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Eleftherios P Diamandis - One of the best experts on this subject based on the ideXlab platform.

  • Human kallikrein-related peptidase 14 (KLK14) is a new activator component of the KLK proteolytic cascade. Possible function in seminal plasma and skin.
    The Journal of biological chemistry, 2007
    Co-Authors: Nashmil Emami, Eleftherios P Diamandis
    Abstract:

    Abstract Human kallikrein-related peptidases (KLKs) are a family of 15 serine proteases mainly known for their biomarker utility in various neoplastic and non-neoplastic diseases. Despite significant progress in understanding their clinical application, little is known about the activation mechanism(s) of this important family of enzymes. Emerging evidence indicates that KLKs are activated in a stepwise manner, which is a characteristic of proteolytic cascades. Thus far, KLK cascades have been implicated in semen liquefaction and skin desquamation. Many members of the KLK family have been reported to be active in seminal plasma and/or skin, suggesting their involvement in common proteolytic cascades. KLK14, in particular, is highly active and has recently been proposed as one of the key trypsin-like proteases involved in skin desquamation. This study aims to elucidate a probable cascade-mediated role of KLK14 by 1) examining KLK14-mediated cleavage of a heptapeptide library encompassing activation sites of the 15 KLKs and 2) verifying activation of certain candidate downstream targets of KLK14 (i.e. pro-KLK1, -KLK3, and -KLK11). Heptapeptides encompassing activation motifs of KLK2, -3, -5, and -11 were cleaved with a high (≥85%) cleavage efficiency. Activation of these candidates was confirmed using full-length recombinant proteins. Pro-KLK11, -KLK3, and -KLK1 were rapidly activated in a concentration-dependent manner. Pro-KLK3 regulation was bidirectional because activation was followed by inactivation via internal cleavage of active KLK3. We are proposing a putative cascade model, operating through multiple KLKs. Identification of novel members of such proteolytic cascades will aid in further defining mechanisms involved in seminal/skin homeostasis.

  • human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients
    Experimental Dermatology, 2007
    Co-Authors: Nahoko Komatsu, Eleftherios P Diamandis, Kiyofumi Saijoh, Fumiaki Shirasaki, Kazuhiko Takehara, Saba Khan
    Abstract:

    :  Human tissue kallikreins are a family of 15 trypsin- or chymotrypsin-like secreted serine proteases (KLK1–KLK15). Many KLKs have been identified in normal stratum corneum (SC) and sweat, and are candidate desquamation-related proteases. We report quantification by enzyme-linked immunosorbent assay (ELISA) of KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of atopic dermatitis (AD) patients by ELISA, and examine their variation with clinical phenotype, correlation with blood levels of eosinophils, lactate dehydrogenase (LDH) and immunoglobulin E. The overall SC serine protease activities were also measured. In the SC of AD, all KLKs, except KLK11, were significantly elevated. The elevation of chymotrypsin-like KLK7 was predominant, compared with trypsin-like KLKs. The SC overall plasmin- and furin-like activities were significantly elevated, while trypsin- and chymotrypsin-like activities did not differ significantly. In the serum of AD patients, KLK8 was significantly elevated and KLK5 and KLK11 were significantly decreased. However, their serum levels were not modified by corticosteroid topical agents. The alterations of KLK levels in the SC of AD were more pronounced than those in the serum. KLK7 in the serum was significantly correlated with eosinophil counts in the blood of AD patients, while KLK5, KLK8 and KLK11 were significantly correlated with LDH in the serum. In conclusion, we report abnormal kallikrein levels in the SC and the serum of AD patients. KLKs might be involved in skin manifestation and/or focal/systemic inflammatory reactions in AD. Our data may contribute to a better understanding of the pathogenesis of AD.

  • aberrant human tissue kallikrein levels in the stratum corneum and serum of patients with psoriasis dependence on phenotype severity and therapy
    British Journal of Dermatology, 2007
    Co-Authors: Nahoko Komatsu, Eleftherios P Diamandis, Kiyofumi Saijoh, Cynthia Kuk, Fumiaki Shirasaki, Kazuhiko Takehara
    Abstract:

