Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets

Background: Dual-specificity phosphatase-5 (DUSP5) plays a central role in vascular development and disease. We present a p-nitrophenol phosphate (pNPP) based enzymatic assay to screen for inhibitors of the phosphatase domain of DUSP5. Methods: pNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds the DUSP5 phosphatase domain with a Km of 7.6 ± 0.4 mM. Docking followed by inhibitor verification using the pNPP assay identified a series of polysulfonated aromatic inhibitors that occupy the DUSP5 active site in the region that is likely occupied by the dual-phosphorylated ERK2 substrate tripeptide (pThr-Glu-pTyr). Secondary assays were performed with full length DUSP5 with ERK2 as substrate. Results: The most potent inhibitor has a naphthalene trisulfonate (NTS) core. A search for similar compounds in a drug database identified suramin, a dimerized form of NTS. While suramin appears to be a potent and competitive inhibitor (25 ± 5 μM), binding to the DUSP5 phosphatase domain more tightly than the monomeric ligands of which it is comprised, it also aggregates. Further ligand-based screening, based on a pharmacophore derived from the 7 Å separation of sulfonates on inhibitors and on sulfates present in the DUSP5 crystal structure, identified a disulfonated and phenolic naphthalene inhibitor (CSD3 _2320) with IC50 of 33 μM that is similar to NTS and does not aggregate. Conclusions: The new DUSP5 inhibitors we identify in this study typically have sulfonates 7 Å apart, likely positioning them where the two phosphates of the substrate peptide (pThr-Glu-pTyr) bind, with one inhibitor also positioning a phenolic hydroxyl where the water nucleophile may reside. Polysulfonated aromatic compounds do not commonly appear in drugs and have a tendency to aggregate. One FDA-approved polysulfonated drug, suramin, inhibits DUSP5 and also aggregates. Docking and modeling studies presented herein identify polysulfonated aromatic inhibitors that do not aggregate, and provide insights to guide future design of mimics of the dual-phosphate loops of the ERK substrates for DUSPs. Keywords: DUSP5, Phosphatase, Drug discovery, Docking, Suramin, Vascular anomalie

Hanging Out, Messing Around, and Geeking Out

The tenth-anniversary edition of a foundational text in digital media and learning, examining new media practices that range from podcasting to online romantic breakups. Hanging Out, Messing Around, and Geeking Out, first published in 2009, has become a foundational text in the field of digital media and learning. Reporting on an ambitious three-year ethnographic investigation into how young people live and learn with new media in varied settings—at home, in after-school programs, and in online spaces—it presents a flexible and useful framework for understanding the ways that young people engage with and through online platforms: hanging out, messing around, and geeking out, otherwise known as HOMAGO. Integrating twenty-three case studies—which include Harry Potter podcasting, video-game playing, music sharing, and online romantic breakups—in a unique collaborative authorship style, Hanging Out, Messing Around, and Geeking Out combines in-depth descriptions of specific group dynamics with conceptual analysis. Since its original publication, digital learning labs in libraries and museums around the country have been designed around the HOMAGO mode and educators have created HOMAGO guidebooks and toolkits. This tenth-anniversary edition features a new introduction by Mizuko Ito and Heather Horst that discusses how digital youth culture evolved in the intervening decade, and looks at how HOMAGO has been put into practice. This book was written as a collaborative effort by members of the Digital Youth Project, a three-year research effort funded by the John D. and Catherine T. MacArthur Foundation and conducted at the University of California, Berkeley, and the University of Southern California

Mechanism of human PINK1 activation at the TOM complex in a reconstituted system

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson's disease (PD). Stabilization of PINK1 at the translocase of outer membrane (TOM) complex of damaged mitochondria is critical for its activation. The mechanism of how PINK1 is activated in the TOM complex is unclear. Here, we report that co-expression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit toward PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modeling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These findings will aid in the development of small-molecule activators of PINK1 as a therapeutic strategy for PD.</p

Mechanism of human PINK1 activation at the TOM complex by reconstitution

Loss of function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of earlyonset Parkinson’s disease (PD). Stabilisation of PINK1 at the Translocase of Outer Membrane (TOM) complex of damaged mitochondria is a critical step for its activation. To date the mechanism of how PINK1 is activated in the TOM complex is unclear. Herein we report coexpression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit towards PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modelling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These molecular findings will aid in the development of small molecule activators of PINK1 as a therapeutic strategy for PD

