Portrait of an Enzyme, a Complete Structural Analysis of a Multimodular β-N-Acetylglucosaminidase from Clostridium perfringens

Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens micro-toxin, which was determined using a combination of small angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction.

βCaMKII Regulates Actin Assembly and Structure

Ca(2+)-Calmodulin-dependent protein kinase II (CaMKII) is an abundant synaptic protein that was recently shown to regulate the organization of actin filaments leading to structural modifications of synapses. CaMKII is a dodecameric complex with a special architecture that provides it with unique potential for organizing the actin cytoskeleton. We report using biochemical assays that the beta isoform of CaMKII binds to and bundles actin filaments, and the disposition of betaCaMKII within the actin bundles was revealed by cryoelectron tomography. In addition, betaCaMKII was found to inhibit actin polymerization, suggesting that it either serves as a capping protein or binds monomeric actin, reducing the amount of freely available monomers to nucleate polymer assembly. By means of fluorescent cross-correlation spectroscopy, we determined that betaCaMKII does indeed bind to monomeric actin, reaching saturation at a stoichiometry of 12:1 actin monomers per betaCaMKII holoenzyme with a binding constant of 2.4 x 10(5) m(-1). In cells, betaCaMKII has a dual functional role; it can sequester monomeric actin to reduce actin polymerization and can also bundle actin filaments. Together, these effects would impact both the dynamics of actin filament assembly and enhance the rigidity of the filaments once formed, significantly impacting the structure of synapses.

Reduced Expression of CD45 Protein-tyrosine Phosphatase Provides Protection against Anthrax Pathogenesis

The modulation of cellular processes by small molecule inhibitors, gene inactivation, or targeted knockdown strategies combined with phenotypic screens are powerful approaches to delineate complex cellular pathways and to identify key players involved in disease pathogenesis. Using chemical genetic screening, we tested a library of known phosphatase inhibitors and identified several compounds that protected Bacillus anthracis infected macrophages from cell death. The most potent compound was assayed against a panel of sixteen different phosphatases of which CD45 was found to be most sensitive to inhibition. Testing of a known CD45 inhibitor and antisense phosphorodiamidate morpholino oligomers targeting CD45 also protected B. anthracis-infected macrophages from cell death. However, reduced CD45 expression did not protect anthrax lethal toxin (LT) treated macrophages, suggesting that the pathogen and independently added LT may signal through distinct pathways. Subsequent, in vivo studies with both gene-targeted knockdown of CD45 and genetically engineered mice expressing reduced levels of CD45 resulted in protection of mice after infection with the virulent Ames B. anthracis. Intermediate levels of CD45 expression were critical for the protection, as mice expressing normal levels of CD45 or disrupted CD45 phosphatase activity or no CD45 all succumbed to this pathogen. Mechanism-based studies suggest that the protection provided by reduced CD45 levels results from regulated immune cell homeostasis that may diminish the impact of apoptosis during the infection. To date, this is the first report demonstrating that reduced levels of host phosphatase CD45 modulate anthrax pathogenesis.

p90 Ribosomal S6 Kinase and p70 Ribosomal S6 Kinase Link Phosphorylation of the Eukaryotic Chaperonin Containing TCP-1 to Growth Factor, Insulin, and Nutrient Signaling

Chaperonin containing TCP-1 (CCT) is a large multisubunit complex that mediates protein folding in eukaryotic cells. CCT participates in the folding of newly synthesized polypeptides, including actin, tubulin, and several cell cycle regulators; therefore, CCT plays an important role in cytoskeletal organization and cell division. Here we identify the chaperonin CCT as a novel physiological substrate for p90 ribosomal S6 kinase (RSK) and p70 ribosomal S6 kinase (S6K). RSK phosphorylates the beta subunit of CCT in response to tumor promoters or growth factors that activate the Ras-mitogen-activated protein kinase (MAPK) pathway. CCTbeta Ser-260 was identified as the RSK site by mass spectrometry and confirmed by site-directed mutagenesis. RSK-dependent Ser-260 phosphorylation was sensitive to the MEK inhibitor UO126 and the RSK inhibitor BID-1870. Insulin weakly activates RSK but strongly activates the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway and utilizes S6K to regulate CCTbeta phosphorylation. Thus, the Ras-MAPK and PI3K-mTOR pathways converge on CCTbeta Ser-260 phosphorylation in response to multiple agonists in various mammalian cells. We also show that RNA interference-mediated knockdown of endogenous CCTbeta causes impaired cell proliferation that can be rescued with ectopically expressed murine CCTbeta wild-type or phosphomimetic mutant S260D, but not the phosphorylation-deficient mutant S260A. Although the molecular mechanism of CCTbeta regulation remains unclear, our findings demonstrate a link between oncogene and growth factor signaling and chaperonin CCT-mediated cellular activities.

