A Role for Mammalian Diaphanous-Related Formins in Complement Receptor (CR3)-Mediated Phagocytosis in Macrophages

SummaryMacrophages, dendritic cells, and neutrophils use phagocytosis to capture and clear off invading pathogens. The process is triggered by the interaction of ligands on the pathogens’ surface with specific phagocytic receptors, including immunoglobulin (FcR) and complement C3bi (CR3) receptors (integrin αMβ2, Mac1) [1]. Localized actin-filament assembly that acts as the driving force for particle engulfment is controlled by Rho-family small GTPases [2, 3]. RhoA regulates CR3-mediated phagocytosis through a mechanism that is still unclear [4–6]. Mammalian Diaphanous-related (mDia) formins participate in the generation of a diverse set of actin-remodeling events downstream of RhoA [7], and mDia1 is recruited around fibronectin-coated beads in a RhoA-dependent manner in fibroblasts [8]. Here, we set out to examine whether mDia proteins are involved in CR3-mediated phagocytosis in macrophages. We show that the RhoA effector mDia1 is recruited early during CR3-mediated phagocytosis and colocalizes with polymerized actin in the phagocytic cup. Interfering with mDia activity inhibits CR3-mediated phagocytosis while having no effect on FcR-mediated phagocytosis. These results indicate a new function for mDia proteins in the regulation of actin polymerization during CR3-mediated phagocytosis

Probing the Role of Asn 152 in the Class C β-lactamase AmpC

AmpC, a class C β-lactamase, is a main cause of antibiotic resistance to cephalosporins in many species of bacteria. The current proposed mechanism of action involves an acyl-intermediate, where the enzyme becomes covalently attached to the drug at serine-64, before an activated water hydrolyzes the bond and regenerates the enzyme. Although this mechanism is generally accepted, the exact roles that the other active site residues play in recognition and breakdown of the substrate are not fully understood. Here, we investigate the role of the active site residue asparagine-152 (Asn152) in E. coli AmpC by mutating it to a glycine, serine, or threonine residue and examining the effect that these mutations have on kinetic and structural properties, when acting upon three different β-lactam drugs: cefotaxime, cefoxitin, and oxacillin. We found that the mutations cause 50 to 200 times higher kcat values against cefotaxime and also allow the enzyme to break down oxacillin, which is not hydrolyzed at a detectable rate by wild type AmpC. We obtained the crystal structure of wild type AmpC and AmpC N152G bound to cefotaxime and found a rotation of glutamine-120 and lysine-67 to be the only significant differences in the active site residues as well as a slight conformational change in the drug itself. Uncovering the specific role of Asn152 in the function of AmpC in conjunction with work done to understand the roles of other active site residues will be useful in the development of inhibitors to these enzymes that may help combat antibiotic resistance

Studying Potential Drug Interactions Involved in the Regulation of the Diaphanous-related Formins

Diaphanous-related formins (DRFs) are a conserved family of proteins that are involved in the regulation of cellular shape, motility, and cell division by regulating the structure of the cellular “skeleton” (cytoskeleton); any disruption of this regulation can result in cell death. Normally, the DRFs are kept in an inactive state by the intramolecular binding of two regions of the protein: the Diaphanous Autoregulatory Domain (DAD) and Diaphanous Inhibitory Domain (DID). This binding can be alleviated by various naturally-occurring mechanisms and proper regulation of the activity of these proteins is vital to cell survival because prolonged activation of DRFs can result in cell death. In a recent scan of 10,000 chemical compounds, two compounds (I and II) were identified to bind to the DID region and activate DRFs indefinitely resulting in the killing of breast and colon cancer cells. In this study, we intend to prove that these compounds are actually alleviating DID-DAD binding (the essential step in DRF activation) by directly binding to DID. In addition, the specific location on DID that binds these compounds must be elucidated. We have hypothesized three amino acid residues that contribute to the binding of these compounds, have generated them using site-directed mutagenesis, and have evaluated their ability to bind the compounds using fluorescence anisotropy. Further experiments will test how these DID mutations affect the ability for these compounds to directly bind to DID using isothermal titration calorimetry. By characterizing the DID-compound bound structure, these studies will allow for the design of new compounds that could bind more tightly to DID, thereby resulting in a drug that could efficiently kill cancer cells

