And AS602801 also sensitized glioma cells to TMZ/VCR by blocking the gap junction communication between glioma cells and astrocytes via down-regulating CX43, indicating its potential role as a novel adjuvant chemotherapeutic agent in the treatment of glioma.     

Involvement of caspases in cytotoxic cytokine-mediated oligodendrocyte cell death

Summary Oligodendrocytes are myelin-forming cells in the mammalian central nervous system. About 50% of oligodendrocytes undergo cell death in normal development. In addition, massive oligodendrocyte cell death has been observed in multiple sclerosis. Tumor necrosis factor (TNF) is thought to be one of the mediators responsible for the damage of oligodendrocytes in multiple sclerosis. The addition of TNF- to primary cultures of oligodendrocytes significantly decreased the number of live cells in 72 h. DNA fragmentation was detected in TNF-treated oligodendrocytes at 36 h by TUNEL assay. Chemical inhibitors Ac-YVAD-CHO (a specific inhibitor of caspase-1 [ICE]-like proteases) as well as Ac-DEVDCHO (a specific inhibitor of caspase-3[CPP32]-like proteases) enhanced the survival of oligodendrocytes treated with TNF-, indicating that caspase-1- and the caspase-3-mediated cell-death pathways are activated in TNF-induced oligodendrocyte cell death. Caspase-11 is involved in activation of caspase-1. Oligodendrocytes fromCASP-11-deficient mice are partially resistant to TNF-induced oligodendrocyte cell death. Our results suggest that the inhibition of caspases may be a novel approach to treat multiple sclerosis.     

Enhanced expression of NLRP3 inflammasome components by monocytes of patients with pulmonary paracoccidioidomycosis is associated with smoking and intracellular hypoxemia

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermally dimorphic fungi of the genus Paracoccidioides that affects predominantly 30-60-year-old male rural workers. The main clinical forms of the disease are acute/subacute, chronic (CF); almost all CF patients develop pulmonary fibrosis, and they also exhibit emphysema due to smoke. An important cytokine in this context, IL-1β, different from the others, is produced by an intracellular multimolecular complex called inflammasome that is activated by pathogens and/or host signs of damage. Inflammasome has been recognized for its contribution to chronic inflammatory diseases, from that, we hypothesized that this activation could be involved in paracoccidioidomycosis, contributing to chronic inflammation. While inflammasome activation has been demonstrated in experimental models of Paracoccidioides brasiliensis infection, no information is available in patients, leading us to investigate the participation of NLRP3-inflammasome machinery in CF/PCM patients from a Brazilian endemic area. Our findings showed increased priming in mRNA levels of NLRP3 inflammasome genes by monocytes of PCM patients in vitro than healthy controls. Similar intracellular protein expression of NLRP3, CASP-1, ASC, and IL-1β were also observed in freshly isolated monocytes of PCM patients and smoker controls. Increased expression of NLRP3 and ASC was observed in monocytes from PCM patients under hypoxia in comparison with smoker controls. For the first time, we showed that primed monocytes of CF-PCM patients were associated with enhanced expression of components of NLRP3-inflammasome due to smoke. Also, hypoxemia boosted this machinery. These findings reinforce the systemic low-grade inflammation activation observed in PCM during and after treatment.     

A human CTL recognizes a caspase-8-derived peptide on autologous HLA-B*3503 molecules and two unrelated peptides on allogeneic HLA-B*3501 molecules

A CTL clone that recognizes autologous tumor cells was previously isolated from the blood of a head-and-neck cancer patient. The Ag was identified as peptide FPSDSWCYF presented by autologous HLA-B*3503 molecules. This peptide was encoded by a mutated CASP-8 gene, which is implicated in the triggering of apoptosis. Here, we show that this CTL clone, which expresses a single TCR, also recognizes two unrelated peptides on allogeneic HLA-B*3501 molecules. One peptide, HIPDVITY, is encoded by squalene synthase, and the other one, QFADVIVLF, is encoded by 2-hydroxyphytanoyl-CoA lyase. Both genes are expressed ubiquitously. These antigenic peptides are processed and presented by HLA-B*3501 cells. The two HLA-B35 alleles are closely related. Our results might reinforce the notion that the recognition of allogeneic HLA molecules depends on the presence in their groove of a limited number of peptides processed from ubiquitous proteins.     

USP7 limits CDK1 activity throughout the cell cycle

Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53‐independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.     

