Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase.

Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes

Oxidative footprinting in the study of structure and function of membrane proteins: Current state and perspectives

Membrane proteins, such as receptors, transporters and ion channels, control the vast majority of cellular signalling and metabolite exchange processes and thus are becoming key pharmacological targets. Obtaining structural information by usage of traditional structural biology techniques is limited by the requirements for the protein samples to be highly pure and stable when handled in high concentrations and in nonnative buffer systems, which is often difficult to achieve for membrane targets. Hence, there is a growing requirement for the use of hybrid, integrative approaches to study the dyna mic and functional aspects of membrane proteins in physiologically relevant conditions. In recent years, significant progress has been made in the field of oxidative labelling techniques and in particular the X-ray radiolytic footprinting in combination with mass spectrometry (MS) (XF-MS), which provide residue-specific information on the solvent accessibility of proteins. In combination with both low- and high-resolution data from other structural biology approaches, it is capable of providing valuable insights into dynamics of membrane proteins, which have been difficult to obtain by other structural techniques, proving a highly complementary technique to address structure and function of membrane targets. XF-MS has demonstrated a unique capability for identification of structural waters and conformational changes in proteins at both a high degree of spatial and a high degree of temporal resolution. Here, we provide a perspective on the place of XF-MS among other structural biology methods and showcase some of the latest developments in its usage for studying water-mediated transmembrane (TM) signalling, ion transport and ligand-induced allosteric conformational changes in membrane proteins

Structural Analysis of Gelsolin Using Synchrotron Protein Footprinting

Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron white beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its inactive and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation

Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins

Protein Footprinting: Auxiliary Engine to Power the Structural Biology Revolution

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins are determined regularly. These advances have been driven by over fifteen years of technology advancements, first in macromolecular crystallography, and recently in Cryo-electron microscopy. These structures are allowing detailed questions about functional mechanisms of the structures, and the biology enabled by these structures, to be addressed for the first time. At the same time, mass spectrometry technologies for protein structure analysis, “footprinting” studies, have improved their sensitivity and resolution dramatically and can provide detailed sub-peptide and residue level information for validating structures and interactions or understanding the dynamics of structures in the context of ligand binding or assembly. In this perspective, we review the use of protein footprinting to extend our understanding of macromolecular systems, particularly for systems challenging for analysis by other techniques, such as intrinsically disordered proteins, amyloidogenic proteins, and other proteins/complexes so far recalcitrant to existing methods. We also illustrate how the availability of high-resolution structural information can be a foundation for a suite of hybrid approaches to divine structure-function relationships beyond what individual techniques can deliver

Multiple Glycosaminoglycan-binding Epitopes of Monocyte Chemoattractant Protein-3/CCL7 Enable it to Function as a Non-oligomerizing Chemokine

The interaction of chemokines with glycosaminoglycans (GAGs) facilitates the formation of localized chemokine gradients that provide directional signals for migrating cells. In this study, we set out to understand the structural basis and impact of the differing oligomerization propensities of the chemokines monocyte chemoattractant protein (MCP)-1/CCL2 and MCP- 3/CCL7 on their ability to bind GAGs. These chemokines provide a unique comparison set because CCL2 oligomerizes and oligomerization is required for its full in vivo activity, whereas CCL7 functions as a monomer. To identify the GAG-binding determinants of CCL7, an unbiased hydroxyl radical footprinting approach was employed, followed by a focused mutagenesis study. Compared with the size of the previously defined GAGbinding epitope of CCL2, CCL7 has a larger binding site, consisting of multiple epitopes distributed along its surface. Furthermore, surface plasmon resonance (SPR) studies indicate thatCCL7 is able to bind GAGs with an affinity similar to CCL2 but higher than the non-oligomerizing variant, CCL2(P8A), suggesting that, in contrast to CCL2, the large cluster of GAG-binding residues in CCL7 renders oligomerization unnecessary for high affinity binding. However, the affinity of CCL7 is more sensitive than CCL2 to the density of heparan sulfate on the SPR surfaces; this is likely due to the inability of CCL7 to oligomerize because CCL2(P8A) also binds significantly less tightly to low than high density heparan sulfate surfaces compared with CCL2. Together, the data suggest that CCL7 and CCL2 are non-redundant chemokines and that GAG chain density may provide a mechanism for regulating the accumulation of chemokines on cell surfaces

Structurally Distinct External Solvent-Exposed Domains Drive Replication of Major Human Prions

There is a limited understanding of structural attributes that encode the iatrogenic transmissibility and various phenotypes of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). Here we report the detailed structural differences between major sCJD MM1, MM2, and VV2 prions determined with two complementary synchrotron hydroxyl radical footprinting techniques—mass spectrometry (MS) and conformation dependent immunoassay (CDI) with a panel of Europium-labeled antibodies. Both approaches clearly demonstrate that the phenotypically distant prions differ in a major way with regard to their structural organization, and synchrotron-generated hydroxyl radicals progressively inhibit their seeding potency in a strain and structure-specific manner. Moreover, the seeding rate of sCJD prions is primarily determined by strain-specific structural organization of solvent-exposed external domains of human prion particles that control the seeding activity. Structural characteristics of human prion strains suggest that subtle changes in the organization of surface domains play a critical role as a determinant of human prion infectivity, propagation rate, and targeting of specific brain structures

