Crystal structure of a family 45 endoglucanase from Melanocarpus albomyces: mechanistic implications based on the free and cellobiose-bound forms

Cellulose, a polysaccharide of beta-1,4-linked D-glucosyl units, is the major component of plant cell walls and one of the most abundant biopolymers in nature. Cellulases (cellobiohydrolases and endoglucanases) are enzymes that catalyse the hydrolysis of cellulose to smaller oligosaccharides, a process of paramount importance in biotechnology. The thermophilic fungus Melanocarpus albomyces produces a 20 kDa endoglucanase known as 20K-cellulase that has been found particularly useful in the textile industry. The crystal structures of free 20K-cellulase and its complex with cellobiose have been determined at 2.0 A resolution. The enzyme, classified into the glycoside hydrolase family 45, exhibits the characteristic six-stranded beta-barrel found before in Humicola insolens endoglucanase V structure. However, the active site in the 20K-cellulase shows a closing of approximately 2.5-3.5A while a mobile loop identified previously in Humicola insolens endoglucanase V and implicated in the catalytic mechanism is well-defined in 20K-cellulase. In addition, the crystal structure of the cellobiose complex shows a shift in the cellobiose position at the substrate-binding cleft. It is therefore proposed that these alterations may reflect differences in the binding mechanism and catalytic action of the enzyme.     

Phorbol 12-myristate 13-acetate-induced ectodomain shedding and phosphorylation of the human meprinbeta metalloprotease

Shedding of proteins localized at the cell surface is an important regulatory step in the function of many of these proteins. Human meprin (N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase, PPH, EC 3.4.24.18) a zinc-metalloendopeptidase of the astacin family is an oligomeric protein complex of alpha- and beta-subunits and is expressed abundantly in the intestine and kidney as well as in leukocytes of the lamina propria and in cancer cells. In transfected cells intracellular proteolytic removal of the membrane anchor results in the secretion of the meprin alpha-subunit. In rats and mice, the beta-subunit exists in a membrane-anchored form. In contrast, human meprinbeta is constitutively converted into a secretable form. We now show that phorbol 12-myristate 13-acetate (PMA) stimulates an increased release of hmeprinbeta from transfected COS-1 cells, whereas hmeprinalpha secretion is not influenced. This stimulatory effect is inhibited by the protein kinase C (PKC) inhibitor staurosporine, suggesting that activation of PKC mediates PMA-induced hmeprinbeta shedding. The use of different protease inhibitors shows that two different metalloprotease activities are responsible for the constitutive and the PMA-stimulated hmeprinbeta shedding. We identified tumor necrosis factor alpha-converting enzyme (TACE or ADAM17) as the protease that mediates the PMA-induced release. We also demonstrate that hmeprinbeta is phosphorylated by PMA treatment on Ser687 within a PKC consensus sequence in the cytosolic domain of the protein. This phosphorylation of hmeprinbeta is not, however, implicated in the enhanced secretion by PMA treatment.     

Allosteric communication between signal peptides and the SecA protein DEAD motor ATPase domain

SecA, the preprotein translocase ATPase is built of an amino-terminal DEAD helicase motor domain bound to a regulatory C-domain. SecA recognizes mature and signal peptide preprotein regions. We now demonstrate that the amino-terminal 263 residues of the ATPase subdomain of the DEAD motor are necessary and sufficient for high affinity signal peptide binding. Binding is abrogated by deletion of residues 219-244 that lie within SSD, a novel substrate specificity element of the ATPase subdomain. SSD is essential for protein translocation, is unique to SecA, and is absent from other DEAD proteins. Signal peptide binding to the DEAD motor is controlled in trans by the C-terminal intramolecular regulator of ATPase (IRA1) switch. IRA1 mutations that activate the DEAD motor ATPase also enhance signal peptide affinity. This mechanism coordinates signal peptide binding with ATPase activation. Signal peptide binding causes widespread conformational changes to the ATPase subdomain and inhibits the DEAD motor ATPase. This involves an allosteric mechanism, since binding occurs at sites that are distinct from the catalytic ATPase determinants. Our data reveal the physical determinants and sophisticated intramolecular regulation that allow signal peptides to act as allosteric effectors of the SecA motor.     

Crystal structure of Streptococcus suis Dps-like peroxide resistance protein Dpr: implications for iron incorporation

The Dps-like peroxide resistance protein (Dpr) is an aerotolerance and hydrogen peroxide resistance agent found in the meningitis-associated pathogen Streptococcus suis. Dpr is believed to act by binding free intracellular iron to prevent Fenton chemistry-catalysed formation of toxic hydroxyl radicals. The crystal structure of Dpr has been determined to 1.95 A resolution. The final model has an Rcyst value of 18.5% (Rfree = 22.4%) and consists of 12 identical monomers (each of them comprising a four alpha-helix bundle) that form a hollow sphere obeying 23 symmetry. Structural features show that Dpr belongs to the Dps family of bacterial proteins. Twelve putative ferroxidase centers, each formed at the interface of neighboring monomer pairs, were identified in the Dpr structure with structural similarities to those found in other Dps family members. Dpr was crystallized in the absence of iron, hence no bound iron was found in the structure in contrast to other Dps family members. A novel metal-binding site approximately 6A from the ferroxidase centre was identified and assigned to a bound calcium ion. Two residues from the ferroxidase centre (Asp63 and Asp74) were found to be involved in calcium binding. Structural comparison with other family members revealed that Asp63 and Asp74 adopt different conformation in the Dpr structure. The structure of Dpr presented here shows potential local conformational changes that may occur during iron incorporation. A role for the metal-binding site in iron uptake is proposed.     

