Signal peptide of FadA adhesin from Fusobacterium nucleatum plays a novel structural role by modulating the filament's length and width

FadA, a novel adhesin of periodontal pathogen Fusobacterium nucleatum is composed of two forms, pre-FadA and mature FadA (mFadA), constituting the functional FadA complex (FadAc). By electron microscopy, we observed that mFadA formed uniformly long and thin filaments, while FadAc formed heterogeneous filaments of varying lengths and widths, as well as "knots". Mutants in signal peptide or in the non-alpha-helical loop retaining heterogeneous structures had binding activity while those forming aggregates or long filaments lost activity. These observations suggest short filaments and knots may be the active forms of FadA. This is the first demonstration that a signal peptide is required for the assembly of a bacterial adhesin.     

δ-Tocopherol Reduces Lipid Accumulation in Niemann-Pick Type C1 and Wolman Cholesterol Storage Disorders

Niemann-Pick disease type C (NPC) and Wolman disease are two members of a family of storage disorders caused by mutations of genes encoding lysosomal proteins. Deficiency in function of either the NPC1 or NPC2 protein in NPC disease or lysosomal acid lipase in Wolman disease results in defective cellular cholesterol trafficking. Lysosomal accumulation of cholesterol and enlarged lysosomes are shared phenotypic characteristics of both NPC and Wolman cells. Utilizing a phenotypic screen of an approved drug collection, we found that δ-tocopherol effectively reduced lysosomal cholesterol accumulation, decreased lysosomal volume, increased cholesterol efflux, and alleviated pathological phenotypes in both NPC1 and Wolman fibroblasts. Reduction of these abnormalities may be mediated by a δ-tocopherol-induced intracellular Ca²⁺ response and subsequent enhancement of lysosomal exocytosis. Consistent with a general mechanism for reduction of lysosomal lipid accumulation, we also found that δ-tocopherol reduces pathological phenotypes in patient fibroblasts from other lysosomal storage diseases, including NPC2, Batten (ceroid lipofuscinosis, neuronal 2, CLN2), Fabry, Farber, Niemann-Pick disease type A, Sanfilippo type B (mucopolysaccharidosis type IIIB, MPSIIIB), and Tay-Sachs. Our data suggest that regulated exocytosis may represent a potential therapeutic target for reduction of lysosomal storage in this class of diseases.     

The mechanism of proton transfer between adjacent sites on the molecular surface

The surface of a protein, or a membrane, is spotted with a multitude of proton binding sites, some of which are only few Å apart. When a proton is released from one site, it propagates through the water by a random walk under the bias of the local electrostatic potential determined by the distribution of the charges on the protein. Eventually, the released protons are dispersed in the bulk, but during the first few nanoseconds after the dissociation, the protons can be trapped by encounter with nearby acceptor sites. While the study of this reaction on the surface of a protein suffers from experimental and theoretical difficulties, it can be investigated with simple model compounds like derivatives of fluorescein. In the present study, we evaluate the mechanism of proton transfer reactions that proceed, preferentially, inside the Coulomb cage of the dye molecules. Kinetic analysis of the measured dynamics reveals the role of the dimension of the Coulomb cage on the efficiency of the reaction and how the ordering of the water molecules by the dye affects the kinetic isotope effect.     

The mechanism of proton transfer between adjacent sites on the molecular surface

The surface of a protein, or a membrane, is spotted with a multitude of proton binding sites, some of which are only few A apart. When a proton is released from one site, it propagates through the water by a random walk under the bias of the local electrostatic potential determined by the distribution of the charges on the protein. Eventually, the released protons are dispersed in the bulk, but during the first few nanoseconds after the dissociation, the protons can be trapped by encounter with nearby acceptor sites. While the study of this reaction on the surface of a protein suffers from experimental and theoretical difficulties, it can be investigated with simple model compounds like derivatives of fluorescein. In the present study, we evaluate the mechanism of proton transfer reactions that proceed, preferentially, inside the Coulomb cage of the dye molecules. Kinetic analysis of the measured dynamics reveals the role of the dimension of the Coulomb cage on the efficiency of the reaction and how the ordering of the water molecules by the dye affects the kinetic isotope effect.     