    Summary Background  Human tissue kallikreins (KLKs) are a family of 15 trypsin-like or chymotrypsin-like secreted serine proteases (KLK1–KLK15). Multiple KLKs have been quantitatively identified in normal stratum corneum (SC) and sweat as candidate desquamation-related proteases. Objectives  To quantify KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of patients with psoriasis, and their variation between lesional and nonlesional areas and with phenotype, therapy and severity. The overall SC serine protease activities were also measured. Methods  Enzyme-linked immunosorbent assays and enzymatic assays were used. Results  The lesional SC of psoriasis generally contained significantly higher levels of all KLKs. KLK6, KLK10 and KLK13 levels were significantly elevated even in the nonlesional SC. The overall trypsin-like, plasmin-like and furin-like activities were significantly elevated in the lesional SC. Plasmin-like activity was significantly elevated also in the nonlesional SC. The SC chymotrypsin-like activity was only slightly elevated in psoriasis. KLK7 serum levels did not differ between normal volunteers and patients with psoriasis. Serum KLK6, KLK8, KLK10 and KLK13 levels in patients with untreated psoriasis significantly correlated with Psoriasis Area and Severity Index score. Serum KLK5 and KLK11 levels decreased in patients with psoriasis after therapy, especially with etretinate. Patients with erythrodermic psoriasis exhibited significantly higher serum KLK levels than normal subjects or patients with psoriasis vulgaris or arthropathic psoriasis. Conclusions  We found aberrant KLK levels in the SC and serum of patients with psoriasis and suggest that KLKs might be involved in the pathogenesis of this disease.

  • New nomenclature for the human tissue kallikrein gene family
    Clinical Chemistry, 2000
    Co-Authors: Eleftherios P Diamandis, Shigetaka Yoshida, Sheila P. Little, George M Yousef, Linda K Ashworth, Peter S Nelson, Torbjörn Egelrud, Judith A. Clements, Sadao Shiosaka, Hans Lilja
    Abstract:

    The human kallikrein gene family is important to the discipline of clinical chemistry because it contains genes that encode for valuable cancer biomarkers, including the best tumor marker available today, prostate-specific antigen (PSA). Despite reports of numerous kallikrein-like genes in the mouse (1), until 2–3 years ago, only three human kallikrein genes were recognized: pancreatic/renal kallikrein (KLK1) , human glandular kallikrein 2 (KLK2) , and prostate-specific antigen (KLK3) (1)(2). The proteins encoded by the three kallikrein genes are now known as hK1, hK2, and hK3 (PSA). These three genes encode for serine proteases with either trypsin-like (hK1, hK2) or chymotrypsin-like (hK3) activity. Traditionally, kallikreins have been defined as enzymes that can act on high-molecular weight substrates and release bioactive peptides, known as kinins (3). Among the known kallikrein enzymes, only plasma kallikrein (encoded by a single gene localized on human chromosome 4q35; official symbol KLKB1 ) and pancreatic/renal kallikrein (hK1) have significant kininogenase activity. The proteins encoded by the KLK2 and the KLK3 genes have minimal or no kininogenase activity (4)(5). Why then are the KLK1 , KLK2 , and KLK3 genes classified together into one gene family (tissue kallikrein gene family), when two of the three enzymes have no significant kallikrein enzymatic activity? This grouping is justified, based on the extensive homologies between the three genes at both the DNA …

Dario R Alessi - One of the best experts on this subject based on the ideXlab platform.

  • deficiency of lkb1 in skeletal muscle prevents ampk activation and glucose uptake during contraction
    The EMBO Journal, 2005
    Co-Authors: Kei Sakamoto, Afshan Mccarthy, Darrin P Smith, Kevin A Green, Grahame D Hardie, Alan Ashworth, Dario R Alessi
    Abstract:

    Recent studies indicate that the LKB1 tumour suppressor protein kinase is the major 9upstream9 activator of the energy sensor AMP‐activated protein kinase (AMPK). We have used mice in which LKB1 is expressed at only ∼10% of the normal levels in muscle and most other tissues, or that lack LKB1 entirely in skeletal muscle. Muscle expressing only 10% of the normal level of LKB1 had significantly reduced phosphorylation and activation of AMPKα2. In LKB1‐lacking muscle, the basal activity of the AMPKα2 isoform was greatly reduced and was not increased by the AMP‐mimetic agent, 5‐aminoimidazole‐4‐carboxamide riboside (AICAR), by the antidiabetic drug phenformin, or by muscle contraction. Moreover, phosphorylation of acetyl CoA carboxylase‐2, a downstream target of AMPK, was profoundly reduced. Glucose uptake stimulated by AICAR or muscle contraction, but not by insulin, was inhibited in the absence of LKB1. Contraction increased the AMP:ATP ratio to a greater extent in LKB1‐deficient muscles than in LKB1‐expressing muscles. These studies establish the importance of LKB1 in regulating AMPK activity and cellular energy levels in response to contraction and phenformin.