Probing the Binding Site in the Antibiotic Resistance Enzyme, AmpC β-lactamase

β-lactam drugs, such as penicillins and cephalosporins, are widely used to treat bacterial infections, but resistance to these drugs is increasingly becoming a problem. One of the main causes of resistance to these β-lactam drugs is the bacterial production of β-lactamase enzymes, such as AmpC. These enzymes are capable of breaking down the drug within their active sites, rendering the antibiotic unable to harm the bacteria. The exact roles that the active site amino acid residues play in the recognition and breakdown of the drug are not fully understood. Here, we investigate the role of the active site residue asparagine-152 (Asn152) in AmpC by mutating it to a glycine, serine, or threonine residue and examining the effect that these mutations have on kinetic and structural properties. Uncovering the specific role of Asn152 in the function of AmpC will be useful in the development of inhibitors to these enzymes in order to combat bacterial resistance

Identification of a Potent Inhibitor for the Extended Spectrum Class C Beta-Lactamase, ADC-7

Resistance to b-lactam antibiotics in the pathogenic bacteria, Acinetobacter baumannii, presents one of the greatest challenges to current antimicrobial chemotherapy. Majority of resistance is due to expression of class C β-lactamase enzymes, known as Acinetobacter-Derived Cephalosporinases (ADCs). The enzyme ADC-7 is a broad-spectrum class C b-lactamase, capable of deactivating multiple types of antibiotics. Boronic acid transition state inhibitors (BATSIs) are compounds that bind covalently and reversibly to class C b-lactamases. Enzyme kinetic studies of one BATSI, designated S02030, demonstrated a greater affinity for binding than a common cephalosporin substrate. After expression and purification of ADC-7, the first known X-ray crystal structure of ADC-7 with inhibitor complex was solved at 2.03 Å resolution. The ADC-7/S02030 complex provides insight into ADC enzyme structure and offers a novel starting point for the structure-based optimization of b-lactamase inhibitors

Probing the role of N152 in the class C beta-lactamase AmpC

AmpC, a class C ²-lactamase, is a main cause of antibiotic resistance to cephalosporins in many species of bacteria. Although a mechanism involving acylation by S64 followed by hydrolytic cleavage has been generally accepted, the exact roles that some active site residues play in recognition and breakdown of the substrate are not fully understood. Here, we investigate the role of the active site residue asparagine-152 (N152) in E. coli AmpC by mutating it to a G, S, or T residue and examining the effect that these mutations have on kinetic and structural properties with four different ²-lactam drugs: cefotaxime, cefoxitin, oxacillin, and a derivative of cephalothin (CENTA). We discovered that although the mutations cause higher Km values with all substrates, they result in 50 to 150 times higher kcat values against cefotaxime. In addition, the N152 mutations provided the enzyme the ability to break down oxacillin, which is not a viable substrate for the wild type AmpC. To probe the mechanism behind the observed kinetics changes, crystal structures were obtained of AmpC WT or N125G in acyl-enzyme complexes with cefotaxime, cefoxitin, and oxacillin. The small structural differences in the active site have been associated with the changes in the Km and kcat kinetic values as a way to uncover the specific role of N152 in the function of AmpC. This work was supported by the Office of Undergraduate Research and Scholarship at GVSU

Probing the role of Asn 152 in the class C ²-lactamase AmpC

AmpC, a class C ²-lactamase, is a main cause of antibiotic resistance to cephalosporins in many species of bacteria. In the hydrolytic cleavage of antibiotics by AmpC, the current proposed mechanism involves an acyl-intermediate, where the enzyme becomes covalently attached to the drug at serine-64, before an activated water molecule hydrolyzes the bond and regenerates the enzyme. Although this mechanism is generally accepted, the exact roles that the other active site residues play in recognition and breakdown of the substrate are not fully understood. Here, we investigated the role of the active site residue asparagine-152 (Asn152) in E. coli AmpC by mutating it to a glycine, serine, or threonine residue and examining the effect that these mutations have on kinetic and structural properties with four different ²-lactam drugs: cefotaxime, cefoxitin, oxacillin, and a derivative of cephalothin (CENTA). We discovered that although the mutations cause higher Km values with all substrates, they result in 50 to 150 times higher kcat values against cefotaxime. In addition, the N152 mutations provided the enzyme the ability to break down oxacillin, which is not a viable substrate for the wild type AmpC