Novel signatures of cancer-associated fibroblasts.

Increasing evidence indicates the importance of the tumor microenvironment, in particular cancer-associated fibroblasts, in cancer development and progression. In our study, we developed a novel, visually based method to identify new immunohistochemical signatures of these fibroblasts. The method employed a protein list based on 759 protein products of genes identified by RNA profiling from our previous study, comparing fibroblasts with differential growth-modulating effect on human cancers cells, and their first neighbors in the human protein interactome. These 2,654 proteins were analyzed in the Human Protein Atlas online database by comparing their immunohistochemical expression patterns in normal versus tumor-associated fibroblasts. Twelve new proteins differentially expressed in cancer-associated fibroblasts were identified (DLG1, BHLHE40, ROCK2, RAB31, AZI2, PKM2, ARHGAP31, ARHGAP26, ITCH, EGLN1, RNF19A and PLOD2), four of them can be connected to the Rho kinase signaling pathway. They were further analyzed in several additional tumor stromata and revealed that the majority showed congruence among the different tumors. Many of them were also positive in normal myofibroblast-like cells. The new signatures can be useful in immunohistochemical analysis of different tumor stromata and may also give us an insight into the pathways activated in them in their true in vivo context. The method itself could be used for other similar analysis to identify proteins expressed in other cell types in tumors and their surrounding microenvironment

A kalpainok szerkezete, működése, élettani és kórtani szerepe = Structure and function of calpains, physiological and pathological roles

Calpains, the intracellular calcium-activated regulatory proteases are at crossroads of cellular pathways. Our aim has been to unravel the molecular interactions underlying function, both physiological and pathological. In resting cells calpains are inactive, but they are switched on by a rise in calcium concentration. We described the intramolecular structural changes accompanying these transitions. Mammalian calpain have a specific endogenous inhibitor protein, calpastatin. Based on the interaction of calpain and calpastatin, we developed a synthetic, membrane-permeable activator for calpains in intact cells. With this unique reactant, we succeded in activating calpain in living nerve cells, without a gross rise in calcium concentration. | A kalpainok, az intracelluláris kalcium aktivált szabályozó proteázok, sok sejtszintű folyamatban részt vesznek. Célul tűztük ki, hogy feltárjuk az enzim molekuláris kölcsönhatásait, ezáltal mind fiziológiás és patológiás funkcióját. A nyugalmi sejtekben a kalpain inaktív, de a kalcium koncentráció növekedésének hatására aktívvá válik. Munkánk során leírtuk ezen átalakulás intramolekuláris szerkezeti vonatkozásait. Az emlős kalpainok specifikus endogén inhibítora a kalpasztain. A kalpain-kalpasztatin kölcsönhatáson alapulva kifejlesztettünk egy intakt sejtekben is működő szintetikus, membrán-permeábilis aktivátort. Ennek az egyedülálló reaktánsnak a segítségével, képesek vagyunk élő idegsejtekben a kalpain aktiválására, az intracelluláris kalcium koncentráció megemelése nélkül

Jelfeldolgozás a sejtben: a kalpain és protein kináz rendszerek és kölcsönhatásaik = Intracellular signal processing: the calpain and protein kinase/phosphatase systems, and their interactions

1. Bizonyítottuk, hogy a kalpain B aktív és inaktív formája foszforilálható PKA, ERK1 és ERK2 kinázokkal, azonosítottuk az in vitro foszforilációs helyeket, és meghatároztuk a foszforilált fehérje megváltozott enzimológiai paramétereit. 2. Előállítottuk az összes Drosophila kalpain elleni antitestet. 3. Kidolgoztunk egy háromdimenziós elektroforetikus eljáráson alapuló, in vivo szubsztrát azonosításra használható módszert, amelynek segítségével számos kalpain szubsztrátot azonosítottunk. 4. Kifejlesztettünk egy sejtpenetráns, szintetikus kalpain szubsztrátot, amely a jelenleg ismert szubsztrátok közül a legérzékenyebb. 5. Kifejlesztettünk egy sejtpenetráns, szintetikus kalpain-aktiváló peptid párt, amely a kalpainok sejten belüli, calcium-mentes és specifikus aktiválására képes. 6. Kimutattuk, hogy patkány hippokampális agyszeletekben a kalpainok specifikus aktiválása az alap-ingerlékenység és az LTP szignifikáns növekedését idézi elő. 7. Kalpain A, kalpain B és kalpain A/B mutáns Drosophila vonalakat állítottunk elő. Elemeztük a fehérjék expresszióját vad és mutáns vonalakban, továbbá vizsgáltuk a fenotípusra gyakorolt hatásukat. A kalpain B fehérjéről megállapítottuk, hogy fontos szerepet játszik a határsejtek vándorlásában. 8. Drosophila kalpain interferencia vonalakat állítottunk elő: a kalpain B csendesített vonalak egyedei csökkent fertilitásúak voltak, a kettős mutánsok szintetikus szemiletalitást mutattak. A kalpain C hiánya letalitást okozott. | 1. We provided evidence that both the active and inactive forms of calpain B can be phosphorylated by PKA, ERK1 and ERK2. Phosphorylation sites were localized and the enzymological parameters of the modified enzymes were determined. 2. Antibodies have been generated against all forms of Drosophila calpain. 3. A new three-dimensional electrophoresis method has been developed to identify calpain substrates in vivo. Several novel substrates have been identified. 4. A synthetic, cell-permeable synthetic calpain susbtrate has been developed. This compound is more sensitive to the enzyme than any other known calpain substrate. 5. A peptide pair capable of calpain activation have also been developed. These sythetic peptides can penetrate cells and activate the enzyme in a calcium-independent manner. 6. It has been shown that specific activation of calpain(s) leads to a significantly incerased basal excitability and long-term potentiation (LTP) in rat hippocampal slices. 7. Drosophila lines mutant in calpain A, calpain B and calpain A/B have been generated. The effect of changes in calpain expression on phenotype was tested and it was found that calpain B plays a major role in regulating the migration of border cells. 8. Interference strains of Drosophila have also been generated. We found that silencing calpain B causes reduced fertility whereas double mutation causes synthetic semilethality. Calpain C deficit was found to be lethal

Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: Structural and mechanistic insights

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-alpha, the karyopherin molecule responsible for 'classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-alpha-wild-type and the importin-alpha-hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the posttranslational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme

Calpain-Catalyzed Proteolysis of Human dUTPase Specifically Removes the Nuclear Localization Signal Peptide

Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca(2+)-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase.Limited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II) ions, inducing calpain action. The presence or absence of the dUTP-analogue α,β-imido-dUTP did not show any effect on Ca(2+)-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca(2+)-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues (4)SE(5); (7)TP(8); and (31)LS(32)). The cleavage between the (31)LS(32) peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization.Results argue for a mechanism where Ca(2+)-calpain may regulate nuclear availability and degradation of dUTPase