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The prolyl-specific peptidase fibroblast activation protein-α (FAP-α) is expressed at very low or undetectable levels in nondiseased human tissues but is selectively induced in activated (myo)fibroblasts at sites of tissue remodeling in fibrogenic processes. In normal regenerative processes involving transient fibrosis FAP-α+(myo)fibroblasts disappear from injured tissues, replaced by cells with a normal FAP-α- phenotype. In chronic uncontrolled pathological fibrosis FAP-α+(myo)fibroblasts permanently replace normal tissues. The mechanisms of regulation and elimination of FAP-α expression in(myo)fibroblasts are unknown. According to a yeast two-hybrid screen and protein databanks search, we propose that the intracellular (co)-chaperone BAG6/BAT3 can interact with FAP-α, mediated by the BAG6/BAT3 Pro-rich domain, inducing proteosomal degradation of FAP-α protein under tissue homeostasis. In this Perspective, we discuss our findings in the context of current knowledge on the regulation of FAP-α expression and comment potential therapeutic strategies for uncontrolled fibrosis, including small molecule degraders (PROTACs)-modified FAP-α targeted inhibitors.

Actin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of protrusions or limits positive signals to allow them to split or retract. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interactor). CYRI binds specifically to activated Rac1 via a common motif that is also found in CYFIP, the Domain of Unknown Function DUF1394; we demonstrate that DUF1394 is a new class of Rac1 binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but exhibit reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation are larger and longer-lived in the absence of CYRI. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE complex to the cell edge, resulting in short-lived, unproductive protrusions. CYRI’s role in cell behaviour is therefore to focus positive protrusion signals and regulate pseudopod complexity and dynamics by inhibiting Scar/WAVE induced actin. As such it behaves like a “local inhibitor” predicted and described in widely accepted mathematical models, but not previously identified in living cells. CYRI is important for biological processes requiring polarity and plasticity of protrusions, including directional migration and polarization of epithelial cysts.

Actin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of a protrusion or limits positive signals to cause splitting or retraction. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interacting) protein. CYRI shows sequence similarity to the Scar/WAVE complex member CYFIP in a Domain of Unknown Function, DUF1394. CYRI binds specifically to activated Rac1 via a common motif shared with CYFIP, establishing DUF1394 as a new Rac1 binding domain. CYRI-depleted cells have broad, Scar/WAVE-enriched lamellipodia and enhanced Rac1 signaling. Conversely, CYRI overexpression suppresses spreading and dramatically sharpens protrusions into unproductive needles. CYRI proteins use dynamic inhibition of Scar/WAVE induced actin to focus positive protrusion signals and regulate pseudopod complexity. CYRI behaves like a “local inhibitor” predicted and described in widely accepted mathematical models, but not previously identified in living cells.

Fibroblast activation protein-α (FAP-α) belongs to the family of prolyl-specific serine proteases. FAP-α displays both exopeptidase and endopeptidase/gelatinase/collagenase activities. FAP-α protein and/or activity have been associated with fibrosis, inflammation and cancer, but the protein is undetectable in most normal tissues. FAP-α is selectively expressed at sites of tissue remodeling and repair and enhances tumor progression, suggesting that this protease may be a therapeutic target to treat human disorders associated with fibrotic dysregulation. Areas covered: In this review, we summarize the mechanisms driving tissue fibrosis and describe some of the enzymes involved in fibrosis, concentrating on FAP-α. We describe its enzymatic properties, discuss the tools developed to control its activity and the problem of selectivity toward the other proteases of the family and outline its potential biological substrates. We also consider non-enzymatic functions of this protein and suggest that repression of FAP-α expression may represent therapeutic options. Expert opinion: Questions remain regarding the biological functions of FAP-α, either dependent or independent of its enzyme activity. However, as progress is underway to develop FAP-α-specific inhibitors and therapeutic antibodies, its role in diseases associated with fibrosis is starting to emerge, ultimately leading to novel therapeutic options for inflammatory and oncologic diseases.

Fruit ripening is a complex developmental process responsible for the transformation of the seed-containing organ into a tissue attractive to seed dispersers and agricultural consumers. The coordinated regulation of the different biochemical pathways necessary to achieve this change receives considerable research attention. The MADS-box transcription factor RIPENING INHIBITOR (RIN) is an essential regulator of tomato (Solanum lycopersicum) fruit ripening but the exact mechanism by which it influences the expression of ripening-related genes remains unclear. Using a chromatin immunoprecipitation approach, we provide evidence that RIN interacts with the promoters of genes involved in the major pathways associated with observed and well-studied ripening phenotypes and phenomena, including the transcriptional control network involved in overall ripening regulation, ethylene biosynthesis, ethylene perception, downstream ethylene response, cell wall metabolism, and carotenoid biosynthesis. Furthermore, in the cases of ethylene and carotenoid biosynthesis, RIN interacts with the promoters of genes encoding rate-limiting activities. We also show that RIN recruitment to target loci is dependent on a normally functioning allele at the ripening-specific transcription factor COLORLESS NONRIPENING gene locus, further clarifying the relationship between these two ripening regulators.

