CLEC12B Is a Melanocytic Gene Regulating the Color of the Skin.

Pigmentation of the human skin is a complex process regulated by many genes. However, only a few have a profound impact on melanogenesis. Transcriptome analysis of pigmented skin compared with analysis of vitiligo skin devoid of melanocytes allowed us to unravel CLEC12B as a melanocytic gene. We showed that CLEC12B, a C-type lectin receptor, is highly expressed in melanocytes and that its expression is decreased in dark skin compared with that in white skin. CLEC12B directly recruits and activates SHP1 and SHP2 through its immunoreceptor tyrosine-based inhibitory motif domain and promotes CRE-binding protein degradation, leading to the downregulation of the downstream MITF pathway. CLEC12B ultimately controls melanin production and pigmentation in vitro and in a model of reconstructed human epidermis. The identification of CLEC12B in melanocytes shows that C-type lectin receptors exert function beyond immunity and inflammation. It also provides insights into the understanding of melanocyte biology and regulation of melanogenesis.

Design and Development of a Two-Color Emissive FRET Pair Based on a Photostable Fluorescent Deoxyuridine Donor Presenting a Mega-Stokes Shift.

We report the synthesis and site-specific incorporation in oligodeoxynucleotides (ODNs) of an emissive deoxyuridine analog electronically conjugated on its C5-position with a 3-methoxychromone moiety acting as a fluorophore. When incorporated in ODNs, this fluorescent deoxyuridine analog exhibits remarkable photostability and good quantum yields. This deoxyuridine analog also displays a mega-Stokes shift, which allows for its use as an efficient donor for FRET-based studies when paired with the yellow emissive indocarbocyanine Cy3 acceptor.

Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance.

We have discovered and developed a series of molecules (thiazole benzenesulfonamides). HA15, the lead compound of this series, displayed anti-cancerous activity on all melanoma cells tested, including cells isolated from patients and cells that developed resistance to BRAF inhibitors. Our molecule displayed activity against other liquid and solid tumors. HA15 also exhibited strong efficacy in xenograft mouse models with melanoma cells either sensitive or resistant to BRAF inhibitors. Transcriptomic, proteomic, and biochemical studies identified the chaperone BiP/GRP78/HSPA5 as the specific target of HA15 and demonstrated that the interaction increases ER stress, leading to melanoma cell death by concomitant induction of autophagic and apoptotic mechanisms.

Characterisation of Structural Proteins from Chronic Bee Paralysis Virus (CBPV) Using Mass Spectrometry.

Chronic bee paralysis virus (CBPV) is the etiological agent of chronic paralysis, an infectious and contagious disease in adult honeybees. CBPV is a positive single-stranded RNA virus which contains two major viral RNA fragments. RNA 1 (3674 nt) and RNA 2 (2305 nt) encode three and four putative open reading frames (ORFs), respectively. RNA 1 is thought to encode the viral RNA-dependent RNA polymerase (RdRp) since the amino acid sequence derived from ORF 3 shares similarities with the RdRP of families Nodaviridae and Tombusviridae. The genomic organization of CBPV and in silico analyses have suggested that RNA 1 encodes non-structural proteins, while RNA 2 encodes structural proteins, which are probably encoded by ORFs 2 and 3. In this study, purified CBPV particles were used to characterize virion proteins by mass spectrometry. Several polypeptides corresponding to proteins encoded by ORF 2 and 3 on RNA 2 were detected. Their role in the formation of the viral capsid is discussed.

Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy.