  • mo25α β interact with stradα β enhancing their ability to bind activate and localize lkb1 in the cytoplasm
    The EMBO Journal, 2003
    Co-Authors: Jerome Boudeau, Hans Clevers, Maria Deak, Agnieszka Kieloch, Nick A Morrice, Annette F Baas, Mike Schutkowski, Alan R Prescott, Dario R Alessi
    Abstract:

    Mutations in the LKB1 protein kinase result in the inherited Peutz Jeghers cancer syndrome. LKB1 has been implicated in regulating cell proliferation and polarity although little is known about how this enzyme is regulated. We recently showed that LKB1 is activated through its interaction with STRADα, a catalytically deficient pseudokinase. Here we show that endogenous LKB1–STRADα complex is associated with a protein of unknown function, termed MO25α, through the interaction of MO25α with the last three residues of STRADα. MO25α and STRADα anchor LKB1 in the cytoplasm, excluding it from the nucleus. Moreover, MO25α enhances the formation of the LKB1–STRADα complex in vivo, stimulating the catalytic activity of LKB1 ∼10-fold. We demonstrate that the related STRADβ and MO25β isoforms are also able to stabilize LKB1 in an active complex and that it is possible to isolate complexes of LKB1 bound to STRAD and MO25 isoforms, in which the subunits are present in equimolar amounts. Our results indicate that MO25 may function as a scaffolding component of the LKB1–STRAD complex and plays a crucial role in regulating LKB1 activity and cellular localization.

  • complexes between the lkb1 tumor suppressor stradα β and mo25α β are upstream kinases in the amp activated protein kinase cascade
    Journal of Biology, 2003
    Co-Authors: Simon A Hawley, Jerome Boudeau, Dario R Alessi, Tomi P Makela, Jennifer L Reid, Kirsty J Mustard, Lina Udd, Grahame D Hardie
    Abstract:

    The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs) that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRADα/β and MO25α/β. We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRADα and MO25α, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRADα/β and MO25α/β activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRADα or STRADβ and MO25α or MO25β are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1), but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos. These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

  • heat shock protein 90 and cdc37 interact with lkb1 and regulate its stability
    Biochemical Journal, 2003
    Co-Authors: Jerome Boudeau, Maria Deak, Nick A Morrice, Margaret A Lawlor, Dario R Alessi
    Abstract:

    LKB1 is a widely expressed serine/threonine protein kinase that is mutated in the inherited Peutz-Jeghers cancer syndrome. Recent findings indicate that LKB1 functions as a tumour suppressor, but little is known regarding the detailed mechanism by which LKB1 regulates cell growth. In this study we have purified LKB1 from cells and establish that it is associated with the heat-shock protein 90 (Hsp90) chaperone and the Cdc37 kinase-specific targetting subunit for Hsp90. We demonstrate that Cdc37 and Hsp90 bind specifically to the kinase domain of LKB1. We also perform experiments using Hsp90 inhibitors, which indicate that the association of Hsp90 and Cdc37 with LKB1 regulates LKB1 stability and prevents its degradation by the proteasome. Hsp90 inhibitors are being considered as potential anti-cancer agents. However, our observations indicate that prolonged usage of these drugs could possibly lead to tumour development by decreasing cellular levels of LKB1.

  • functional analysis of lkb1 stk11 mutants and two aberrant isoforms found in peutz jeghers syndrome patients
    Human Mutation, 2003
    Co-Authors: Jerome Boudeau, Agnieszka Kieloch, Dario R Alessi, Alessandro Stella, Ginevra Guanti, Nicoletta Resta
    Abstract:

    Peutz-Jeghers Syndrome (PJS) is thought to be caused by mutations occurring in the widely expressed serine/threonine protein kinase named LKB1/STK11. Recent work has led to the identification of four mutants (R304W, I177N, K175-D176del, L263fsX286) and two novel aberrant LKB1/STK11 cDNA isoforms (r291-464del, r485-1283del) in a group of PJS Italian patients. Three of the four mutations only change 1 or 2 amino acids in the LKB1/STK11 catalytic domain. Here we demonstrate that all six LKB1/STK11 variants analysed are completely inactive in vitro as they were unable to autophosphorylate at Thr336, the major LKB1/STK11 autophosphorylation site, and to phosphorylate the p53 tumour suppressor protein. We also show that 5 out of the 6 variants are entirely localised in the nucleus in contrast to the wild type LKB1/STK11, which is detected in both the nucleus and cytoplasm. Finally we demonstrate that all 6 LKB1/STK11 variants, in contrast to wild type LKB1/STK11, are unable to suppress the growth of melanoma G361 cells. Taken together, these results demonstrate that the LKB1 mutations investigated in this study lead to the loss of serine/threonine kinase activity and are therefore likely to be the primary cause of PJS development in the patients that they were isolated from.