To identify host factors that play critical roles in processes, including cell-to-cell movement of plant-adapted rhabdoviruses, we constructed and validated a high-resolution Nicotiana benthamiana yeast two-hybrid library. The library was screened with the putative movement protein (sc4), nucleocapsid (N), and matrix (M) proteins of Sonchus yellow net virus (SYNV). This resulted in identification of 31 potential host factors. Steady-state localization studies using autofluorescent protein fusions to full-length clones of interactors were conducted in transgenic N. benthamiana marker lines. Bimolecular fluorescence complementation assays were used to validate two-hybrid interactions. The sc4 interactor, sc4i21, localized to microtubules. The N interactor, Ni67, localized to punctuate loci on the endoplasmic reticulum. These two proteins are 84% identical homologues of the Arabidopsis phloem-associated transcription activator AtVOZ1, and contain functional nuclear localization signals. Sc4i17 is a microtubule-associated motor protein. The M interactor, Mi7, is a nuclear-localized transcription factor. Combined with a binary interaction map for SYNV proteins, our data support a model in which the SYNV nucleocapsids are exported from the nucleus and moved cell-to-cell by transcription activators tethered in the cytoplasm.

International audience; In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT 1 and MT 2 receptors, belonging to the G protein-coupled receptor (GPCR) super-family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT 1 and MT 2 receptors, which comprises 378 individual proteins. Among the proteins interacting with MT 1 , but not with MT 2 , we identified several presynaptic proteins, suggesting a potential role of MT 1 in neurotransmission. Presynaptic localization of MT 1 receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT 1 physically interacts with the voltage-gated calcium channel Ca v 2.2 and inhibits Ca v 2.2-promoted Ca 2+ entry in an agonist-independent manner. In conclusion, we show that MT 1 is part of the presynaptic protein network and negatively regulates Ca v 2.2 activity, providing a first hint for potential synaptic functions of MT 1 .

Cytochrome c peroxidase (I) from Saccharomyces cerevisiae was subjected to directed mol. evolution to generate mutants with increased activity against ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)]. Using a combination of DNA shuffling and satn. mutagenesis, mutants were isolated which possessed >20-fold increased activity against ABTS and a 70-fold increased specificity toward ABTS compared to the natural substrate. In contrast, activities against another small org. mol., guaiacol, were not significantly affected. Mutations at residues Asp-224 and Asp-217 were responsible for this increase in activity. These 2 residues are located on the surface of the protein and not in the direct vicinity of the distal cavity of the peroxidase, where small org. substrates are believed to be oxidized. Mutations at position Asp-224 also lead to an increased amt. of the active holoenzyme expressed in Escherichia coli, favoring the selection of these mutants in the employed colony screen. Possible explanations for the effect of the mutations on the in vitro activity of I as well as the increased amt. of holoenzyme were discussed. (c) 2001 Academic Press. [on SciFinder (R)]

International audience; Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rapidly emerging human disease in which mycolactone, a cytotoxic and immunosuppressive macrocyclic polyketide, is responsible for massive skin destruction. The genome sequencing of M. ulcerans has recently been accomplished (http://genolist.pasteur.fr/BuruList/) enabling the first proteome study of this important human pathogen. Here, we present a comprehensive proteome analysis of different subcellular fractions and culture supernatant of in vitro grown M. ulcerans. By a combination of gel-based and gel-free techniques for protein and peptide separation with subsequent analysis by MS, we identified 1074 different proteins, corresponding to 25% of the protein-coding DNA sequence. Interestingly, new information was obtained about central metabolism and lipid biosynthesis, and as many as 192 conserved hypothetical proteins were found. Comparative analysis of the wild-type strain and an isogenic mycolactone-deficient mutant, by 2-DE and iTRAQ labeling of the cytoplasmic fraction, revealed differences in the expression profiles of proteins involved in lipid metabolism and information pathways, as well as stress responses.

