Cytosine Deaminase as a Negative Selection Marker for Gene Disruption and Replacement in the Genus Streptomyces and Other Actinobacteria

We developed a novel negative selection system for actinobacteria based on cytosine deaminase (CodA). We constructed vectors that include a synthetic gene encoding the CodA protein from Escherichia coli optimized for expression in Streptomyces species. Gene disruption and the introduction of an unmarked in-frame deletion were successfully achieved with these vectors.     

Control of Promatrilysin (MMP7) Activation and Substrate-specific Activity by Sulfated Glycosaminoglycans

Matrix metalloproteinases are maintained in an inactive state by a bond between the thiol of a conserved cysteine in the prodomain and a zinc atom in the catalytic domain. Once this bond is disrupted, MMPs become active proteinases and can act on a variety of extracellular protein substrates. In vivo, matrilysin (MMP7) activates pro-α-defensins (procryptdins), but in vitro, processing of these peptides is slow, with about 50% conversion in 8–12 h. Similarly, autolytic activation of promatrilysin in vitro can take up to 12–24 h for 50% conversion. These inefficient reactions suggest that natural cofactors enhance the activation and activity of matrilysin. We determined that highly sulfated glycosaminoglycans (GAG), such as heparin, chondroitin-4,6-sulfate (CS-E), and dermatan sulfate, markedly enhanced (>50-fold) the intermolecular autolytic activation of promatrilysin and the activity of fully active matrilysin to cleave specific physiologic substrates. In contrast, heparan sulfate and less sulfated forms of chondroitin sulfate did not augment matrilysin activation or activity. Chondroitin-2,6-sulfate (CS-D) also did not enhance matrilysin activity, suggesting that the presentation of sulfates is more important than the overall degree of sulfation. Surface plasmon resonance demonstrated that promatrilysin bound heparin (K_D, 400 nm) and CS-E (K_D, 630 nm). Active matrilysin bound heparin (K_D, 150 nm) but less so to CS-E (K_D, 60 μm). Neither form bound heparan sulfate. These observations demonstrate that sulfated GAGs regulate matrilysin activation and its activity against specific substrates.Matrix metalloproteinases (MMPs)³ comprise a family of endopeptidases that act on a variety of extracellular proteins, such as chemokines, antimicrobial peptides, matrix components, and more, to effect numerous repair, immune, and disease processes (1–3). For many substrates, MMP cleavage results in gain-of-function processing, such as the activation of latent antimicrobial peptides (4, 5) and cytokines (1), or altered biologic activity, as with limited proteolysis of chemokines (6, 7) and shedding of cell surface proteins (8). Thus, the mechanisms controlling zymogen activation and proteinase activity against specific substrates would sit high in the hierarchy of events controlling many host response pathways. As for all proteinases, the activity of MMPs is regulated at four points: gene expression, compartmentalization (i.e. pericellular accumulation of enzyme), proenzyme (or zymogen) activation, and enzyme inactivation, and is further controlled by substrate availability, concentration, and affinity.ProMMPs are kept in a catalytically inactive state by the interaction between the thiol of the conserved prodomain cysteine and the zinc ion of the catalytic site. To become active, the thiol-Zn²⁺ interaction, commonly called the “cysteine switch,” must be disrupted (9), which can be mediated by proteolysis of the prodomain, post-translational modification of the thiol, allosteric interactions with other macromolecules, or other possible mechanisms (10). About one-third of proMMPs contains a furin-recognition sequence and are activated in the secretion pathway by furin proprotein convertase cleavage of the prodomain. However, with the possible exception of proMMP2 activation by MMP14, the physiologic activation mechanism of most MMPs is not known (10).Matrilysin (28 kDa zymogen, 19 kDa active enzyme) is expressed by mucosal epithelia and some macrophages and functions as a key effector of repair and immunity. Established functions of matrilysin include facilitating re-epithelialization (11, 12), cleaving Fas ligand to promote apoptosis (13, 14), shedding syndecan-1 to control neutrophil influx (15), and macrophage-mediated elastolysis (16).In mice, matrilysin activates pro-α-defensins (procryptdins), a family of structurally similar 3–4 kDa antimicrobial peptides found in the granules of Paneth cells at the base of the crypts of Lieberkühn (17). Because of the lack of mature cryptdins, matrilysin-null (Mmp7^−/−) mice have an impaired ability to battle enteric pathogens (4). Cryptdins are packaged as pro-proteins of 7–8 kDa and are cleaved at a conserved site by matrilysin within the secretion granules (4, 18). In resting Paneth cells, the steady-state levels of pro- and activated cryptdins are roughly equivalent. Upon stimulation, the balance of procryptdins is rapidly activated indicating efficient proteolysis by matrilysin within the secretory pathway (18, 19). However, in defined in vitro reactions containing just substrate and proteinase, activation of procryptdins by matrilysin is slow, with only 50% of the precursor cleaved in 8 h or longer (4). Furthermore, both pro- and active matrilysin are present in Paneth cells granules (4) indicating that this MMP is activated in vivo by prodomain cleavage. The inefficient cleavage of procryptdins in vitro, their rapid processing in vivo, and the presence of activated matrilysin in Paneth cell granules led us to hypothesize that other factors regulate both the activation of promatrilysin and its activity against physiologic substrates.Yu et al. (20, 21) reported that heparin increases matrilysin activity about 2–4-fold in a transferrin zymogram assay, and they reported that matrilysin colocalizes to heparan sulfate molecules in tissue. However, transferrin is not a physiologic substrate of this MMP, and it is not known how heparin and other glycosaminoglycans affect matrilysin activity against established substrates, such as procryptdins. Therefore, we assessed matrilysin activity in vitro in the presence of various glycosaminoglycans (GAGs), and we found that both zymogen activation and activity against specific substrates are markedly enhanced by highly sulfated molecules. Our findings suggest that specific GAGs function to control matrilysin proteolysis.     