BACKGROUND: Idiopathic membranous nephropathy is an autoimmune disease. In approximately 70% of patients, it is associated with autoantibodies against the phospholipase A2 receptor 1 (PLA2R1). Antigenic targets in the remaining patients are unknown. METHODS: Using Western blotting, we screened serum samples from patients with idiopathic membranous nephropathy, patients with other glomerular diseases, and healthy controls for antibodies against human native glomerular proteins. We partially purified a putative new antigen, identified this protein by means of mass spectrometry of digested peptides, and validated the results by analysis of recombinant protein expression, immunoprecipitation, and immunohistochemical analysis. RESULTS: Serum samples from 6 of 44 patients in a European cohort and 9 of 110 patients in a Boston cohort with anti-PLA2R1-negative idiopathic membranous nephropathy recognized a glomerular protein that was 250 kD in size. None of the serum samples from the 74 patients with idiopathic membranous nephropathy who were seropositive for anti-PLA2R1 antibodies, from the 76 patients with other glomerular diseases, and from the 44 healthy controls reacted against this antigen. Although this newly identified antigen is clearly different from PLA2R1, it shares some biochemical features, such as N-glycosylation, membranous location, and reactivity with serum only under nonreducing conditions. Mass spectrometry identified this antigen as thrombospondin type-1 domain-containing 7A (THSD7A). All reactive serum samples recognized recombinant THSD7A and immunoprecipitated THSD7A from glomerular lysates. Moreover, immunohistochemical analyses of biopsy samples from patients revealed localization of THSD7A to podocytes, and IgG eluted from one of these samples was specific for THSD7A. CONCLUSIONS: In our cohort, 15 of 154 patients with idiopathic membranous nephropathy had circulating autoantibodies to THSD7A but not to PLA2R1, a finding that suggests a distinct subgroup of patients with this condition. (Funded by the French National Center for Scientific Research and others.).

Black mamba venom peptides target acid-sensing ion channels to abolish pain.

Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia.

Evidence that the amyloid-ß protein precursor intracellular domain, AICD, derives from ß-secretase-generated C-terminal fragment.

One of the major pathological hallmarks of brains affected with Alzheimer's disease (AD) is the senile plaque, an extracellular deposit mainly composed of a set of highly insoluble peptides of various lengths (39-43 amino acids) referred to as amyloid-ß (Aß) peptides. Aß peptides are derived from combined proteolytic cleavages undergone on the amyloid-ß protein precursor (AßPP) by a set of enzymes called secretases. Several lines of anatomical and biological evidence suggest that Aß peptides would not account for all pathological stigmata and molecular dysfunctions taking place in AD. In amyloidogenic and non-amyloidogenic pathways, AßPP first undergoes ß- or α-secretases-mediated cleavages yielding C99 and C83, respectively. These two membrane-embedded C-terminal fragments are both potential targets of subsequent γ-secretase-mediated proteolysis. The latter cleavage not only generates either p3 or Aß peptides but similarly gives rise to an AßPP IntraCellular Domain (AICD fragment) that could modulate the transcription of several genes linked to AD pathology. It is therefore striking that AICD theoretically derives from both amyloidogenic and non-amyloidogenic AßPP processing pathways. Here we show that AICD predominantly derives from C99 by means of recombinant substrates and transiently transfected cells expressing C99. Our data suggest a preferred pathogenic pathway for AICD production and suggests that this fragment, in addition to C99 and Aß peptides, could contribute to AD pathology.

Group X secreted phospholipase A2 proenzyme is matured by a furin-like proprotein convertase and releases arachidonic acid inside of human HEK293 cells.

Among mammalian secreted phospholipases A(2) (sPLA(2)s), group X sPLA(2) has the most potent hydrolyzing activity toward phosphatidylcholine and is involved in arachidonic acid (AA) release. Group X sPLA(2) is produced as a proenzyme and contains a short propeptide of 11 amino acids ending with a dibasic motif, suggesting cleavage by proprotein convertases. Although the removal of this propeptide is clearly required for enzymatic activity, the cellular location and the protease(s) involved in proenzyme conversion are unknown. Here we have analyzed the maturation of group X sPLA(2) in HEK293 cells, which have been extensively used to analyze sPLA(2)-induced AA release. Using recombinant mouse (PromGX) and human (ProhGX) proenzymes; HEK293 cells transfected with cDNAs coding for full-length ProhGX, PromGX, and propeptide mutants; and various permeable and non-permeable sPLA(2) inhibitors and protease inhibitors, we demonstrate that group X sPLA(2) is mainly converted intracellularly and releases AA before externalization from the cell. Most strikingly, the exogenous proenzyme does not elicit AA release, whereas the transfected proenzyme does elicit AA release in a way insensitive to non-permeable sPLA(2) inhibitors. In transfected cells, a permeable proprotein convertase inhibitor, but not a non-permeable one, prevents group X sPLA(2) maturation and partially blocks AA release. Mutations at the dibasic motif of the propeptide indicate that the last basic residue is required and sufficient for efficient maturation and AA release. All together, these results argue for the intracellular maturation of group X proenzyme in HEK293 cells by a furin-like proprotein convertase, leading to intracellular release of AA during secretion.

