SynopsisDeimination (or citrullination) is a recently described post-translational modification, but its consequences are not yet well understood. It is catalysed by peptidylarginine deiminases (PADs). These enzymes transform arginyl residues involved in a peptidyl link into citrullyl residues in a calciumdependent manner. Several PAD substrates have already been identified like filaggrin and keratins K1 and K10 in the epidermis, trichohyalin in hair follicles, but also ubiquitous proteins like histones. PADs act in a large panel of physiological functions as cellular differentiation or gene regulation. It has been suggested that deimination plays a role in many major diseases such as rheumatoid arthritis, multiple sclerosis, Alzheimer's disease and psoriasis. Five human genes (PADIs), encoding five highly conserved paralogous enzymes (PAD1-4 and 6), have been characterized. These genes are clustered in a single locus, at 1p35-36 in man. Only PAD1-3 are expressed in human epidermis. PADs seem to be controlled at transcriptional, translational and activity levels and they present particular substrate specificities. In this review, we shall discuss these main biochemical, genetic and functional aspects of PADs together with their pathophysiological implications. Ré suméLa désimination (ou citrullination) est une modification post-traductionnelle catalysée par les peptidylarginine désiminases (PADs), décrite depuis peu et dont les conséquences sont encore mal comprises. Ces enzymes transforment, de façon dépendante du calcium, les résidus arginyl engagés dans un lien peptidique en résidus citrullyl. Plusieurs substrats ont été identifiés: la filaggrine et les cytokératines K1 et K10 de l'épiderme, la trichohyaline dans le follicule pileux mais aussi des protéines ubiquistes comme les histones. Les PADs interviennent dans de nombreuses fonctions physiologiques telles que la différenciation cellulaire ou la régulation génique. La désimination pourrait jouer un rôle dans plusieurs maladies sévères et fréquentes comme la polyarthrite rhumatoïde, la sclérose en plaque, la maladie d'Alzheimer ou encore le psoriasis. Cinq gènes humains (PADIs) codant pour 5 enzymes paralogues conservées (PAD1-4 et 6) ont été caractérisés. Ils sont regroupés en un seul locus, en 1p35-36 chez l'homme. Seules les PAD1-3 sont exprimées dans l'épiderme humain. Les PADs semblent contrôlées aux niveaux transcriptionnel et Correspondence: Marie-Claire Méchin, UMR5165, Faculté de Médecine,

Vascular calcification is a hallmark of advanced atherosclerosis, but the underlying mechanisms remain unknown. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells (vSMCs) of Low Density Lipoprotein receptor (LDLr) deficient mice fed an atherogenic high-cholesterol diet results in accelerated vascular calcification with chondrogenic metaplasia within the lesions. We demonstrate that vascular calcification in the absence of PPARγ requires the transmembrane receptor Low Density Lipoprotein receptor-related protein-1 (LRP1). LRP1 promotes a previously unknown Wnt5a dependent prochondrogenic pathway that activates the chondrogenic program. PPARγ protects against vascular calcification by activating sFRP2, which we show functions as a Wnt5a antagonist. Thus, targeting this signaling pathway has important clinical implications, impacting on common complications of atherosclerosis including coronary artery calcification and valvular sclerosis.

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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.

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 PKC␤1, PKC␣, PKC, and PKC␦, resulting in their accumulation at the entire plasma membrane (PKC␤ and -␦) or selectively at the cell-cell contacts (PKC␣ and -). The duration of activation ranged from 20 s for PKC␣ to 20 min for PKC. PKC␣ and -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 PKC␣ localization is controlled by PKC␤1 activity and is calcium independent, while PKC localization is dependent on PKC␣ activity. PKC␦ was independent of the cascade linking PKC␤1, -␣, and -. Furthermore, PKC␣, but not PKC, is involved in the TRH-induced ␤-catenin relocation at cell-cell contacts, suggesting that PKC is not the unique functional effector of the cascade. Thus, TRH receptor activation results in PKC␤1 activation, which in turn initiates a calcium-independent but PKC␤1 activity-dependent sequential translocation of PKC␣ and -. These results challenge the current understanding of PKC signaling and raise the question of a functional dependence between isoforms.Space and time parameters are intimately linked to the biological function of proteins and are pivotal in the organization and functioning of living matter. Particularly important in the case of intracellular signaling networks, this spatiotemporal constraint determines the modalities by which a given protein interacts with its partners in order to exchange information. Efforts are currently being made to translate the experimental evidence of this plasticity into a visualization concept of dynamic signaling networks (4,22). A degree of complexity is added when different isoforms of a protein coexist within the same cell and when they all potentially respond to the same stimulus, which is the case of the protein kinase C (PKC) family.The PKC family is composed of at least 11 isoforms. Several isoforms usually coexist within a given cell type, and each isoform is thought to mediate distinct cellular functions leading to proliferation, differentiation, apoptosis, or secretion. PKC function requires its translocation to a membrane compartment. The current understanding of PKC translocation/ activation has been largely based on the work of Oancea and Meyer (34), who presented conventional PKCs (cPKCs) and novel PKCs (nPKCs) as molecular machines responsible for decoding calcium and/or diacylglycerol (DAG)-mediated signals. Several observations suggest, however, that other, as yet unknown, parameters are involved in the temporal organization of PKC signaling. Indeed, despite similarities in sequence and cofactor regulation by DAG and Ca 2ϩ , the conventional P...

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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.

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