No abstract

The fraction of pyruvate dehydrogenase complex (PDC) in the active form is reduced by the activities of dedicated PD kinase isozymes (PDK1, PDK2, PDK3 and PDK4). Via binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase (E2 60mer), PDK rapidly access their E2-bound PD substrate. The E2-enhanced activity of the widely distributed PDK2 is limited by dissociation of ADP from its C-terminal catalytic domain, and this is further slowed by pyruvate binding to the N-terminal regulatory (R) domain. Via the reverse of the PDC reaction, NADH and acetyl-CoA reductively acetylate lipoyl group of L2, which binds to the R domain and stimulates PDK2 activity by speeding up ADP dissociation. Activation of PDC by synthetic PDK inhibitors binding at the pyruvate or lipoyl binding sites decreased damage during heart ischemia and lowered blood glucose in insulin-resistant animals. PDC activation also triggers apoptosis in cancer cells that selectively convert glucose to lactate.

SummaryExtracellular DNA (eDNA), a by-product of cell lysis, was recently established as a critical structural component of the Enterococcus faecalis biofilm matrix. Here, we describe fratricide as the governing principle behind gelatinase (GelE)-mediated cell death and eDNA release. GFP reporter assays confirmed that GBAP (gelatinase biosynthesis-activating pheromone) quorum non-responders (GelE Finally, we address a mechanism by which GelE and SprE may modify the cell wall affinity of proteolytically processed AtlA resulting in either a pro-or antilytic outcome.

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the Cterminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2⅐E3BP was thought to be 60 E2 plus ϳ12 E3BP. We have prepared homogenous human components. E2 and E2⅐E3BP have s 20,w values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2⅐E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2⅐E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound ϳ60 E1 and maximally sedimented 64.4 ؎ 1.5 S faster than E2, whereas E1-saturated E2⅐E3BP maximally sedimented 49.5 ؎ 1.4 S faster than E2⅐E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (ϳ12) were bound by E2⅐E3BP than by E2. The findings of a smaller E2⅐E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E2 48 ⅐E3BP 12 mass, which is consistent with this model.The mitochondrial pyruvate dehydrogenase complex (PDC) 1 catalyzes the irreversible conversion of pyruvate to acetyl-CoA along with the reduction of NAD ϩ . PDCs from all known sources contain the pyruvate dehydrogenase (E1), the dihydrolipoyl acetyltransferase (E2), and the dihydrolipoyl dehydrogenase (E3) components. Mammalian PDC has a highly organized structure in which the E2 component plays a central role in the organization, integrated chemical reactions, and regulation of the complex (1-5). Besides those universal components, the mammalian and subsequently some other eukaryotic PDCs were shown to contain another component, called E3-binding protein (E3BP); this protein was originally designated protein X (6, 7). Mammalian E3BP was first characterized as a component with a reactive lipoyl group on a single lipoyl domain (7-11) that, alone, could support the overall reaction (12). E3BP was tightly bound to E2 (7) by its C-terminal region (10). The E3BP component was then shown to contribute to the organization of the complex by binding the E3 component (13)(14)(15)(16)(17)(18). This work provides new insights into the integration of E3BP into the central framework of the mammalian complex.Although first characterized in the bo...

Peptide-based packaging systems show great potential as safer drug delivery systems. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. Here, we describe a set of 15 & 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. These peptides undergo supramolecular self-assembly and form solvent-filled, bilayer delimited spheres with 50–200 nm diameters as confirmed by TEM, STEM and DLS. Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these all-peptide structures are stabilized potentially by both hydrophobic interactions and hydrogen bonds and remain intact at low micromolar concentrations and higher temperatures. A linear peptide lacking the branch point showed no self-assembly properties. We have observed that these peptide vesicles can trap fluorescent dye molecules within their interior and are taken up by N/N 1003A rabbit lens epithelial cells grown in culture. These assemblies are thus potential drug delivery systems that can overcome some of the key limitations of the current packaging systems.