Medfly promoters relevant to the sterile insect technique

This review summarizes structural and functional studies on medfly promoters and regulatory elements that can be used for driving sex-specific, conditional and constitutive gene expression in this species. Sex-specific and conditional promoters are important for generating transgenic sexing strains that could increase the performance of the Sterile Insect Technique while strong constitutive promoters are necessary for developing sensitive transgenic marker systems. The review focuses on the functional analysis of the promoters of two male-specific and heat shock medfly genes. A special emphasis is put on the potential utility of these promoters for developing transgenic sexing strains.     

c-Jun N-Terminal Kinase Phosphorylation of MARCKSL1 Determines Actin Stability and Migration in Neurons and in Cancer Cells

Cell migration is a fundamental biological function, critical during development and regeneration, whereas deregulated migration underlies neurological birth defects and cancer metastasis. MARCKS-like protein 1 (MARCKSL1) is widely expressed in nervous tissue, where, like Jun N-terminal protein kinase (JNK), it is required for neural tube formation, though the mechanism is unknown. Here we show that MARCKSL1 is directly phosphorylated by JNK on C-terminal residues (S120, T148, and T183). This phosphorylation enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers and dynamics, and retard migration in neurons. Conversely, when MARCKSL1 phosphorylation is inhibited, actin mobility increases and filopodium formation is compromised whereas lamellipodium formation is enhanced, as is cell migration. We find that MARCKSL1 mRNA is upregulated in a broad range of cancer types and that MARCKSL1 protein is strongly induced in primary prostate carcinomas. Gene knockdown in prostate cancer cells or in neurons reveals a critical role for MARCKSL1 in migration that is dependent on the phosphorylation state; phosphomimetic MARCKSL1 (MARCKSL1(S120D,T148D,T183D)) inhibits whereas dephospho-MARCKSL1(S120A,T148A,T183A) induces migration. In summary, these data show that JNK phosphorylation of MARCKSL1 regulates actin homeostasis, filopodium and lamellipodium formation, and neuronal migration under physiological conditions and that, when ectopically expressed in prostate cancer cells, MARCKSL1 again determines cell movement.     

SecA-mediated targeting and translocation of secretory proteins

More than 30 years of research have revealed that the dynamic nanomotor SecA is a central player in bacterial protein secretion. SecA associates with the SecYEG channel and transports polypeptides post-translationally to the trans side of the cytoplasmic membrane. It comprises a helicase-like ATPase core coupled to two domains that provide specificity for preprotein translocation. Apart from SecYEG, SecA associates with multiple ligands like ribosomes, nucleotides, lipids, chaperones and preproteins. It exerts its essential contribution in two phases. First, SecA, alone or in concert with chaperones, helps mediate the targeting of the secretory proteins from the ribosome to the membrane. Next, at the membrane it converts chemical energy to mechanical work and translocates preproteins through the SecYEG channel. SecA is a highly dynamic enzyme, it exploits disorder–order kinetics, swiveling and dissociation of domains and dimer to monomer transformations that are tightly coupled with its catalytic function. Preprotein signal sequences and mature domains exploit these dynamics to manipulate the nanomotor and thus achieve their export at the expense of metabolic energy. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Effects of 2‐year calorie restriction on circulating levels of IGF‐1, IGF‐binding proteins and cortisol in nonobese men and women: a randomized clinical trial

Young‐onset calorie restriction (CR) in rodents decreases serum IGF‐1 concentration and increases serum corticosterone levels, which have been hypothesized to play major roles in mediating its anticancer and anti‐aging effects. However, little is known on the effects of CR on the IGF‐1 system and cortisol in humans. To test the sustained effects of CR on these key hormonal adaptations, we performed a multicenter randomized trial of a 2‐year 25% CR intervention in 218 nonobese (body mass index between 22 and 27.8 kg m^?2) young and middle‐aged (20–50 years age range) men and women. Average CR during the first 6 months was 19.5 ± 0.8% and 9.1 ± 0.7% over the next 18 months of the study. Weight loss averaged 7.6 ± 0.3 kg over the 2‐years period of which 71% was fat mass loss (P < 0.0001). Average CR during the CR caused a significant 21% increase in serum IGFBP‐1 and a 42% reduction in IGF‐1:IGFBP‐1 ratio at 2 years (P < 0.008), but did not change IGF‐1 and IGF‐1:IGFBP‐3 ratio levels. Serum cortisol concentrations were slightly but significantly increased by CR at 1 year only (P = 0.003). Calorie restriction had no effect on serum concentrations of PDGF‐AB and TGFβ‐1. We conclude, on the basis of the present and previous findings, that, in contrast to rodents, humans do not respond to CR with a decrease in serum IGF‐1 concentration or with a sustained and biological relevant increase in serum cortisol. However, long‐term CR in humans significantly and persistently increases serum IGFBP‐1 concentration.     

Arc/Arg3.1: linking gene expression to synaptic plasticity and memory

Arc/Arg3.1 is an effector immediate-early gene implicated in the consolidation of memories. Although cloned a decade ago, the physiological role of Arc/Arg3.1 in the brain has remained elusive. Four papers in this issue of Neuron address this function. These studies show that Arc/Arg3.1 regulates endophilin 3 and dynamin 2, two components of the endocytosis machinery. Genetic ablation of Arc/Arg3.1 in mice or overexpression in culture suggest that Arc/Arg3.1 regulates AMPA receptor trafficking and synaptic plasticity. Finally, Arc/Arg3.1 knockout mice show memory retention deficits. These recent developments provide new insights into the function of this popular activity-dependent neuronal marker.