Nathalie Vigneron

Towards a new classification of the Arthoniales (Ascomycota) based on a three-gene phylogeny focussing on the genus Opegrapha

A multi-locus phylogenetic study of the order Arthoniales is presented here using the nuclear ribosomal large subunit (nuLSU), the second largest subunit of RNA polymerase II (RPB2) and the mitochondrial ribosomal small subunit (mtSSU). These genes were sequenced from 43 specimens or culture isolates representing 33 species from this order, 16 of which were from the second largest genus, Opegrapha. With the inclusion of sequences from GenBank, ten genera and 35 species are included in this study, representing about 18% of the genera and ca 3% of the species of this order. Our study revealed the homoplastic nature of morphological characters traditionally used to circumscribe genera within the Arthoniales, such as exciple carbonization and ascomatal structure. The genus Opegrapha appears polyphyletic, species of that genus being nested in all the major clades identified within Arthoniales. The transfer of O. atra and O. calcarea to the genus Arthonia will allow this genus and family Arthoniaceae to be recognized as monophyletic. The genus Enterographa was also found to be polyphyletic. Therefore, the following new combinations are needed: Arthonia calcarea (basionym: O. calcarea), and O. anguinella (basionym: Stigmatidium anguinellum); and the use of the names A. atra and Enterographa zonata are proposed here. The simultaneous use of a mitochondrial gene and two nuclear genes led to the detection of what seems to be a case of introgression of a mitochondrion from one species to another (mitochondrion capture; cytoplasmic gene flow) resulting from hybridization.

Proprotein convertases process Pmel17 during secretion.

Pmel17 is a melanocyte/melanoma-specific protein that traffics to melanosomes where it forms a fibrillar matrix on which melanin gets deposited. Before being cleaved into smaller fibrillogenic fragments the protein undergoes processing by proprotein convertases, a class of serine proteases that typically recognize the canonical motif RX(R/K)R↓. The current model of Pmel17 maturation states that this processing step occurs in melanosomes, but in light of recent reports this issue has become controversial. We therefore addressed this question by thoroughly assessing the processing kinetics of either wild-type Pmel17 or a secreted soluble Pmel17 derivative. Our results demonstrate clearly that processing of Pmel17 occurs during secretion and that it does not require entry of the protein into the endocytic system. Strikingly, processing proceeds even in the presence of the secretion inhibitor monensin, suggesting that Pmel17 is an exceptionally good substrate. In line with this, we find that newly synthesized surface Pmel17 is already quantitatively cleaved. Moreover, we demonstrate that Pmel17 function is independent of the sequence identity of its unconventional proprotein convertase-cleavage motif that lacks arginine in P4 position. The data alter the current view of Pmel17 maturation and suggest that the multistep processing of Pmel17 begins with an early cleavage during secretion that primes the protein for later functional processing.

Functional significance of tapasin membrane association and disulfide linkage to ERp57 in MHC class I presentation.

Tapasin is disulfide linked to ERp57 within the peptide loading complex. In cell‐free assays, a soluble variant of the tapasin/ERp57 dimer recruits MHC class I molecules and promotes peptide binding to them, whereas soluble tapasin alone does not. Here we show that within cells, tapasin conjugation with ERp57 is as critical as its integration into the membrane for efficient MHC class I assembly, surface expression, and Ag presentation to CD8+ T cells. Elimination of both of these properties severely compromises tapasin function, in keeping with predictions from in vitro studies.

Proteasome Subtypes and Regulators in the Processing of Antigenic Peptides Presented by Class I Molecules of the Major Histocompatibility Complex

The proteasome is responsible for the breakdown of cellular proteins. Proteins targeted for degradation are allowed inside the proteasome particle, where they are cleaved into small peptides and released in the cytosol to be degraded into amino acids. In vertebrates, some of these peptides escape degradation in the cytosol, are loaded onto class I molecules of the major histocompatibility complex (MHC) and displayed at the cell surface for scrutiny by the immune system. The proteasome therefore plays a key role for the immune system: it provides a continued sampling of intracellular proteins, so that CD8-positive T-lymphocytes can kill cells expressing viral or tumoral proteins. Consequently, the repertoire of peptides displayed by MHC class I molecules at the cell surface depends on proteasome activity, which may vary according to the presence of proteasome subtypes and regulators. Besides standard proteasomes, cells may contain immunoproteasomes, intermediate proteasomes and thymoproteasomes. Cells may also contain regulators of proteasome activity, such as the 19S, PA28 and PA200 regulators. Here, we review the effects of these proteasome subtypes and regulators on the production of antigenic peptides. We also discuss an unexpected function of the proteasome discovered through the study of antigenic peptides: its ability to splice peptides.

Critical residues in the PMEL/Pmel17 N-terminus direct the hierarchical assembly of melanosomal fibrils

Asp-73, Pro-75, Trp-153, and Trp-160 are essential residues in the PMEL NTR that are required for functional fibril formation. The NTR is necessary in cis to drive the downstream PKD into an amyloid core matrix, which subsequently incorporates and stabilizes the RPT domain–containing, MαC fibril–associated fragment.

Endoplasmic Reticulum Export, Subcellular Distribution, and Fibril Formation by Pmel17 Require an Intact N-terminal Domain Junction

Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes.

Inefficient exogenous loading of a tapasin-dependent peptide onto HLA-B*44:02 can be improved by acid treatment or fixation of target cells: Antigen processing

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by “cancer‐germline” genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE‐A1‐encoded protein (160‐169) and presented to CTLs by HLA‐B*44:02. Although this peptide is encoded by MAGE‐A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide‐receptive form of HLA‐B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide‐receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide‐based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.

Exogenous loading of a tapasin-dependent peptide onto HLA-B*44:02 can be restored by acid treatment or fixation of target cells

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by “cancer‐germline” genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE‐A1‐encoded protein (160‐169) and presented to CTLs by HLA‐B*44:02. Although this peptide is encoded by MAGE‐A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide‐receptive form of HLA‐B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide‐receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide‐based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.

Natural layer-by-layer photonic structure in the scales of Hoplia coerulea (Coleoptera)

Hoplia coerulea is known for its spectacular blue-violet iridescence. The blue coloration is caused by the presence of a photonic structure inside the scales which cover the dorsal parts of the insects body, including the head, the thorax, and the wing cases. The structure can be described by a stack of chitin plates wearing arrays of parallel rods. This arrangement leads to a multilayer structure which only uses a single solid material. The shift of the reflected wavelength to the ultraviolet (passing through violet iridescence) is described and explained on the basis of the optical properties of this structured metamaterial.