A review. Directed evolution consists of repetitive cycles of random mutagenesis of the protein/peptide sequence followed by screening or selection of candidates with desired properties. Many different approaches are used to introduce mutations into a gene; most of the currently used are error-prone polymerase chain reaction (PCR), DNA shuffling, and satn. mutagenesis. On the other hand, the techniques used in screening or selection expts. range from facile colony activity screening to yeast two-hybrid systems or in-vitro selection display systems such as phage display, mRNA display, and ribosome display. All these approaches have been used to alter protein function, to increase the activity or soly. of proteins, or to adapt enzymes for industrial applications. And, although none of these approaches provides the ultimate soln. to the directed evolution of proteins, numerous examples of successfully altered and improved proteins clearly show the power of directed evolution for protein design. [on SciFinder (R)]

Protein interaction mapping provides molecular basis to protein functions and signaling pathways. We have developed a high-throughput yeast two-hybrid technology that allows the exhaustive screening of highly complex, domain-based cDNA libraries relevant to the immune system (e.g. leukocytes and activated mononuclear cells, thymocytes CD4+/CD8+ and T-cells). These libraries are screened to saturation thanks to an optimized mating procedure, allowing to test on average 100 million interactions per screen. This corresponds to a 10-fold coverage of the libraries, and ensures exhaustive and reproducible results. Moreover, multiple independent fragments are isolated for each interactant, enabling the immediate delineation of a minimal interacting domain and the computation of a confidence score. A database containing over five thousand cDNA screens realized with this technique permits a rigorous calculation of relative prey connectivity and understanding of true/false positives and sticky proteins. We have developed additional methods to probe and characterize protein interactions in vitro with a medium to high throughput, using Surface Plasmon Resonance and a miniaturized time-resolved FRET assay which has been used for drug discovery projects targeting protein-protein interactions. Here, we will present results that illustrate the strength and benefits of our protein interaction screening and validation technologies to decipher the molecular components of immune responses.

Background Thestate of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown. Methodology/Principal Findings We first confirmed that SMILE mRNA is differentially expressed in the blood of operationally tolerant patients with drug-free long term graft function compared to stable and rejecting patients. Using a yeast two-hybrid approach and a colocalization study by confocal microscopy we furthermore report an interaction of SMILE with PDIA3, a molecule resident in the endoplasmic reticulum (ER). In accordance with this observation, SMILE silencing in HeLa cells correlated with the modulation of several transcripts involved in proteolysis and a decrease in proteasome activity. Finally, SMILE silencing increased HeLa cell sensitivity to the proteasome inhibitor Bortezomib, a drug that induces ER stress via protein overload, and increased transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes. Conclusion/Significance In this study we showed that SMILE is involved in the endoplasmic reticulum stress response, by modulating proteasome activity and XBP-1 transcript expression. This function of SMILE may influence immune cell behavior in the context of transplantation, and the analysis of endoplasmic reticulum stress in transplantation may reveal new pathways of regulation in long-term graft acceptance thereby increasing our understanding of tolerance.

Protein interaction mapping has proven instrumental for the delineation and understanding of cancer signaling pathways, both in Homo sapiens and model organisms. We have recently published a drosophila protein interaction map centered on fly orthologs of human cancer-related and signaling proteins1. We now report the completion of the cognate human interaction map using the same high-throughput, domain-based yeast two-hybrid (Y2H) technology. 149 human homologs of the 102 drosophila proteins, including 29 human oncogenes and tumor suppressors2, were used as entry points to screen a highly complex, random-primed placenta cDNA library comprised of 10 million independent fragments in yeast. In order to strictly parallel the drosophila study, the same domains were selected as baits on human orthologs, resulting in 230 different screens. To ensure reproducible and exhaustive Y2H results, the library was screened at saturation using an optimized mating procedure that allowed for testing on average 50 million interactions per screen, corresponding to a 5-fold coverage of the library. As a consequence, multiple, independent fragments of the same interactant could be isolated, enabling the delineation of a minimal interacting domain and the computation of a confidence score. We identified 4,089 protein-protein interactions (PPI) connecting 2,412 human proteins. 1,212 PPI (30%) are of high confidence and connect 998 proteins. Interestingly, 54 additional human oncogenes or tumor suppressors were identified in the screening, and a sub-network of 1089 PPI linking at least one cancer protein was derived from the full map. The features of the drosophila and human parallel interaction maps were compared. Orthology relationships were combined with Y2H data and the conservation of interaction networks across evolution was examined. Experimentally determined interacting domains were also systematically confronted to known functional domains in both species, pointing to a significant enrichment of domains involved in protein binding. Finally, both experimental protein interaction maps were extended by including protein interaction data from the literature. We believe that this protein interaction map centered on human cancer signaling pathways will be an invaluable resource for researchers in the field. References : 1. Formstecher et al., 2005, Genome Research, 15:376 2. Futreal et al., 2004, Nature Review Cancer, 4(3):177 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4016.

