A new amyloidosis caused by fibrillar aggregates of mutated corneodesmosin.

Heterozygous nonsense mutations in the CDSN gene encoding corneodesmosin (CDSN), an adhesive protein expressed in cornified epithelia and hair follicles, cause hypotrichosis simplex of the scalp (HSS), a nonsyndromic form of alopecia. Truncated mutants of CDSN ((mut)CDSN), which bear the N-terminal adhesive Gly/Ser-rich domain (GS domain) of the protein, abnormally accumulate as amorphous deposits at the periphery of hair follicles and in the papillary dermis of the patient skin. Here, we present evidence that the (mut)CDSN deposits display an affinity for amyloidophilic dyes, namely Congo red and thioflavin T. We also detected the serum amyloid protein component in the dermis of HSS patients. We demonstrated that recombinant forms of (mut)CDSN and of the GS domain assemble in vitro into ring-shaped oligomeric structures and fibrils. The amyloid-like nature of the fibrils was demonstrated by dye binding and Fourier transform infrared spectrometry measurements. We showed that the ring-shaped oligomers of (mut)CDSN, but not the fibrillar forms, are toxic to cultured keratinocytes. Finally, online algorithms predicted the GS domain to be a particularly disordered region of CDSN in agreement with circular dichroism measurements. This identifies HSS as a human amyloidosis related to the aggregation of natively unfolded (mut)CDSN polypeptides into amyloid fibrils.

Deimination and expression of peptidylarginine deiminases during cutaneous wound healing in mice.

Deimination, the conversion of protein-bound arginines into citrullines, is a post-translational modification catalyzed by a peptidylarginine deiminase (Pad). In the epidermis, three Pads are expressed, namely Pad1, 2 and 3, and the major deiminated protein is filaggrin. Deimination of fibrin has been observed in various pathological inflammatory conditions. Here, we analyzed the expression of Pads and citrullination of proteins during cutaneous wound healing, i.e. in a physiological inflammatory condition. Full-thickness punches were performed on adult mouse back skin, and wound recovery was analyzed over 10 days by immunohistology and western blotting. Pad1 was immunodetected in all the neo-epidermis. Pad3, normally expressed in the stratum granulosum, was not detected in the hyperproliferative tongue of the neo-epidermis, but was shown to be co-localized with (pro)filaggrin in a large number of keratinocyte layers in its differentiating part. Deiminated proteins were detected in the stratum corneum of the neo-epidermis in the late phase of re-epithelialization, and in the clot and the clot-derived scab. In the clot where we only detected Pad4, one of the deiminated proteins was shown to be fibrin. Deimination of the clot proteins, and more generally wound healing and keratinocyte differentiation, seemed to be Pad2-independent, as shown using Padi2(-/-) mice.

Deimination is regulated at multiple levels including auto-deimination of peptidylarginine deiminases.

Peptidylarginine deiminases (PADs) catalyze deimination, converting arginyl to citrullyl residues. Only three PAD isotypes are detected in the epidermis where they play a crucial role, targeting filaggrin, a key actor for the tissue hydration and barrier functions. Their expression and activation depends on the keratinocyte differentiation state. To investigate this regulation, we used primary keratinocytes induced to differentiate either by increasing cell-density or by treatment with vitamin D. High cell-density increased PAD1 and 3, but not PAD2, at the mRNA and protein levels, and up-regulated protein deimination. By contrast, vitamin D increased PAD1-3 mRNA amounts, with distinct kinetics, but neither the proteins nor the deimination rate. Furthermore, auto-deimination was shown to decrease PAD activity, increasing the distances between the four major amino acids of the active site. In summary, deimination can be regulated at multiple levels: transcription of the PADI genes, translation of the corresponding mRNAs, and auto-deimination of PADs.

[Deimination or citrullination, a post-translational modification with many physiological and pathophysiological facets]. / La désimination ou citrullination: une modification post-traductionnelle aux multiples facettes.

Deimination or citrullination, is a post-translational modification with many facets. It is involved in several basic cellular processes, including gene regulation, embryonic development and terminal differentiation, and also in various pathophysiological mechanisms linked to severe human diseases such as multiple sclerosis and rheumatoid arthritis. Deimination, the calcium-dependent enzymatic conversion of peptidyl-arginine to peptidyl-citrulline, induces a decrease in the charge of the modified proteins with major consequences on their conformation, stability and/or interactions, and therefore on their functions. Five isotypes of peptidylarginine deiminases (1-4 and 6), exist in humans with a variable tissue expression. These highly conserved enzymes are closely regulated at transcriptional and post-transcriptional levels, probably including auto-deimination.