Involvement of All-trans-retinal in Acute Light-induced Retinopathy of Mice

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.

Regulation of Neuronal Cell Death by MST1-FOXO1 Signaling

The protein kinase mammalian Sterile 20-like kinase 1 (MST1) plays a critical role in the regulation of cell death. Recent studies suggest that MST1 mediates oxidative stress-induced neuronal cell death by phosphorylating the transcription factor FOXO3 at serine 207, a site that is conserved in other FOXO family members. Here, we show that MST1-induced phosphorylation of FOXO1 at serine 212, corresponding to serine 207 in FOXO3, disrupts the association of FOXO1 with 14-3-3 proteins. Accordingly, MST1 mediates the nuclear translocation of FOXO1 in primary rat cerebellar granule neurons that are deprived of neuronal activity. We also find a requirement for MST1 in cell death of granule neurons upon withdrawal of growth factors and neuronal activity, and MST1 induces cell death in a FOXO1-dependent manner. Finally, we show that the MST1-regulatory, scaffold protein Nore1 is required for survival factor deprivation induced neuronal death. Collectively, these findings define MST1-FOXO1 signaling as an important link survival factor deprivation-induced neuronal cell death with implications for our understanding of brain development and neurological diseases.

The Ubiquitin-interacting Motif Protein, S5a, Is Ubiquitinated by All Types of Ubiquitin Ligases by a Mechanism Different from Typical Substrate Recognition

S5a/Rpn10 is a ubiquitin (Ub)-binding protein that is a subunit of the 26S proteasome but also exists free in the cytosol. It binds poly-Ub chains through its two Ub-interacting motifs (UIMs). We discovered that, unlike typical substrates of Ub ligases (E3s), S5a can be ubiquitinated by all E3s tested including multimeric and monomeric Ring finger E3s (MuRF1, Siah2, Parkin, APC, and SCF(betaTRCP1)), the U-box E3, CHIP, and HECT domain E3s (E6AP and Nedd4) when assayed with UbcH5 or related Ub-conjugating enzymes. However, the E2s, UbcH1 and UbcH13/Uev1a, which function by distinct mechanisms, do not support S5a ubiquitination. Thus, S5a can be used for assay of probably all E3s with UbcH5. Ubiquitination of S5a results from its binding to Ub chains on the E3 (after self-ubiquitination) or on the substrate, as a mutant lacking the UIM domain was not ubiquitinated. Furthermore, if the S5a UIM domains were fused to GST, the protein was rapidly ubiquitinated by MuRF1 and CHIP. In addition, polyubiquitination (but not monoubiquitination) of MuRF1 allowed S5a to bind to MuRF1 and accelerated S5a ubiquitination. This tendency of S5a to associate with the growing Ub chain can explain how S5a, unlike typical substrates, which are recognized by certain E3s through specific motifs, is ubiquitinated by all E3s tested and is rapidly degraded in vivo.

Calpain Expression and Activity during Lens Fiber Cell Differentiation

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was alphaII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.

Reply to Xu: Role of Lactate in Lipid Metabolism, Just Always Inhibiting Lipolysis?

Exosite Determinants of Serpin Specificity

Serpins form an enormous superfamily of 40-60-kDa proteins found in almost all types of organisms, including humans. Most are one-use suicide substrate serine and cysteine proteinase inhibitors that have evolved to finely regulate complex proteolytic pathways, such as blood coagulation, fibrinolysis, and inflammation. Despite distinct functions for each serpin, there is much redundancy in the primary specificity-determining residues. However, many serpins exploit additional exosites to generate the exquisite specificity that makes a given serpin effective only when certain other criteria, such as the presence of specific cofactors, are met. With a focus on human serpins, this minireview examines use of exosites by nine serpins in the initial complex-forming phase to modulate primary specificity in either binary serpin-proteinase complexes or ternary complexes that additionally employ a protein or other cofactor. A frequent theme is down-regulation of inhibitory activity unless the exosite(s) are engaged. In addition, the use of exosites by maspin and plasminogen activator inhibitor-1 to indirectly affect proteolytic processes is considered.