Effects of HIV-1 protease on cellular functions and their potential applications in antiretroviral therapy

ABSTRACT: Human Immunodeficiency Virus Type 1 (HIV-1) protease inhibitors (PIs) are the most potent class of drugs in antiretroviral therapies. However, viral drug resistance to PIs could emerge rapidly thus reducing the effectiveness of those drugs. Of note, all current FDA-approved PIs are competitive inhibitors, i.e., inhibitors that compete with substrates for the active enzymatic site. This common inhibitory approach increases the likelihood of developing drug resistant HIV-1 strains that are resistant to many or all current PIs. Hence, new PIs that move away from the current target of the active enzymatic site are needed. Specifically, allosteric inhibitors, inhibitors that block HIV-1 protease active site, should be sought. Another common feature of current PIs is they were all developed based on the structure-based design. Drugs derived from a structure-based strategy may generate target specific and potent inhibitors. However, this type of drug design can only target one site at a time and drugs discovered by this method are often associated with strong side effects such as cellular toxicity, limiting its number of target choices, efficacy, and applicability. In contrast, a cell-based system may provide a useful alternative strategy that can overcome many of the inherited shortcomings associated with structure-based drug designs. For example, allosteric PIs can be sought using a cell-based system without considering the site or mechanism of inhibition. In addition, a cell-based system can eliminate those PIs that have strong cytotoxic effect. Most importantly, a simple, economical, and easy-to-maintained eukaryotic cellular system such as yeast will allow us to search for potential PIs in a large-scaled high throughput screening (HTS) system, thus increasing the chance of success. Based on our many years of experience in using fission yeast as a model system to study HIV-1 Vpr, we propose the use of fission yeast as a possible surrogate system to study the effects of HIV-1 protease on cellular functions and to explore its utility as a HTS system to search for new PIs to battle HIV-1 strains resistant to the current PI drugs.     

Polo and Aurora kinases: lessons derived from chemical biology

During the cell division cycle, mitotic entry, spindle assembly, chromosome segregation, and cytokinesis must all be carefully coordinated to ensure that the two daughter cells inherit all the genetic material required for further growth and development. Central to this coordination are several protein kinases including Aurora A, Aurora B, and the Polo-like kinase, Plk1. A number of small-molecule Aurora and Plk1 inhibitors have been developed because these kinases are seen as attractive anticancer drug targets. These inhibitors are now being widely used as chemical biology tools to understand how these kinases ensure faithful genome transmission.     

The role of immunosuppression of mesenchymal stem cells in tissue repair and tumor growth

Human Immunodeficiency Virus Type 1 (HIV-1) protease inhibitors (PIs) are the most potent class of drugs in antiretroviral therapies. However, viral drug resistance to PIs could emerge rapidly thus reducing the effectiveness of those drugs. Of note, all current FDA-approved PIs are competitive inhibitors, i.e., inhibitors that compete with substrates for the active enzymatic site. This common inhibitory approach increases the likelihood of developing drug resistant HIV-1 strains that are resistant to many or all current PIs. Hence, new PIs that move away from the current target of the active enzymatic site are needed. Specifically, allosteric inhibitors, inhibitors that prohibit PR enzymatic activities through non-competitive binding to PR, should be sought. Another common feature of current PIs is they were all developed based on the structure-based design. Drugs derived from a structure-based strategy may generate target specific and potent inhibitors. However, this type of drug design can only target one site at a time and drugs discovered by this method are often associated with strong side effects such as cellular toxicity, limiting its number of target choices, efficacy, and applicability. In contrast, a cell-based system may provide a useful alternative strategy that can overcome many of the inherited shortcomings associated with structure-based drug designs. For example, allosteric PIs can be sought using a cell-based system without considering the site or mechanism of inhibition. In addition, a cell-based system can eliminate those PIs that have strong cytotoxic effect. Most importantly, a simple, economical, and easy-to-maintained eukaryotic cellular system such as yeast will allow us to search for potential PIs in a large-scaled high throughput screening (HTS) system, thus increasing the chances of success. Based on our many years of experience in using fission yeast as a model system to study HIV-1 Vpr, we propose the use of fission yeast as a possible surrogate system to study the effects of HIV-1 protease on cellular functions and to explore its utility as a HTS system to search for new PIs to battle HIV-1 resistant strains.     