Mechanism of human PINK1 activation at the TOM complex by reconstitution

Loss of function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of earlyonset Parkinson’s disease (PD). Stabilisation of PINK1 at the Translocase of Outer Membrane (TOM) complex of damaged mitochondria is a critical step for its activation. To date the mechanism of how PINK1 is activated in the TOM complex is unclear. Herein we report coexpression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit towards PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modelling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These molecular findings will aid in the development of small molecule activators of PINK1 as a therapeutic strategy for PD

Mechanism of human PINK1 activation at the TOM complex in a reconstituted system

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson's disease (PD). Stabilization of PINK1 at the translocase of outer membrane (TOM) complex of damaged mitochondria is critical for its activation. The mechanism of how PINK1 is activated in the TOM complex is unclear. Here, we report that co-expression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit toward PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modeling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These findings will aid in the development of small-molecule activators of PINK1 as a therapeutic strategy for PD.</p

Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle

Assembly and structure of Lys³³-linked polyubiquitin reveals distinct conformations

Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys(33)-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys(33) chains by combining the HECT (homologous to the E6–AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys(33)- and Lys(11)-linked Ub chains. Intriguingly, the crystal structure of Lys(33)-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys(11)-linked diUb. In contrast, crystallographic analysis of Lys(33)-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys(33)-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys(33)-linked polyUb that will allow further characterization of this atypical chain type

The Oxytocin Receptor (OXTR) Contributes to Prosocial Fund Allocations in the Dictator Game and the Social Value Orientations Task

Background: Economic games observe social decision making in the laboratory that involves real money payoffs. Previously we have shown that allocation of funds in the Dictator Game (DG), a paradigm that illustrates costly altruistic behavior, is partially determined by promoter-region repeat region variants in the arginine vasopressin 1a receptor gene (AVPR1a). In the current investigation, the gene encoding the related oxytocin receptor (OXTR) was tested for association with the DG and a related paradigm, the Social Values Orientation (SVO) task. Methodology/Principal Findings: Association (101 male and 102 female students) using a robust-family based test between 15 single tagging SNPs (htSNPs) across the OXTR was demonstrated with both the DG and SVO. Three htSNPs across the gene region showed significant association with both of the two games. The most significant association was observed with rs1042778 (p = 0.001). Haplotype analysis also showed significant associations for both DG and SVO. Following permutation test adjustment, significance was observed for 2–5 locus haplotypes (p,0.05). A second sample of 98 female subjects was subsequently and independently recruited to play the dictator game and was genotyped for the three significant SNPs found in the first sample. The rs1042778 SNP was shown to be significant for the second sample as well (p = 0.004, Fisher’s exact test). Conclusions: The demonstration that genetic polymorphisms for the OXTR are associated with human prosocial decisio

A framework for healthcare interventions to address maternal morbidity

© 2018 World Health Organization; licensed by John Wiley & Sons Ltd on behalf of International Federation of Gynecology and Obstetrics. The maternal health agenda is undergoing a paradigm shift from preventing maternal deaths to promoting women's health and wellness. A critical focus of this trajectory includes addressing maternal morbidity and the increasing burden of chronic and noncommunicable diseases (NCD) among pregnant women. The WHO convened the Maternal Morbidity Working Group (MMWG) to improve the scientific basis for defining, measuring, and monitoring maternal morbidity. Based on the MMWG's work, we propose paradigms for conceptualizing maternal health and related interventions, and call for greater integration between maternal health and NCD programs. This integration can be synergistic, given the links between chronic conditions, morbidity in pregnancy, and long-term health. Pregnancy should be viewed as a window of opportunity into the current and future health of women, and offers critical entry points for women who may otherwise not seek or have access to care for chronic conditions. Maternal health services should move beyond the focus on emergency obstetric care, to a broader approach that encompasses preventive and early interventions, and integration with existing services. Health systems need to respond by prioritizing funding for developing integrated health programs, and workforce strengthening. The MMWG's efforts have highlighted the changing landscape of maternal health, and the need to expand the narrow focus of maternal health, moving beyond surviving to thriving