Dynamic imaging of protease activity with fluorescently quenched activity-based probes

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.     

Regulation of gap junctions by tyrosine protein kinases

Most of the gap junction proteins are regulated in part by post-translational phosphorylation. Phosphorylation has been shown to be important in gap junction assembly and turnover, and for channel function in the resting state. Connexin phosphorylation may be altered by the activation of intracellular signaling pathways in response to growth factors, tumor promoters, activated oncogenes, hormones and inflammatory mediators. In some instances altered phosphorylation has been associated with changes in connexin function and in other cases appears to be associated with changes in the levels of the connexin protein and/or mRNA. This review focuses on the role of tyrosine protein kinases in the regulation of gap junctions. The literature is most extensive for connexin43 and those studies are reviewed here. A great deal has been learned in recent years about how connexin43 is regulated by tyrosine kinase-dependent signaling pathways. These pathways are often complex and to some extent are cell type- and stimulus-dependent. Although considerable progress has been made in unraveling the cellular pathways that regulate connexin function, significant challenges remain to be addressed in identifying additional phosphorylation sites and determining the stoichiometries of the phosphorylation events that regulate connexin function and it's interaction with other cellular proteins.     

Mutations Altering a Structurally Conserved Loop-Helix-Loop Region of a Viral Packaging Motor Change DNA Translocation Velocity and Processivity

Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage λ gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.     

Predicting transmembrane beta-barrels and interstrand residue interactions from sequence

Transmembrane beta-barrel (TMB) proteins are embedded in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. The cellular location and functional diversity of beta-barrel outer membrane proteins (omps) makes them an important protein class. At the present time, very few nonhomologous TMB structures have been determined by X-ray diffraction because of the experimental difficulty encountered in crystallizing transmembrane proteins. A novel method using pairwise interstrand residue statistical potentials derived from globular (nonouter membrane) proteins is introduced to predict the supersecondary structure of transmembrane beta-barrel proteins. The algorithm transFold employs a generalized hidden Markov model (i.e., multitape S-attribute grammar) to describe potential beta-barrel supersecondary structures and then computes by dynamic programming the minimum free energy beta-barrel structure. Hence, the approach can be viewed as a "wrapping" component that may capture folding processes with an initiation stage followed by progressive interaction of the sequence with the already-formed motifs. This approach differs significantly from others, which use traditional machine learning to solve this problem, because it does not require a training phase on known TMB structures and is the first to explicitly capture and predict long-range interactions. TransFold outperforms previous programs for predicting TMBs on smaller (相似文献   

CD40 expression by microglial cells is required for their completion of a two-step activation process during central nervous system autoimmune inflammation

Microglial cells are monocytic lineage cells that reside in the CNS and have the capacity to become activated during various pathological conditions. Although it was demonstrated that activation of microglial cells could be achieved in vitro by the engagement of CD40-CD40L interactions in combination with proinflammatory cytokines, the exact factors that mediate activation of microglial cells in vivo during CNS autoimmunity are ill-defined. To investigate the role of CD40 in microglial cell activation during experimental autoimmune encephalomyelitis (EAE), we used bone marrow chimera mice that allowed us to distinguish microglial cells from peripheral macrophages and render microglial cells deficient in CD40. We found that the first step of microglial cell activation was CD40-independent and occurred during EAE onset. The first step of activation consisted of microglial cell proliferation and up-regulation of the activation markers MHC class II, CD40, and CD86. At the peak of disease, microglial cells underwent a second step of activation, which was characterized by a further enhancement in activation marker expression along with a reduction in proliferation. The second step of microglial cell activation was CD40-dependent and the failure of CD40-deficient microglial cells to achieve a full level of activation during EAE was correlated with reduced expansion of encephalitogenic T cells and leukocyte infiltration in the CNS, and amelioration of clinical symptoms. Thus, our findings demonstrate that CD40 expression on microglial cells is necessary to complete their activation process during EAE, which is important for disease progression.     

Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: insight into N-linked glycosylation pathways in Archaea

N-linked glycosylation is recognized as an important post-translational modification across all three domains of life. However, the understanding of the genetic pathways for the assembly and attachment of N-linked glycans in eukaryotic and bacterial systems far outweighs the knowledge of comparable processes in Archaea. The recent characterization of a novel trisaccharide [beta-ManpNAcA6Thr-(1-4)-beta-GlcpNAc3NAcA-(1-3)-beta-GlcpNAc]N-linked to asparagine residues in Methanococcus voltae flagellin and S-layer proteins affords new opportunities to investigate N-linked glycosylation pathways in Archaea. In this contribution, the insertional inactivation of several candidate genes within the M. voltae genome and their resulting effects on flagellin and S-layer glycosylation are reported. Two of the candidate genes were shown to have effects on flagellin and S-layer protein molecular mass and N-linked glycan structure. Further examination revealed inactivation of either of these two genes also had effects on flagella assembly. These genes, designated agl (archaeal glycosylation) genes, include a glycosyl transferase (aglA) involved in the attachment of the terminal sugar to the glycan and an STT3 oligosaccharyl transferase homologue (aglB) involved in the transfer of the complete glycan to the flagellin and S-layer proteins. These findings document the first experimental evidence for genes involved in any glycosylation process within the domain Archaea.     

Autoinducer 2: a concentration-dependent signal for mutualistic bacterial biofilm growth

4,5-Dihydroxy-2,3-pentanedione (DPD), a product of the LuxS enzyme in the catabolism of S-ribosylhomocysteine, spontaneously cyclizes to form autoinducer 2 (AI-2). AI-2 is proposed to be a universal signal molecule mediating interspecies communication among bacteria. We show that mutualistic and abundant biofilm growth in flowing saliva of two human oral commensal bacteria, Actinomyces naeslundii T14V and Streptococcus oralis 34, is dependent upon production of AI-2 by S. oralis 34. A luxS mutant of S. oralis 34 was constructed which did not produce AI-2. Unlike wild-type dual-species biofilms, A. naeslundii T14V and an S. oralis 34 luxS mutant did not exhibit mutualism and generated only sparse biofilms which contained a 10-fold lower biomass of each species. Restoration of AI-2 levels by genetic or chemical (synthetic AI-2 in the form of DPD) complementation re-established the mutualistic growth and high biomass characteristic for the wild-type dual-species biofilm. Furthermore, an optimal concentration of DPD was determined, above and below which biofilm formation was suppressed. The optimal concentration was 100-fold lower than the detection limit of the currently accepted AI-2 assay. Thus, AI-2 acts as an interspecies signal and its concentration is critical for mutualism between two species of oral bacteria grown under conditions that are representative of the human oral cavity.     

Biosynthetic diversity in plant triterpene cyclization

Plants produce a wealth of terpenoids, many of which have been the tools of healers and chiefs for millennia. Recent research has led to the identification and characterization of many genes that are responsible for the biosynthesis of triterpenoids. Cyclases that generate sterol precursors can be recognized with some confidence on the basis of sequence; several catalytically important residues are now known, and the product profiles of sterol-generating cyclases typically reflect their phylogenetic position. By contrast, the phylogenetic relationships of cyclases that generate nonsteroidal triterpene alcohols do not consistently reflect their catalytic properties and might indicate recent and rapid catalytic evolution.     

Cathepsin B localizes to plasma membrane caveolae of differentiating myoblasts and is secreted in an active form at physiological pH

Our in vitro studies support a functional link between the induction of cathepsin B gene expression and the catabolic restructuring associated with myotube formation during myogenesis in vivo. We have tested two predictions that are basic to this hypothesis: (1) that active cathepsin B is localized to plasma membrane caveolae of fusing myoblasts; and (2) that active cathepsin B is secreted from fusing myoblasts at physiological pH. During differentiation, L6 rat myoblasts demonstrated a fusion-related increase in activity associated with the 25/26-kDa, fully processed, active form of cathepsin B. Immunocytochemical studies demonstrated a redistribution of lysosomal cathepsin B protein toward the membrane of fusing myoblasts, and a colocalization of cathepsin B with caveolin-3, the muscle-specific structural protein of membrane caveolae. Sucrose density fractionation and Western blot analysis demonstrated that an active form of cathepsin B localizes to caveolar fractions along with caveolin-3, annexin-VII, beta-dystroglycan and dystrophin. Finally, 'real-time' activity assays and Western blot analysis demonstrated that active cathepsin B is secreted from fusing myoblasts at physiological pH. Collectively, these studies support an association of active cathepsin B with plasma membrane caveolae and the secretion of active cathepsin B from differentiating myoblasts during myoblast fusion.