Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles. © 2012 Meng et al

Molecular mechanisms of the interplay between Vpx and SAMHD1

SAMHD1, a dGTP-regulated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase, down-regulates dNTP pools in terminally differentiated and quiescent cells, thereby inhibiting HIV-1 infection at the reverse transcription step. HIV-2 and SIV counteract this restriction via a virion-associated virulence accessory factor, Vpx (Vpr in some SIVs), which loads SAMHD1 onto CRL4-DCAF1 E3 ubiquitin ligase for poly-ubiquitination, programming it for proteasome-dependent degradation. However, the detailed molecular mechanisms of SAMHD1 recruitment to the E3 ligase have not been defined. Further, whether divergent, orthologous Vpr/Vpx proteins, encoded by distinct HIV/SIV strains, bind SAMHD1 in a similar manner, at a molecular level, is not known

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis

Surface-Binding to Cardiolipin Nanodomains Triggers Cytochrome c Pro-apoptotic Peroxidase Activity via Localized Dynamics

The peroxidation of cardiolipins by reactive oxygen species, which is regulated and enhanced by cytochrome c (cyt c), is a critical signaling event in mitochondrial apoptosis. We probe the molecular underpinnings of this mitochondrial death signal through structural and functional studies of horse heart cyt c binding to mixed-lipid membranes containing cardiolipin with mono- and polyunsaturated acyl chains. Lipidomics reveal the selective oxidation of polyunsaturated fatty acid (PUFA) cardiolipin (CL), while multidimensional solid-state NMR probes the structure and dynamics of the membrane and the peripherally bound protein. The hydrophilic milieu at the membrane interface stabilizes a native-like fold, but also leads to localized flexibility at the membrane-interacting protein face. PUFA CL acts as both a preferred substrate and a dynamic regulator by affecting the dynamics of the cyt c N70-I85 Ω loop, which covers the heme cavity

Enhanced weathering in the U.S. Corn Belt delivers carbon removal with agronomic benefits

Enhanced weathering (EW) with crushed basalt on farmlands is a promising scalable atmospheric carbon dioxide removal strategy that urgently requires performance assessment with commercial farming practices. Our large-scale replicated EW field trial in the heart of the U.S. Corn Belt shows cumulative time-integrated carbon sequestration of 15.4 +/- 4.1 t CO2 ha-1 over four years, with additional emissions mitigation of ~0.1 - 0.4 t CO2,e ha-1 yr-1 for soil nitrous oxide, a potent long-lived greenhouse gas. Maize and soybean yields increased 12-16% with EW following improved soil fertility, decreased soil acidification, and upregulation of root nutrient transport genes. Our findings suggest that widespread adoption of EW across farming sectors has the potential to contribute significantly to net-zero greenhouse gas emissions goals and global food and soil security

Análise da atividade e gasto fisiológico dos catadores de resíduos sólidos: a percepção da terapia ocupacional

The world’s transformation in the market has been causing changes in the working class. As those changes have come, the market calls for a high capacity from the workers. Due to that, the unemployment rate has increased and induced a new type of class worker: the Recyclable Material Collectors. In this job, the role of the collector is to return the materials to their commodity chain, and to accomplish this, two methods of collecting are used: one with a truck and the other with a manual collector car. This is one type of occupation which has an extenuating work time, where the worker is exposed to a high ergonomic risk. This study, which was labored from a quantitative and qualitative approach to exploratory method and field research, is intended to compare which collecting technique can be less weariness to the worker. To the purpose of comparison of the physiological work weight, the participants used the Polar FT7 frequency counter, which generated, in graphic form, the performance of the collector in both methods. According to the values presented, was concluded that three of the four participants had a higher physiological weariness by using the manual car collecting approach, concluding that this method is the one that brings most effort to the worker. Considering the results obtained in this research, it was possible to observe that the use of the manual car causes a higher damage to the health of the recyclable material collector.A transformação no mundo do trabalho tem causado mudanças no perfil da classe trabalhadora. Cada vez mais, o mercado de trabalho requer exigências e, como decorrência disso, a taxa de desemprego aumentou e fez surgir uma nova profissão, a de Catadores de Materiais Recicláveis. A função do catador é a de devolver os materiais à sua cadeia produtiva e, para isso, existem dois métodos de coleta: com o caminhão e com o carro manual de coleta. Essa é uma profissão que possui jornadas de trabalho extenuantes, nas quais o trabalhador está exposto a riscos ergonômicos. Esse estudo foi elaborado a partir de uma abordagem quantitativa e qualitativa de método explorátorio e pesquisa de campo; pretende comparar qual método de coleta causa menos desgaste ao trabalhador. Para fins de comparação da carga fisiológica do trabalho, os participantes utilizaram o frequencímetro Polar FT7 que gerou, em forma de gráfico, o desempenho do catador em ambos os métodos de coleta. De acordo com os valores expostos, avaliou-se que, dos 4 participantes, 3 apresentaram um maior desgaste fisiológico no método de coleta com o carro manual, sendo esta a forma pela qual o trabalhador realiza mais esforços. Considerando os resultados obtidos nessa pesquisa, foi possível observar que o uso do carro manual de coleta causa um maior prejuízo à saúde do catador de materiais recicláveis

The impact of Lesson Study on teacher attitude and beliefs as a form of professional development

This qualitative research analyzed a series of three interviews from each of six purposefully selected mathematics teachers who participated in a Lesson Study project, under the auspices of Middlesex County College, in New Jersey. Additionally, direct observations of professional practices and researcher reflections were factored into the analyses. The three cohorts were two teachers from the elementary grades, middles-level grades, and high school grades, and they worked together for a period of four-months; engaging in professional collaboration, self-reflection, sharing of content knowledge, crafting specific lessons around self-selected concepts, peer observation, and critically discussing how to further enhance student learning. The conclusions were that: Lesson Study allowed for creating a strikingly positive experience in professional development, with all participants citing the benefits to being involved with the activities of topic selection, location selection for study, and freedom to engage in critical commentary. Also it was determined that Lesson Study would be a desirable and sustainable means for meaningful professional development, but there was the caveat of needing to convince school administrators of its value when the cost factor was recognized. Finally it was stated that despite the inherent cost for implementing Lesson Study, to not consider it as a viable means for furthering teachers\u27 professional development, especially mathematics teacher, was imprudent and fiscal irresponsible