Unlatched BAX pairs for death.

Self-interacting BAX proteins permeabilize outer mitochondrial membranes to trigger apoptotic cell death. Czabotar et al. present two revealing structures of BAX dimers: one dimer has an activator BH3 helix bound into its canonical cleft, and the other dimer exposes a planar hydrophobic face potentially critical for membrane interactions.

The Dark Side of Estrogen Stops Translation to Induce Apoptosis.

The role of hormones in triggering cell death has been controversial. In this issue of Molecular Cell, Li et al. (2019) have defined a molecular pathway where an unexpected estrogen receptor, phosphodiesterase 3A, allows its partner Schlafen-12 to inhibit survival pathways, ultimately leading to apoptosis.

Co-ideation and co-design in co-creation research: Reflections from the 'Co-Creating Safe Spaces' project.

INTRODUCTION: Numerous frameworks for defining and supporting co-created research exist. The practicalities of designing and conducting co-created research are clearly important, yet the utility of these frameworks and their operationalisation within local contexts and involving a diversity of stakeholders and interests are currently not well-researched. METHODS: Using an instrumental case study approach, we examined the utility of a published systematic framework designed to improve clarity about co-creation as a concept and approach. The framework is explored based on the first two processes that correspond to our own work to date: co-ideation and co-design. RESULTS: Our study showed that diverse stakeholders bring challenges regarding research priorities, methods, language and the distribution of power within co-creation processes. Co-creation activities were incremental, adaptable, responsive and made best use of established relationships, structures and collective leadership to meet the competing demands of funders and human research ethics committees, while ensuring the meaningful participation of multiple stakeholders. CONCLUSION: The findings highlight the iterative, fluid and deeply relational nature of co-created research. Rather than seeking to categorise these processes, we argue that the social relations of research production that provide the structures within which all co-created knowledge is generated are more important drivers of effective knowledge mobilisation and implementation. Thus, close attention to these social relations is needed in co-created research. PATIENT OR PUBLIC CONTRIBUTION: People with lived experience of emotional distress and/or suicidal crisis, including academic researchers, service and peer workers, carers and advocates were involved in the co-ideation and co-design of this research. All authors identify as people with lived experience, from both academic and nonresearch backgrounds.

Crystal structure of NucB, a biofilm-degrading endonuclease.

Bacterial biofilms are a complex architecture of cells that grow on moist interfaces, and are held together by a molecular glue of extracellular proteins, sugars and nucleic acids. Biofilms are particularly problematic in human healthcare as they can coat medical implants and are thus a potential source of disease. The enzymatic dispersal of biofilms is increasingly being developed as a new strategy to treat this problem. Here, we have characterized NucB, a biofilm-dispersing nuclease from a marine strain of Bacillus licheniformis, and present its crystal structure together with the biochemistry and a mutational analysis required to confirm its active site. Taken together, these data support the categorization of NucB into a unique subfamily of the ßßα metal-dependent non-specific endonucleases. Understanding the structure and function of NucB will facilitate its future development into an anti-biofilm therapeutic agent.

KCTD7 deficiency defines a distinct neurodegenerative disorder with a conserved autophagy-lysosome defect.

OBJECTIVE: Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function. METHODS: Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples. RESULTS: Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology. INTERPRETATION: Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774-788.

Mechanistic insight into the reaction catalysed by bacterial type II dehydroquinases.

DHQ2 (type II dehydroquinase), which is an essential enzyme in Helicobacter pylori and Mycobacterium tuberculosis and does not have any counterpart in humans, is recognized to be an attractive target for the development of new antibacterial agents. Computational and biochemical studies that help understand in atomic detail the catalytic mechanism of these bacterial enzymes are reported in the present paper. A previously unknown key role of certain conserved residues of these enzymes, as well as the structural changes responsible for triggering the release of the product from the active site, were identified. Asp89*/Asp88* from a neighbouring enzyme subunit proved to be the residue responsible for the deprotonation of the essential tyrosine to afford the catalytic tyrosinate, which triggers the enzymatic process. The essentiality of this residue is supported by results from site-directed mutagenesis. For H. pylori DHQ2, this reaction takes place through the assistance of a water molecule, whereas for M. tuberculosis DHQ2, the tyrosine is directly deprotonated by the aspartate residue. The participation of a water molecule in this deprotonation reaction is supported by solvent isotope effects and proton inventory studies. MD simulation studies provide details of the required motions for the catalytic turnover, which provides a complete overview of the catalytic cycle. The product is expelled from the active site by the essential arginine residue and after a large conformational change of a loop containing two conserved arginine residues (Arg109/Arg108 and Arg113/Arg112), which reveals a previously unknown key role for these residues. The present study highlights the key role of the aspartate residue whose blockage could be useful in the rational design of inhibitors and the mechanistic differences between both enzymes.