Cytosolic activity of SPINDLY implies the existence of a DELLA-independent gibberellin-response pathway

Specific plant developmental processes are modulated by cross-talk between gibberellin (GA)- and cytokinin-response pathways. Coordination of the two pathways involves the O-linked N -acetylglucosamine transferase SPINDLY (SPY) that suppresses GA signaling and promotes cytokinin responses in Arabidopsis. Although SPY is a nucleocytoplasmic protein, its site of action and targets are unknown. Several studies have suggested that SPY acts in the nucleus, where it modifies nuclear components such as the DELLA proteins to regulate signaling networks. Using chimeric GFP–SPY fused to a nuclear-export signal or to a glucocorticoid receptor, we show that cytosolic SPY promotes cytokinin responses and suppresses GA signaling. In contrast, nuclear-localized GFP–SPY failed to complement the spy mutation. To examine whether modulation of cytokinin activity by GA and spy is mediated by the nuclear DELLA proteins, cytokinin responses were studied in double and quadruple della mutants lacking the activities of REPRESSOR OF GA1-3 (RGA) and GA-INSENSITIVE (GAI) or RGA, GAI, RGA Like1 (RGL1) and RGL2. Unlike spy , the della mutants were cytokinin-sensitive. Moreover, when GA was applied to a cytokinin-treated quadruple della mutant it was able to suppress various cytokinin responses. These results suggest that cytosolic SPY and GA regulate cytokinin responses via a DELLA-independent pathway(s).     

Viral adaptation to host: a proteome‐based analysis of codon usage and amino acid preferences

Viruses differ markedly in their specificity toward host organisms. Here, we test the level of general sequence adaptation that viruses display toward their hosts. We compiled a representative data set of viruses that infect hosts ranging from bacteria to humans. We consider their respective amino acid and codon usages and compare them among the viruses and their hosts. We show that bacteria‐infecting viruses are strongly adapted to their specific hosts, but that they differ from other unrelated bacterial hosts. Viruses that infect humans, but not those that infect other mammals or aves, show a strong resemblance to most mammalian and avian hosts, in terms of both amino acid and codon preferences. In groups of viruses that infect humans or other mammals, the highest observed level of adaptation of viral proteins to host codon usages is for those proteins that appear abundantly in the virion. In contrast, proteins that are known to participate in host‐specific recognition do not necessarily adapt to their respective hosts. The implication for the potential of viral infectivity is discussed.     

Design of multispecific protein sequences using probabilistic graphical modeling

In nature, proteins partake in numerous protein– protein interactions that mediate their functions. Moreover, proteins have been shown to be physically stable in multiple structures, induced by cellular conditions, small ligands, or covalent modifications. Understanding how protein sequences achieve this structural promiscuity at the atomic level is a fundamental step in the drug design pipeline and a critical question in protein physics. One way to investigate this subject is to computationally predict protein sequences that are compatible with multiple states, i.e., multiple target structures or binding to distinct partners. The goal of engineering such proteins has been termed multispecific protein design. We develop a novel computational framework to efficiently and accurately perform multispecific protein design. This framework utilizes recent advances in probabilistic graphical modeling to predict sequences with low energies in multiple target states. Furthermore, it is also geared to specifically yield positional amino acid probability profiles compatible with these target states. Such profiles can be used as input to randomly bias high‐throughput experimental sequence screening techniques, such as phage display, thus providing an alternative avenue for elucidating the multispecificity of natural proteins and the synthesis of novel proteins with specific functionalities. We prove the utility of such multispecific design techniques in better recovering amino acid sequence diversities similar to those resulting from millions of years of evolution. We then compare the approaches of prediction of low energy ensembles and of amino acid profiles and demonstrate their complementarity in providing more robust predictions for protein design. Proteins 2010. © 2009 Wiley‐Liss, Inc.