A arte de endurecer sem perder a ternura: o uso de tecnologias por obstetrizes na atenção ao parto domiciliar planejado

Resumo Introdução: Diante da intensa medicalização do parto, evidencia-se a necessidade de profissionais orientadas pela normalidade desse evento, como parteiras diplomadas, sejam elas enfermeiras obstetras ou obstetrizes. Apesar da reconhecida importância, certas especificidades da atuação dessas profissionais ainda permanecem pouco elucidadas. Objetivo: Buscou-se identificar as ferramentas presentes na valise de parteiras diplomadas formadas pelo curso de Obstetrícia da Universidade de São Paulo. Metodologia: Trata-se de estudo descrito-exploratório, com abordagem qualitativa. Foram realizadas entrevistas semiestruturadas com cinco obstetrizes entre agosto de 2020 e maio de 2021, em ambiente virtual. O material foi analisado por meio de mapas dialógicos. Resultados: Na valise das entrevistadas, identificou-se tecnologias leves, leve-duras e duras. Entre as tecnologias leves encontram-se o afeto, a comunicação (saber falar, saber ouvir) e o vínculo. As tecnologias leve-duras incluem disciplinas básicas, evidências científicas, experiência e intuição. Por fim, as tecnologias duras contemplam instrumentos produzidos pela atividade humana, além do uso da materialidade do corpo. Conclusões: Verificou-se que as obstetrizes recorrem a tecnologias duras à medida que não conseguem atingir o fim desejado com tecnologias leves, embora utilizem os recursos relacionais de maneira transversal a todo o processo. Para empregar tais recursos tecnológicos, as profissionais utilizam saberes muito distintos, sem necessariamente hierarquizá-los

As mulheres como objeto das práticas jurídicas: uma análise do Projeto de Lei 478/2007

Once life itself was included in the operations of power, the female body has been positioned as an object of disciplinary devices and governmentality. The issue of abortion condenses in itself such complex relations of power. Given the problematic correlation between the regulation of the practice and its incidence in Brazil, our aim is to identify the notions of women's rights present in the bill 478/2007, considering the changes proposed by its substitute. Through discursive analysis, we identified three ways of understanding and performing women's rights: women are positioned as objects of legal practices; they are not understood as subjects of law; and, finally, they have their own right to life brought into question. It is evident that the proposition prioritizes the rights of the embryo/fetus while anchored in a specific notion of human nature.Cuando la vida se incluye en las operaciones del poder, el cuerpo feminino se convierte en objeto de disciplinarización y control, de manera que el tema del aborto condensa em sí mismo complejas relaciones de poder. En vista de la relación problemática entre la regulación de la práctica y su incidencia en Brasil, nuestro objetivo es identificar las nociones de derechos de las mujeres presentes en el proyecto de ley 478/2007. A través del análisis discursivo, identificamos tres formas de entender y performar los derechos de las mujeres: las mujeres están posicionadas como objeto de prácticas legales; no son reconocidas como sujetos de derecho; y, finalmente, se les cuestiona su derecho a la vida. En este contexto, es evidente que la propuesta prioriza los derechos del embrión/feto anclada en una noción específica de la naturaleza humana.Quando a vida é incluída nas operações de poder o corpo feminino passa a ser posicionado como objeto de disciplinarização e controle, de modo que a questão do aborto condensa em si relações complexas de poder. Tendo em vista a relação problemática entre a regulamentação da prática e sua incidência no Brasil, nosso objetivo é identificar as noções de direitos das mulheres presentes no Projeto de Lei 478/2007, considerando as mudanças propostas por seu substitutivo. Por meio da análise discursiva, identificamos três modos de entender e performar os direitos femininos: as mulheres são posicionadas como objeto de práticas jurídicas; não são compreendidas como sujeitos de direito; e, por fim, têm seu próprio direito à vida colocado em questão. Nesse contexto, fica evidente que a proposição prioriza os direitos do embrião/feto ancorando-se em uma noção específica de natureza humana

Small Molecule Inhibitors of 15-PGDH Exploit a Physiologic Induced-Fit Closing System

15-prostaglandin dehydrogenase (15-PGDH) is a negative regulator of tissue stem cells that acts via enzymatic activity of oxidizing and degrading PGE2, and related eicosanoids, that support stem cells during tissue repair. Indeed, inhibiting 15-PGDH markedly accelerates tissue repair in multiple organs. Here we have used cryo-electron microscopy to solve the solution structure of native 15-PGDH and of 15-PGDH individually complexed with two distinct chemical inhibitors. These structures identify key 15-PGDH residues that mediate binding to both classes of inhibitors. Moreover, we identify a dynamic 15-PGDH lid domain that closes around the inhibitors, and that is likely fundamental to the physiologic 15-PGDH enzymatic mechanism. We furthermore identify two key residues, F185 and Y217, that act as hinges to regulate lid closing, and which both inhibitors exploit to capture the lid in the closed conformation, thus explaining their sub-nanomolar binding affinities. These findings provide the basis for further development of 15-PGDH targeted drugs as therapeutics for regenerative medicine