Detection of matrix metalloproteinase active forms in complex proteomes: evaluation of affinity versus photoaffinity capture.

Various attempts to detect matrix metalloproteinase (MMP) active forms from complex proteomes, based on the use of specific photoactivatable affinity probes, have up to now failed. To overcome this failure, an affinity approach has been evaluated as an alternative to the photoaffinity one. For this purpose, two probes were synthesized to interact specifically with the active site of MMPs and allow isolation of MMP/probe complexes on magnetic beads through a biotin linker. Using phosphinic peptide chemistry, we prepared an affinity probe displaying picomolar potency toward several MMPs, and a related photoaffinity probe incorporating a photoactivatable azido group exhibiting subnanomolar affinity toward these targets. By a combination of silver-staining detection and MALDI peptide mass fingerprints, a systematic comparison was made of both strategies in terms of hMMP-12 and hMMP-8 recovery and identification when present in mixtures of different complexity. The results obtained show that the affinity protocol is superior to the photoaffinity strategy in terms of quantity of captured MMPs and number of MMP tryptic fragments detected in MALDI-MS. The specificity and efficiency of the affinity capture protocol developed in this study allowed easy, fast, and unambiguous detection by MALDI-MS of three hMMPs (2, 8, and 12), from a single affinity capture experiment, when added (10-36 ng of MMPs) to a tumor extract (10 microg). Thus, the tools and approaches reported should enable us to progress in the detection of endogenous active forms of MMPs in complex proteomes, an important objective with many diagnostic applications.

Covalent modification of matrix metalloproteinases by a photoaffinity probe: influence of nucleophilicity and flexibility of the residue in position 241.

A photoaffinity probe, developed for the specific labeling of matrix metalloproteinase (MMP) active sites, was recently shown to covalently modify a single residue in human MMP-12, namely, Lys(241), by reacting selectively with the side chain epsilon-amino group of that residue. The residue in position 241 of MMPs is not conserved; thus, variability in this position may be responsible for the dispersion in cross-linking yield observed between MMPs when labeled by this photoaffinity probe. By studying the pH dependence of the labeling properties of this probe toward different MMPs (MMP-12, MMP-3, MMP-9, and various mutants of human MMP-12) and identifying the site of covalent modification of MMP-3 by this probe, our new data demonstrated that the nucleophilicity of the residue in position 241 plays a key role in determining the cross-linking yield of MMP modification by the probe. However, these studies also reveal that subtle additional structural parameters, including local conformation and flexibility, of the residue in position 241 should also be taken into consideration, a property adding a further degree of complexity in our understanding of the photolabeling probe reactivity and in designing optimal photoaffinity probes for performing functional proteomic studies of zinc proteinases like MMPs.

Molecular determinants of matrix metalloproteinase-12 covalent modification by a photoaffinity probe: insights into activity-based probe development and conformational variability of matrix metalloproteinases.

Mass spectroscopy, microsequencing, and site-directed mutagenesis studies have been performed to identify in human matrix metalloelastase (hMMP-12) residues covalently modified by a photoaffinity probe, previously shown to be able to covalently label specifically the active site of matrix metalloproteinases (MMPs). Results obtained led us to conclude that photoactivation of this probe in complex with hMMP-12 affects a single residue in human MMP-12, Lys(241), through covalent modification of its side chain epsilon NH(2) group. Because x-ray and NMR studies of hMMP-12 indicate that Lys(241) side chain is highly flexible, our data reveal the existence of particular Lys(241) side-chain conformation in which the epsilon NH(2) group points toward the photolabile group of the probe, an event explaining the high levels of cross-linking yield between hMMP-12 and the probe. Lys(241) is not conserved in MMPs, thus differences in cross-linking yields observed with this probe between MMP members may be linked to the residue variability observed at position 241 in this family.