Mycobacterium tuberculosis shikimate kinase inhibitors: design and simulation studies of the catalytic turnover.

Shikimate kinase (SK) is an essential enzyme in several pathogenic bacteria and does not have any counterpart in human cells, thus making it an attractive target for the development of new antibiotics. The key interactions of the substrate and product binding and the enzyme movements that are essential for catalytic turnover of the Mycobacterium tuberculosis shikimate kinase enzyme (Mt-SK) have been investigated by structural and computational studies. Based on these studies several substrate analogs were designed and assayed. The crystal structure of Mt-SK in complex with ADP and one of the most potent inhibitors has been solved at 2.15 Å. These studies reveal that the fixation of the diaxial conformation of the C4 and C5 hydroxyl groups recognized by the enzyme or the replacement of the C3 hydroxyl group in the natural substrate by an amino group is a promising strategy for inhibition because it causes a dramatic reduction of the flexibility of the LID and shikimic acid binding domains. Molecular dynamics simulation studies showed that the product is expelled from the active site by three arginines (Arg117, Arg136, and Arg58). This finding represents a previously unknown key role of these conserved residues. These studies highlight the key role of the shikimic acid binding domain in the catalysis and provide guidance for future inhibitor designs.

Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase: factors influencing their inhibition potency.

Several 3-alkylaryl mimics of the enol intermediate in the reaction catalyzed by type II dehydroquinase were synthesized to investigate the effect on the inhibition potency of replacing the oxygen atom in the side chain by a carbon atom. The length and the rigidity of the spacer was also studied. The inhibitory properties of the reported compounds against type II dehydroquinase from Mycobacterium tuberculosis and Helicobacter pylori are also reported. The binding modes of these analogs in the active site of both enzymes were studied by molecular docking using GOLD 5.0 and dynamic simulations studies.

Co-creating safe spaces: Study protocol for translational research on innovative alternatives to the emergency department for people experiencing emotional distress and/or suicidal crisis.

INTRODUCTION: Safe spaces are an alternative to emergency departments, which are often unable to provide optimum care for people experiencing emotional distress and/or suicidal crisis. At present, there are several different safe space models being trialled in Australia. However, research examining the effectiveness of safe space models, especially in community settings, is rare. In this paper, we present a protocol for a study in which we will investigate the implementation, effectiveness, and sustainability of safe space models as genuine alternatives for people who might usually present to the emergency department or choose not to access help due to past negative experiences. MATERIAL AND METHODS: We will use a mixed methods, co-designed study design, conducted according to the principles of community-based participatory research to obtain deep insights into the benefits of different safe space models, potential challenges, and facilitators of effective practice. We developed the study plan and evaluation framework using the RE-AIM framework, and this will be used to assess key outcomes related to reach, effectiveness, adoption, implementation, and maintenance. Data collection will comprise quantitative measures on access, use, satisfaction, (cost) effectiveness, distress, and suicidal ideation; and qualitative assessments of service implementation, experience, feasibility, acceptability, community awareness, and the fidelity of the models to service co-design. Data will be collected and analysed concurrently throughout the trial period of the initiatives. DISCUSSION: This study will enable an extensive investigation of safe spaces that will inform local delivery and provide a broader understanding of the key features of safe spaces as acceptable and effective alternatives to hospital-based care for people experiencing emotional distress and/or suicidal crisis. This study will also contribute to a growing body of research on the role and benefits of peer support and provide critical new knowledge on the successes and challenges of service co-design to inform future practice.

Are the parameters of VO2, heart rate and muscle deoxygenation kinetics affected by serial moderate-intensity exercise transitions in a single day?