Yarrowia lipolytica as a model for bio-oil production

The yeast Yarrowia lipolytica has developed very efficient mechanisms for breaking down and using hydrophobic substrates. It is considered an oleaginous yeast, based on its ability to accumulate large amounts of lipids. Completion of the sequencing of the Y. lipolytica genome and the existence of suitable tools for genetic manipulation have made it possible to use the metabolic function of this species for biotechnological applications. In this review, we describe the coordinated pathways of lipid metabolism, storage and mobilization in this yeast, focusing in particular on the roles and regulation of the various enzymes and organelles involved in these processes. The physiological responses of Y. lipolytica to hydrophobic substrates include surface-mediated and direct interfacial transport processes, the production of biosurfactants, hydrophobization of the cytoplasmic membrane and the formation of protrusions. We also discuss culture conditions, including the mode of culture control and the culture medium, as these conditions can be modified to enhance the accumulation of lipids with a specific composition and to identify links between various biological processes occurring in the cells of this yeast. Examples are presented demonstrating the potential use of Y. lipolytica in fatty-acid bioconversion, substrate valorization and single-cell oil production. Finally, this review also discusses recent progress in our understanding of the metabolic fate of hydrophobic compounds within the cell: their terminal oxidation, further degradation or accumulation in the form of intracellular lipid bodies.     

N-glycan trimming by glucosidase II is essential for Arabidopsis development

Glucosidase II, one of the early N-glycan processing enzymes and a major player in the glycoprotein folding quality control, has been described as a soluble heterodimer composed of α and β subunits. Here we present the first characterization of a plant glucosidase II α subunit at the molecular level. Expression of the Arabidopsis α subunit restored N-glycan maturation capacity in Schizosaccharomyces pombe α− or αβ−deficient mutants, but with a lower efficiency in the last case. Inactivation of the α subunit in a temperature sensitive Arabidopsis mutant blocked N-glycan processing after a first trimming by glucosidase I and strongly affected seedling development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Cecilia D’Alessio and Thomas Paccalet have equal contributions to this work An erratum to this article can be found at     

Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3′-side on a 31 nt ssDNA.The replication protein A (RPA) is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes (1–3). RPA is one of the key players in various essential processes of DNA metabolism including replication, recombination, DNA repair and telomere maintenance (1,2,4–9). The functions of this protein are based on its DNA-binding activity and specific protein–protein interactions. Its ssDNA binding properties depend on DNA length and nucleotide sequence (6,10–13). RPA is a heterotrimeric protein, composed of 70-, 32- and 14-kDa subunits, commonly referred to as RPA1, RPA2 and RPA3, respectively. There are four DNA-binding domains (DBD) located in RPA1 (DBD A, DBD B, DBD C and DBD F), one located in RPA2 (DBD D) and one belongs to RPA3 (DBD E). RPA interacts with ssDNA via four DBD: DBD A, DBD B, DBD C and DBD D (14).It is now accepted (11) that RPA binds to ssDNA in a sequential pathway with a defined polarity (15–17). RPA binds ssDNA with three different binding modes. First, binding initially involves an unstable recognition site of 8–10 nt with the high-affinity DBD A and DBD B domains on the 5′-side of the occluded ssDNA; it is designated ‘compact conformation’ or 8–10 nt binding mode. Second, this step is followed by the weaker binding of DBD C, on the 3′-side, leading to an intermediate or ‘elongated contracted’ (13–22 nt) binding mode (18–19). Finally binding of DBD D on the 3′-side forms a stable ‘elongated extended’ complex characterized by a 30 nt long occluded binding site (30 nt binding mode). Although RPA3 contains an Oligonucleotide-Binding (OB)-fold motif found in the other DBDs, there is presently no biochemical evidence that this subunit directly contacts DNA. Thus positioning of the RPA3 subunit relative to the other domains is still speculative (11,20). It has been clearly demonstrated that RPA3 is crucial for RPA function (1,2): RPA3 is involved in heterotrimer formation and is responsible for the polarity of binding to DNA (11,21,22). The scope of the data indicates that either RPA3 participates only in protein–protein interactions or that putative interaction of RPA3 with ssDNA is unstable and too transient to be detected by standard biochemical experiments. This latter possibility is likely if such interaction is provided by the 3′-side of the ssDNA, since it has been suggested that this region might be transiently accessible to the RPA DBD domains (23,24).In the past few years, thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded molecules and to identify transient contacts within nucleic acids and/or between nucleic acids and proteins, in nucleoprotein assemblies (25). Thio residues such as 4-thiothymine and 6-thioguanine absorb light at wavelengths longer than 320 nm, and thus can be selectively photo-activated. Owing to the high photo-reactivity of their triplet state, they exhibit high photo-cross-linking ability towards nucleic acid bases as well as towards amino acid residues. Here we used a combination of approaches including gel retardation assays, chemical cross-linking and cross-linking with photoreactive ssDNA probes containing 4-thiothymine, introduced at a defined site in the sequence of the ssDNA, to study interactions present in human RPA (hRPA): ssDNA complexes. These studies coupled with the identification of cross-linked targets using specific antibodies revealed that in the elongated extended hRPA:ssDNA complex RPA3 closely contacts the 3′-end positioned nucleotide and yields a covalent adduct with zero-length photolabel.     

Clustering of C-Terminal Stromal Domains of Tha4 Homo-oligomers during Translocation by the Tat Protein Transport System

The chloroplast Twin arginine translocation (Tat) pathway uses three membrane proteins and the proton gradient to transport folded proteins across sealed membranes. Precursor proteins bind to the cpTatC-Hcf106 receptor complex, triggering Tha4 assembly and protein translocation. Tha4 is required only for the translocation step and is thought to be the protein-conducting component. The organization of Tha4 oligomers was examined by substituting pairs of cysteine residues into Tha4 and inducing disulfide cross-links under varying stages of protein translocation. Tha4 formed tetramers via its transmembrane domain in unstimulated membranes and octamers in membranes stimulated by precursor and the proton gradient. Tha4 formed larger oligomers of at least 16 protomers via its carboxy tail, but such C-tail clustering only occurred in stimulated membranes. Mutational studies showed that transmembrane domain directed octamers as well as C-tail clusters require Tha4's transmembrane glutamate residue and its amphipathic helix, both of which are necessary for Tha4 function. A novel double cross-linking strategy demonstrated that both transmembrane domain directed- and C-tail directed oligomerization occur in the translocase. These results support a model in which Tha4 oligomers dock with a precursor–receptor complex and undergo a conformational switch that results in activation for protein transport. This possibly involves accretion of additional Tha4 into a larger transport-active homo-oligomer.     

Origin and evolution of the Notch signalling pathway: an overview from eukaryotic genomes

Background  

Of the 20 or so signal transduction pathways that orchestrate cell-cell interactions in metazoans, seven are involved during development. One of these is the Notch signalling pathway which regulates cellular identity, proliferation, differentiation and apoptosis via the developmental processes of lateral inhibition and boundary induction. In light of this essential role played in metazoan development, we surveyed a wide range of eukaryotic genomes to determine the origin and evolution of the components and auxiliary factors that compose and modulate this pathway.