A spatiotemporally coordinated cascade of protein kinase C activation controls isoform-selective translocation.

In pituitary GH3B6 cells, signaling involving the protein kinase C (PKC) multigene family can self-organize into a spatiotemporally coordinated cascade of isoform activation. Indeed, thyrotropin-releasing hormone (TRH) receptor activation sequentially activated green fluorescent protein (GFP)-tagged or endogenous PKCbeta1, PKCalpha, PKCepsilon, and PKCdelta, resulting in their accumulation at the entire plasma membrane (PKCbeta and -delta) or selectively at the cell-cell contacts (PKCalpha and -epsilon). The duration of activation ranged from 20 s for PKCalpha to 20 min for PKCepsilon. PKCalpha and -epsilon selective localization was lost in the presence of Gö6976, suggesting that accumulation at cell-cell contacts is dependent on the activity of a conventional PKC. Constitutively active, dominant-negative PKCs and small interfering RNAs showed that PKCalpha localization is controlled by PKCbeta1 activity and is calcium independent, while PKCepsilon localization is dependent on PKCalpha activity. PKCdelta was independent of the cascade linking PKCbeta1, -alpha, and -epsilon. Furthermore, PKCalpha, but not PKCepsilon, is involved in the TRH-induced beta-catenin relocation at cell-cell contacts, suggesting that PKCepsilon is not the unique functional effector of the cascade. Thus, TRH receptor activation results in PKCbeta1 activation, which in turn initiates a calcium-independent but PKCbeta1 activity-dependent sequential translocation of PKCalpha and -epsilon. These results challenge the current understanding of PKC signaling and raise the question of a functional dependence between isoforms.

Peptidylarginine deiminases and deimination in biology and pathology: relevance to skin homeostasis.

Deimination corresponds to the transformation of arginine residues within a peptide sequence into citrulline residues. Catalyzed by peptidylarginine deiminases, it decreases the net positive charge of proteins, alters intra and intermolecular ionic interactions and probably the folding of target proteins. Deimination has recently been implicated in several physiological and pathological processes. Here, we describe the enzymes involved in this post-translational modification, focusing on their expression, location and roles in skin, as well as their known protein substrates in the epidermis and hair follicles. We discuss also the potential involvement of deimination in human diseases including cutaneous disorders.

The nuclear hormone receptor PPARγ counteracts vascular calcification by inhibiting Wnt5a signalling in vascular smooth muscle cells.

Vascular calcification is a hallmark of advanced atherosclerosis. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPARγ requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. We show that PPARγ protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist. Targeting this signalling pathway may have clinical implications in the context of common complications of atherosclerosis, including coronary artery calcification and valvular sclerosis.

Long-range enhancer differentially regulated by c-Jun and JunD controls peptidylarginine deiminase-3 gene in keratinocytes.

Long-range cis elements are critical regulators of transcription, particularly for clustered paralogous genes. Such are the five PADI genes in 1p35-36 encoding peptidylarginine deiminases, which catalyze deimination, a Ca2+-dependent post-translational modification. Deimination has been implicated in the pathophysiology of severe human diseases such as multiple sclerosis and rheumatoid arthritis. The PADI genes present different expression patterns. PADI1-3 are expressed in the epidermis, with increased expression levels in the most differentiated keratinocytes. Previous studies on PADI proximal promoters failed to explain such specificity of expression. We identified a conserved intergenic sequence in the PADI locus (IG1), which may play a role in PADI transcriptional regulation. In this work, we identified two DNase I.hypersensitive sites located in IG1, PAD intergenic enhancer segment 1 (PIE-S1) and PIE-S2, which act in synergy as a bipartite enhancer of the PADI3 and probably PADI1 promoters in normal human epidermal keratinocytes differentiated by a high-calcium-containing medium (1.5 mM). PIE-S1 and PIE-S2 present all the hallmarks of transcriptional enhancers: orientation-independence, copy-number dependence and cell-type specificity. PIE-S1 and PIE-S2 comprise conserved putative binding sites for MIBP1/RFX1 and activator protein 1, respectively. Deletion mutant screening revealed that these sites are crucial for the enhancer activity. Furthermore, chromatin immunoprecipitation assays evidenced differential binding of JunD or c-Jun on the activator protein 1 site depending on the cell differentiation state. Our results reveal the molecular bases of the expression specificity of PADI1 and PADI3 during keratinocyte differentiation through a long-range enhancer and support a model of PADI gene regulation depending on c-Jun-JunD competition.