The selective inhibition of thrombin by peptides of boroarginine

Peptides containing alpha-aminoboronic acids with neutral side chains are highly effective reaction intermediate analog inhibitors of the serine proteases leukocyte elastase, pancreatic elastase, and chymotrypsin. A protocol has been developed for the synthesis of peptides containing alpha-aminoboronic acids with a basic, 3-guanidinopropyl side chain (boroArg) to extend the range of these compounds to trypsin-like proteases. Ac-(D)Phe-Pro-boroArg-OH, Boc-(D)Phe-Pro-boroArg-OH, and H-(D)Phe-Pro-boroArg-OH were prepared as inhibitors of thrombin based on earlier observations that it has a high affinity for this sequence. All three boronic acids are highly effective, slow-binding inhibitors of thrombin, inhibiting it with final inhibition constants and association rates of: 41 pM, 5.5 x 10(6) M-1 s-1; 3.6 pM, 9.3 x 10(6) M-1 s-1; less than 1 pM, 8.0 x 10(6) M-1 s-1, respectively. Comparison of their binding at equilibrium to thrombin, plasma kallikrein, factor Xa, plasmin, and two-chain tissue plasminogen activator has shown that all three inhibitors have at least 2 orders of magnitude greater affinity for thrombin, with the exception of the acetyl derivative which has a 40-fold greater affinity for thrombin than kallikrein. The boroarginine peptides are effective in inhibiting the action of thrombin in rabbit plasma against its physiological substrates. Activated partial thromboplastin time was significantly prolonged in vitro by all of the inhibitors at concentrations of 50-200 nM. Prolongations of activated partial thromboplastin time were also observed in rabbits after intravenous (40-80 micrograms/kg or subcutaneous (0.20-2 mg/kg) injections of Ac-(D)Phe-Pro-boroArg-OH. Results indicate that this new class of synthetic thrombin inhibitors may be clinically useful as antithrombotic agents.     

Overexpression, Purification, and Characterization of Human and Bovine Mitochondrial ATPase Inhibitors: Comparison of the Properties of Mammalian and Yeast ATPase Inhibitors

Mitochondrial ATP synthase (F₁F₀-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In this study, we overexpressed and purified human and bovine ATPase inhibitors and their properties were compared with those of a yeast inhibitor. The human and bovine inhibitors inhibited bovine ATPase in a similar way. The yeast inhibitor also inhibited bovine F₁F₀-ATPase, although the activity was about three times lower than the mammalian inhibitors. All three inhibitors inhibited yeast F₁F₀-ATPase in a similar way. The activities of all inhibitors decreased at higher pH, but the magnitude of the decrease was different for each combination of inhibitor and ATPase. The results obtained in this study show that the inhibitory mechanism of the inhibitors was basically shared in yeast and mammals, but that mammalian inhibitors require unique residues, which are lacking in the yeast inhibitor, for their maximum inhibitory activity. Common inhibitory sites of mammalian and yeast inhibitors are suggested.     

The role of auxin transport during inflorescence development in maize (Zea mays, Poaceae)

Axillary meristems play a fundamental role in inflorescence architecture. Maize (Zea mays) inflorescences are highly branched panicles because of the production of multiple types of axillary meristems. We used auxin transport inhibitors to show that auxin transport is required for axillary meristem initiation in the maize inflorescence. The phenotype of plants treated with auxin transport inhibitors is very similar to that of barren inflorescence2 (bif2) and barren stalk1 (ba1) mutants, suggesting that these genes function in the same auxin transport pathway. To dissect this pathway, we performed RNA in situ hybridization on plants treated with auxin transport inhibitors. We determined that bif2 is expressed upstream and that ba1 is expressed downstream of auxin transport, enabling us to integrate the genetic and hormonal control of axillary meristem initiation. In addition, treatment of maize inflorescences with auxin transport inhibitors later in development results in the production of single instead of paired spikelets. Paired spikelets are a key feature of the Andropogoneae, a group of over 1000 grasses that includes maize, sorghum, and sugarcane. Because all other grasses bear spikelets singly, these results implicate auxin transport in the evolution of inflorescence architecture. Furthermore, our results provide insight into mechanisms of inflorescence branching that are relevant to all plants.     

Unexpected small molecules as novel SIRT2 suicide inhibitors

A series of sirtuin inhibitor candidates were assembled based on an intermediate ester (1a) our accidently discovered. After screening and evaluation, several SIRT2 selective inhibitors were identified, which can inhibit all the deacetylation, defatty-acylation and debenzoylation of SIRT2. Among these inhibitors, compound 1e was the best SIRT2 selective inhibitors. The primary study on the inhibitory mechanism indicated that compound 1e may be a suicide inhibitor acting as an irreversible way. Given almost all reported sirtuin inhibitors are non-covalent, sirtuin covalent inhibitors are still need to be developed. These findings will facilitate for further development of SIRT2 selective and suicide inhibitors.