The role of biofilms in the pathogenesis of mycobacterial diseases remains largely unknown. Mycobacterium ulcerans, the etiological agent of Buruli ulcer, a disfiguring disease in humans, adopts a biofilm-like structure in vitro and in vivo, displaying an abundant extracellular matrix (ECM) that harbors vesicles. The composition and structure of the ECM differs from that of the classical matrix found in other bacterial biofilms. More than 80 proteins are present within this extracellular compartment and appear to be involved in stress responses, respiration, and intermediary metabolism. In addition to a large amount of carbohydrates and lipids, ECM is the reservoir of the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, and purified vesicles extracted from ECM are highly cytotoxic. ECM confers to the mycobacterium increased resistance to antimicrobial agents, and enhances colonization of insect vectors and mammalian hosts. The results of this study support a model whereby biofilm changes confer selective advantages to M. ulcerans in colonizing various ecological niches successfully, with repercussions for Buruli ulcer pathogenesis., Author Summary Mycobacterium ulcerans is the etiologic agent of Buruli ulcer, a necrotic skin disease affecting humans living close to wetlands in tropical countries. This mycobacteria resides in water where it could colonize many ecological niches such as aquatic plants, herbivorous animals, and water bugs. The latter were shown to be able to transmit the bacteria to mammalian hosts. Here, we described that the bacilli could be structured with a thick envelope called the extracellular matrix (ECM). This peculiar coat contains in small vesicles a toxin named mycolactone, the main virulence factor of M. ulcerans. The ECM confers to the mycobacterium increased resistance to antimicrobial agents and plays a role in virulence. Indeed, a bacteria with ECM is more potent for colonization of insect vectors and mammalian hosts compared to bacteria. Unraveling the regulation of the production of the ECM together with the export of mycolactone will be an important step in developing new pharmacological approaches for the treatment of Buruli ulcer, which has been greatly handicapped by the lack of effectiveness of the current antibiotics.

Split-protein sensors have become an important tool for the analysis of protein-protein interactions in living cells. We present here a combinatorial method for the generation of new split-protein sensors and demonstrate its application toward the (β/α) 8 -barrel enzyme N-(5′-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae . The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan. This powerful selection complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening .

Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae was subjected to directed molecular evolution to generate mutants with increased activity against the classical peroxidase substrate guaiacol, thus changing the substrate specificity of CCP from the protein cytochrome c to a small organic molecule. After three rounds of DNA shuffling and screening, mutants were isolated which possessed a 300-fold increased activity against guaiacol and an up to 1000-fold increased specificity for this substrate relative to that for the natural substrate. In all of the selected mutants, the distal arginine (Arg48), which is fully conserved in the superfamily of peroxidases, was mutated to histidine, showing that this mutation plays a key role in the significant increase in activity against phenolic substrates. The results suggest that, in addition to stabilizing the reactive intermediate compound I, the distal arginine plays an important role as a gatekeeper in the active site of CCP, controlling the access to the ferryl oxygen and the distal histidine. Other isolated mutations increase the general reactivity of the peroxidase or increase the intracellular concentration of the active holo form, allowing their selection under the employed screening conditions. The results illustrate the ability of directed molecular evolution technologies to deliver solutions to biochemical problems that would not be readily predicted by rational design.

Combinatorial mutagenesis was used to investigate the role of three key residues in cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae, Arg48, Trp51, and Trp191, in control of the reactivity and selectivity of the heme-containing enzyme. Libraries were prepared by randomization of these residues and were subsequently screened for activity against the phenolic substrate guaiacol. Screening conditions were employed that favor either mutants with high activity or those with both high activity and stability of the reactive enzyme intermediates. The results obtained suggest a dual role for Arg48 of CCP: in addition to stabilizing reactive enzyme intermediates, the distal arginine residue plays a major role in restriction of access to the ferryl oxygen atom by small molecules and thereby controls reactivity and substrate specificity of the peroxidase. At position 51 of CCP, either a phenylalanine or a tryptophan residue is required both for catalytic and structural reasons. In contrast, either polar or positively charged residues are accepted at the position of Trp191, which is located inside the core of the protein. The variability at position 191 can be interpreted as a reflection of the mechanism of cytochrome c peroxidase, which transforms the nonpolar Trp191 into a transient cation radical.

A review. Directed evolution consists of repetitive cycles of random mutagenesis of the protein/peptide sequence followed by screening or selection of candidates with desired properties. Many different approaches are used to introduce mutations into a gene; most of the currently used are error-prone polymerase chain reaction (PCR), DNA shuffling, and satn. mutagenesis. On the other hand, the techniques used in screening or selection expts. range from facile colony activity screening to yeast two-hybrid systems or in-vitro selection display systems such as phage display, mRNA display, and ribosome display. All these approaches have been used to alter protein function, to increase the activity or soly. of proteins, or to adapt enzymes for industrial applications. And, although none of these approaches provides the ultimate soln. to the directed evolution of proteins, numerous examples of successfully altered and improved proteins clearly show the power of directed evolution for protein design. [on SciFinder (R)]