This study compared the parameter estimates of pulmonary oxygen uptake (VO(2p)), heart rate (HR) and muscle deoxygenation (Δ[HHb]) kinetics when several moderate-intensity exercise transitions (MODs) were performed during a single visit versus several MODs performed during separate visits. Nine subjects (24 ± 5 years, mean ± SD) each completed two successive cycling MODs on six occasions (1-6A and 1-6B) from 20 W to a work rate corresponding to 80% estimated lactate threshold with 6 min recovery at 20 W. During one visit, subjects completed two series of three MODs (6A-F), separated by 20 min rest. VO(2p) time constants (τVO(2p); 27 ± 10 s, 25 ± 12 s, 25 ± 11 s) were similar (p > 0.05) for MODs 1-6A, 1-6B and 6A-F, respectively. τVO(2p) had reproducibility 95% confidence intervals (CI(95)) of 8.3, 8.2, 4.7, 4.9 and 4.7 s when comparing single (1A vs. 2A), the average of two (1-2A vs. 3-4A), three (1-3A vs. 4-6A), four (1-2AB vs. 3-4AB) and six (1-3AB vs. 4-6AB) MODs, respectively. The effective Δ[HHb] response time (τ'Δ[HHb]) was unaffected across conditions (1-6A: 19 ± 2 s, 1-6B: 19 ± 3 s, 6A-F: 17 ± 4 s) with reproducibility CI(95) of 5.3, 4.5, 3.1, 2.9 and 3.3 s when a single, two, three, four and six MODs were compared, respectively. τHR was reduced in MODs 6A-F compared to 1-6A and 1-6B (23 ± 5 s, 25 ± 5 s, 27 ± 6 s, respectively). This study showed that parameter estimates of VO(2p), HR and Δ[HHb] kinetics are largely unaffected by data collection sequence, and the day-to-day reproducibility of τVO(2p) and τ'Δ[HHb] estimates, as determined by the CI(95), was appreciably improved by averaging of at least three MODs.

Noncanonical functions of BCL-2 proteins in the nervous system.

BCL-2 family proteins form heterodimers or homo-oligomers to inhibit or induce apoptotic cell death, respectively. They often relocalize from the cytoplasm to mitochondria to carry out these functions. The traditional model is that in healthy cells, anti-death family members hold pro-death BCL-2 family members in check. Upon receiving a death stimulus, another set of proteins (BH3-only proteins) inactivate the protective BCL-2 proteins, forcing them to release their pro-death partners that are subsequently triggered to oligomerize and porate the mitochondrial outer membrane leading to cell death. In support of this traditional view, there is a preponderance of supporting evidence derived from the study of events that occur following treatment of cells with a death stimulus. Knockout and mutant mice also exhibit many developmental and treatment-induced phenotypes consistent with this model of antagonism between BCL-2 family proteins. Emphasis is logically placed on those phenotypes that support the model. However, this working model of BCL-2 family interactions has become so engrained that alternative, potentially valid interpretations are sometimes dismissed. Therefore, it is useful to consider the evidence that seems contrary to accepted models. In particular, the analysis of BCL-2 family functions in the nervous system has revealed unexpected outcomes that can serve to further stimulate critical probing of the yet unknown biochemical functions of BCL-2 proteins.

Double agents of cell death: novel emerging functions of apoptotic regulators.

Apoptosis is a highly regulated form of cell death that is required for many homeostatic and pathological processes. Recently, alternative cell death pathways have emerged whose regulation is dependent on proteins with canonical functions in apoptosis. Dysregulation of apoptotic signaling frequently underlies the pathogenesis of many cancers, reinforcing the need to develop therapies that initiate alternative cell death processes. This review outlines the convergence points between apoptosis and other death pathways with the purpose of identifying novel strategies for the treatment of apoptosis-refractory cancers. Apoptosis proteins can play key roles in the initiation, regulation, and execution of nonapoptotic death processes that include necroptosis, autophagy, pyroptosis, mPTP-mediated necrosis, and ferroptosis. Notably, recent evidence illustrates that dying cells can exhibit biochemical and molecular characteristics of more than one different type of regulated cell death. Thus, this review highlights the amazing complexity and interconnectivity of cell death processes and also raises the idea that a top-to-bottom approach to describing cell death mechanisms may be inadequate for fully understanding the means by which cells die.

Overview of BCL-2 Family Proteins and Therapeutic Potentials.

