Structure of an Engineered β-Lactamase Maltose Binding Protein Fusion Protein: Insights into Heterotropic Allosteric Regulation

Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn2+ at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn2+ concentration and determined its structure. The structure reveals that the MBP and TEM-1 domains are in close proximity connected via two linkers and a zinc ion bridging both domains. By bridging both TEM-1 and MBP, Zn2+ acts to “twist tie” the linkers thereby partially dislodging a linker between the two domains from its original catalytically productive position in TEM-1. This linker 1 contains residues normally part of the TEM-1 active site including the critical β3 and β4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn2+ site only slightly affected Zn2+ inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn2+ sites. Mutagenesis and molecular modeling of an intact TEM-1 domain near MBP within the RG13 framework indicated a close surface proximity of the two domains with maltose switching being critically dependent on MBP linker anchoring residues and linker length. Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive. Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via steric and/or linker juxtapositioning mechanisms

Identification and manipulation of tumor associated macrophages in human cancers

Evading immune destruction and tumor promoting inflammation are important hallmarks in the development of cancer. Macrophages are present in most human tumors and are often associated with bad prognosis. Tumor associated macrophages come in many functional flavors ranging from what is known as classically activated macrophages (M1) associated with acute inflammation and T-cell immunity to immune suppressive macrophages (M2) associated with the promotion of tumor growth. The role of these functionally different myeloid cells is extensively studied in mice tumor models but dissimilarities in markers and receptors make the direct translation to human cancer difficult. This review focuses on recent reports discriminating the type of infiltrating macrophages in human tumors and the environmental cues present that steer their differentiation. Finally, immunotherapeutic approaches to interfere in this process are discussed

Biochar: pyrogenic carbon for agricultural use: a critical review.

O biocarvão (biomassa carbonizada para uso agrícola) tem sido usado como condicionador do solo em todo o mundo, e essa tecnologia é de especial interesse para o Brasil, uma vez que tanto a ?inspiração?, que veio das Terras Pretas de Índios da Amazônia, como o fato de o Brasil ser o maior produtor mundial de carvão vegetal, com a geração de importante quantidade de resíduos na forma de finos de carvão e diversas biomassas residuais, principalmente da agroindústria, como bagaço de cana, resíduos das indústrias de madeira, papel e celulose, biocombustíveis, lodo de esgoto etc. Na última década, diversos estudos com biocarvão têm sido realizados e atualmente uma vasta literatura e excelentes revisões estão disponíveis. Objetivou-se aqui não fazer uma revisão bibliográfica exaustiva, mas sim uma revisão crítica para apontar alguns destaques na pesquisa sobre biochar. Para isso, foram selecionados alguns temaschave considerados críticos e relevantes e fez-se um ?condensado? da literatura pertinente, mais para orientar as pesquisas e tendências do que um mero olhar para o passad