A systems biology approach reveals the role of a novel methyltransferase in response to chemical stress and lipid homeostasis

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors.     

Introducing shear stress in the study of bacterial adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm(2). Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm(2)). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes (1). On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm(2)(2). The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium (3). To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber (4). Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells (1), to proliferate on the cell surface (5)and to detach to colonize new sites (6) (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience(1) and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.     

The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.     

Key interactions in neocarzinostatin,a protein of the immunoglobulin fold family

Neocarzinostatin (NCS) is a seven-stranded beta-sandwich protein, the folding of which is similar to that of the variable domains of immunoglobulins (Ig). The investigation of the backbone dynamics of apo-NCS [Izadi-Pruneyre et al. (2001) Protein Sci., 10, 2228-2240] enabled us to identify the involvement of long side-chain residues in maintaining the rigidity of this beta-protein. In the perspective of using this protein for drug targeting, this raises the following question: do these residues also play a key role in the stabilization of the beta-sheet? To investigate this problem, various genetically engineered variants were constructed by mutating these residues to amino acids with shorter aliphatic side chains. These substitutions have no effects on the global fold. However, an important destabilization of the protein, higher than that expected for a simple 'large-to-small' substitution of buried hydrophobic residues, is observed for three mutants, V34A, V21A and V95A. Interestingly, the nature of the residues in these positions is highly conserved in the other Ig-like proteins. The absence of an evolutionary relationship between NCS and the other Ig-like proteins strongly suggests that this hydrophobic core is characteristic of the Ig-fold itself.     

A small sequence in the third intracellular loop of the VPAC(1) receptor is responsible for its efficient coupling to the calcium effector

The stimulatory effect of VIP on intracellular calcium concentration ([Ca(2+)](i)) has been investigated in Chinese hamster ovary cells stably transfected with the reporter gene aequorin, and expressing human VPAC(1), VPAC(2), chimeric VPAC(1)/VPAC(2), or mutated receptors. The VIP-induced [Ca(2+)](i) increase was linearly correlated with receptor density and was higher in cells expressing VPAC(1) receptors than in cells expressing a similar VPAC(2) receptor density. The study was performed to establish the receptor sequence responsible for that difference. VPAC(1)/VPAC(2) chimeric receptors were first used for a broad positioning: those having the third intracellular loop (IC(3)) of the VPAC(1) or of the VPAC(2) receptor behaved, in that respect, phenotypically like VPAC(1) and VPAC(2) receptor, respectively. Replacement in the VPAC(2) receptor of the sequence 315-318 (VGGN) within the IC(3) by its VPAC(1) receptor counterpart 328-331 (IRKS) and the introduction of VGGN in state of IRKS in VPAC(1) was sufficient to mimic the VPAC(1) and VPAC(2) receptor characteristics, respectively. Thus, a small sequence in the IC(3) of the VPAC(1) receptor, probably through interaction with G(alphai) and G(alphaq) proteins, is responsible for the efficient agonist-stimulated [Ca(2+)](i) increase.     

No effect of creatine supplementation on human myofibrillar and sarcoplasmic protein synthesis after resistance exercise

Muscle hypertrophy during resistance training is reportedly increased by creatine supplementation. Having previously failed to find an anabolic effect on muscle protein turnover at rest, either fed or fasted, we have now examined the possibility of a stimulatory effect of creatine in conjunction with acute resistance exercise. Seven healthy men (body mass index, 23 +/- 2 kg/m2, 21 +/- 1 yr, means +/- SE) performed 20 x 10 repetitions of leg extension-flexion at 75% one-repetition maximum in one leg, on two occasions, 4 wk apart, before and after ingesting 21 g/day creatine for 5 days. The subjects ate approximately 21 g maltodextrin + 6 g protein/h for 3 h postexercise. We measured incorporation of [1-13C]leucine into quadriceps muscle proteins in the rested and exercised legs. Leg protein breakdown (as dilution of [2H5]phenylalanine) was also assessed in the exercised and rested leg postexercise. Creatine supplementation increased muscle total creatine by approximately 21% (P < 0.01). Exercise increased the synthetic rates of myofibrillar and sarcoplasmic proteins by two- to threefold (P < 0.05), and leg phenylalanine balance became more positive, but creatine was without any anabolic effect.