BCL-2 family proteins interact in a network that regulates apoptosis. The BH3 amino acid sequence motif serves to bind together this conglomerate protein family, both literally and figuratively. BH3 motifs are present in antiapoptotic and proapoptotic BCL-2 homologs, and in a separate group of unrelated BH3-only proteins often appended to the BCL-2 family. BH3-containing helices mediate many of their physical interactions to determine cell death versus survival, leading to the development of BH3 mimetics as therapeutics. Here we provide an overview of BCL-2 family interactions, their relevance in health and disease, and the progress toward regulating their interactions therapeutically.

Development of tools to facilitate palliative and supportive care referral for patients with idiopathic pulmonary fibrosis.

OBJECTIVES: Palliative care is underused in non-malignant respiratory diseases, including interstitial lung diseases (ILDs). We investigated current practices around palliative and supportive care and explored the impact of a supportive care decision aid tool. METHODS: This was a single centre study in a UK ILD centre. Retrospective analysis of hospice referrals and patients with idiopathic pulmonary fibrosis (IPF) under the Bristol ILD (BILD) service were used to identify unmet palliative and supportive care needs. Using quality improvement methodology, we explored the impact of a supportive care decision aid on clinician behaviours for patients with ILD. RESULTS: 108 patients with ILD were referred for hospice care between 2010 and 2015, representing 0.15% of all referrals, compared with a population prevalence of IPF of 0.9%. The median interval between referral and death was 124 days.Records were reviewed for 64 deceased and 89 living patients with IPF seen on July-December 2014. The decision aid was prospectively assessed with 73 patients. The deceased patients had greater markers of severity. There were no other differences between the groups.After introduction, the decision aid tool was completed for 49.3% of patients and resulted in significant increases in documented discussion of referral to palliative care (11.2%vs53.6%, p<0.01) and end-of-life discussions (15.7%vs91.8%, p<0.01). Tool completion led to an increase in referral for palliative care (2.7%vs16.7%, p<0.01). CONCLUSION: Palliative care services are underused in ILD and a supportive care decision aid can prompt consideration of palliative and supportive care needs.

Specialist palliative care, psychology, interstitial lung disease (ILD) multidisciplinary team meeting: a novel model to address palliative care needs.

INTRODUCTION: Patients with progressive idiopathic fibrotic interstitial lung disease (ILD), such as those with idiopathic pulmonary fibrosis (IPF), can have an aggressive disease course, with a median survival of only 3-5 years from diagnosis. The palliative care needs of these patients are often unmet. There are calls for new models of care, whereby the patient's usual respiratory clinician remains central to the integration of palliative care principles and practices into their patient's management, but the optimal model of service delivery has yet to be determined. METHODS: We developed a novel, collaborative, multidisciplinary team (MDT) meeting between our palliative care, psychology and ILD teams with the principal aim of integrating specialist care to ensure the needs of persons with ILD, and their caregivers were identified and met by referral to the appropriate service. The objective of this study was to assess the effectiveness of this novel MDT meeting on the assessment of a patient's palliative care needs. RESULTS: Significant increases in advance care planning discussions were observed, in conjunction with increased referrals to community courses and teams, following introduction of this novel MDT. CONCLUSIONS: Our results suggest that our collaborative MDT is an effective platform to address patients' unmet palliative care needs. Further work is required to explore the effect of our model on achieving the preferred place of death and reductions in unplanned hospital admissions.

Functional analysis of the GTPases EngA and YhbZ encoded by Salmonella typhimurium.

The S. typhimurium genome encodes proteins, designated EngA and YhbZ, which have a high sequence identity with the GTPases EngA/Der and ObgE/CgtAE of Escherichia coli. The wild-type activity of the E. coli proteins is essential for normal ribosome maturation and cell viability. In order to characterize the potential involvement of the Salmonella typhimurium EngA and YhbZ proteins in ribosome biology, we used high stringency affinity chromatography experiments to identify strongly binding ribosomal partner proteins. A combination of biochemical and microcalorimetric analysis was then used to characterize these protein:protein interactions and quantify nucleotide binding affinities. These experiments show that YhbZ specifically interacts with the pseudouridine synthase RluD (KD=2 microM and 1:1 stoichiometry), and we show for the first time that EngA can interact with the ribosomal structural protein S7. Thermodynamic analysis shows both EngA and YhbZ bind GDP with a higher affinity than GTP (20-fold difference for EngA and 3.8-fold for YhbZ), and that the two nucleotide binding sites in EngA show a 5.3-fold difference in affinity for GDP. We report a fluorescence assay for nucleotide binding to EngA and YhbZ, which is suitable for identifying inhibitors specific for this ligand-binding site, which would potentially inhibit their biological functions. The interactions of YhbZ with ribosome structural proteins that we identify may demonstrate a previously unreported additional function for this class of GTPase: that of ensuring delivery of rRNA modifying enzymes to the appropriate region of the ribosome.

Characterization of Salmonella typhimurium YegS, a putative lipid kinase homologous to eukaryotic sphingosine and diacylglycerol kinases.

Salmonella typhimurium YegS is a protein conserved in many prokaryotes. Although the function of YegS is not definitively known, it has been annotated as a potential diacylglycerol or sphingosine kinase based on sequence similarity with eukaryotic enzymes of known function. To further characterize YegS, we report its purification, biochemical analysis, crystallization, and structure determination. The crystal structure of YegS reveals a two-domain fold related to bacterial polyphosphate/ATP NAD kinases, comprising a central cleft between an N-terminal alpha/beta domain and a C-terminal two-layer beta-sandwich domain; conserved structural features are consistent with nucleotide binding within the cleft. The N-terminal and C-terminal domains of YegS are however counter-rotated, relative to the polyphosphate/ATP NAD kinase archetype, such that the potential nucleotide binding site is blocked. There are also two Ca2+ binding sites and two hydrophobic clefts, one in each domain of YegS. Analysis of mutagenesis data from eukaryotic homologues of YegS suggest that the N-terminal cleft may bind activating lipids while the C-terminal cleft may bind the lipid substrate. Microcalorimetry experiments showed interaction between recombinant YegS and Mg2+, Ca2+, and Mn2+ ions, with a weaker interaction also observed with polyphosphates and ATP. However, biochemical assays showed that recombinant YegS is endogenously neither an active diacylglycerol nor sphingosine kinase. Thus although the bioinformatics analysis and structure of YegS indicate that many of the ligand recognition determinants for lipid kinase activity are present, the absence of such activity may be due to specificity for a different lipid substrate or the requirement for activation by an, as yet, undetermined mechanism. In this regard the specific interaction of YegS with the periplasmic chaperone OmpH, which we demonstrate from pulldown experiments, may be of significance. Such an interaction suggests that YegS can be translocated to the periplasm and directed to the outer-membrane, an environment that may be required for enzyme activity.

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins.

The morphology of a population of mitochondria is the result of several interacting dynamical phenomena, including fission, fusion, movement, elimination and biogenesis. Each of these phenomena is controlled by underlying molecular machinery, and when defective can cause disease. New understanding of the relationships between form and function of mitochondria in health and disease is beginning to be unraveled on several fronts. Studies in mammals and model organisms have revealed that mitochondrial morphology, dynamics and function appear to be subject to regulation by the same proteins that regulate apoptotic cell death. One protein family that influences mitochondrial dynamics in both healthy and dying cells is the Bcl-2 protein family. Connecting mitochondrial dynamics with life-death pathway forks may arise from the intersection of Bcl-2 family proteins with the proteins and lipids that determine mitochondrial shape and function. Bcl-2 family proteins also have multifaceted influences on cells and mitochondria, including calcium handling, autophagy and energetics, as well as the subcellular localization of mitochondrial organelles to neuronal synapses. The remarkable range of physical or functional interactions by Bcl-2 family proteins is challenging to assimilate into a cohesive understanding. Most of their effects may be distinct from their direct roles in apoptotic cell death and are particularly apparent in the nervous system. Dual roles in mitochondrial dynamics and cell death extend beyond BCL-2 family proteins. In this review, we discuss many processes that govern mitochondrial structure and function in health and disease, and how Bcl-2 family proteins integrate into some of these processes.

Matriptase Induction of Metalloproteinase-Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms.

OBJECTIVE: To assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA. METHODS: Aggrecan release from human OA cartilage explants and human stem cell-derived cartilage discs was evaluated, and cartilage-conditioned media were used for Western blotting. Gene expression was analyzed by real-time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies. RESULTS: The addition of soluble recombinant matriptase promoted a time-dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease-activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase- and aggrecanase-mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low-density lipoprotein receptor-related protein 1 (LRP-1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase-mediated aggrecan cleavage. CONCLUSION: Matriptase potently induces the release of metalloproteinase-generated aggrecan fragments as well as soluble LRP-1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease-modifying therapy in OA.