Amutha Ramaswamy - One of the best experts on this subject based on the ideXlab platform.

  • effects of peutz jeghers syndrome pjs causing missense mutations l67p l182p g242v and r297s on the structural dynamics of lkb1 liver kinase b1 protein
    Journal of Biomolecular Structure & Dynamics, 2019
    Co-Authors: Ikrormi Rungsung, Amutha Ramaswamy
    Abstract:

    The liver kinase B1 (LKB1) is encoded by LKB1 gene. Several pathogenic mutations of LKB1 causing Peutz–Jeghers syndrome and also cancers in breast, gastric, pancreas, and colon have been reported. The present study is focused to analyze the effects on the structural dynamics of LKB1 caused by the 4 pathogenic missense mutations (L67P, L182P, G242V, and R297S), which are reported to reduce the catalytic activity. In this study, the structural changes of LKB1 in apo- and in heterotrimeric complex (LKB1–STRADα–MO25α) form with wild and mutated LKB1 are investigated using all atomistic molecular dynamic simulation. The present study reveals that these four mutations initiate local structural distortions and the solvent accessibility of the surrounding regions of ATP-binding pocket such as glycine-rich loop, αB and αC loop, activation and catalytic loops. The mutations of L67P, L182P, and G242 V induce distortions of the secondary structure of β1–β3 sheets, π – π interaction (observed between Phe204 of LKB1 an...

  • Effects of Peutz-Jeghers syndrome (PJS) causing missense mutations L67P, L182P, G242V and R297S on the structural dynamics of LKB1 (Liver kinase B1) protein
    2018
    Co-Authors: Ikrormi Rungsung, Amutha Ramaswamy
    Abstract:

    The liver kinase B1 (LKB1) is encoded by LKB1 gene. Several pathogenic mutations of LKB1 causing Peutz–Jeghers syndrome and also cancers in breast, gastric, pancreas, and colon have been reported. The present study is focused to analyze the effects on the structural dynamics of LKB1 caused by the 4 pathogenic missense mutations (L67P, L182P, G242V, and R297S), which are reported to reduce the catalytic activity. In this study, the structural changes of LKB1 in apo- and in heterotrimeric complex (LKB1–STRADα–MO25α) form with wild and mutated LKB1 are investigated using all atomistic molecular dynamic simulation. The present study reveals that these four mutations initiate local structural distortions and the solvent accessibility of the surrounding regions of ATP-binding pocket such as glycine-rich loop, αB and αC loop, activation and catalytic loops. The mutations of L67P, L182P, and G242 V induce distortions of the secondary structure of β1–β3 sheets, π – π interaction (observed between Phe204 of LKB1 and Phe243 of MO25α), and increase the helical properties (both helical twist and length) of the adjacent αH-helix, respectively. The active kinase features like the conformation of catalytic and activation loops, salt bridge and, finally, the formation of stable R- and C-hydrophobic spines are also found to be perturbed by these mutations. Hence, the observed mutation-induced structural distortions fail to coordinate the essential binding nature of LKB1 with STRADα and MO25α, which eventually affects the native function of LKB1. These observations are in line with the experimentally reported reduced kinase activity of LKB1.

  • Insights into the structural dynamics of Liver kinase B1 (LKB1) by the binding of STe20 Related Adapterα (STRADα) and Mouse protein 25α (MO25α) co-activators
    2016
    Co-Authors: Ikrormi Rungsung, Amutha Ramaswamy
    Abstract:

    LKB1, the tumour suppressor, is found mutated in Peutz-Jeghers syndrome (PJS). The LKB1 is a serine-threonine kinase protein that is allosterically activated by the binding of STRADα and MO25α without phosphorylating the Thr212 present at activation loop. The present study aims to highlight the structural dynamics and complexation mechanism during the allosteric activation of LKB1 by these co-activators using molecular dynamics simulations. The all atom simulations performed on the complexes of LKB1 with ATP, STRADα, and MO25α for a period of 30 ns reveal that binding of STRADα and MO25α significantly stabilizes the highly flexible regions of LKB1 such as ATP binding region (β1-β2 loop), catalytic & activation loop segments and αG helix. Also, binding of STRADα and MO25α to LKB1 promotes coordinated motion between N- and C-lobes along with the catalytic & activation loops by forming H-bonds between LKB1 and co-activators, which further facilitate to establish the conserved attributes of active LKB1 such as (i) formation of salt bridge between Lys78 and Glu98, (ii) formation of stable hydrophobic R- and C-spines, and (iii) interaction between both catalytic and activation loops. Especially, the residues of LKB1 interacting with STRADα (Arg74, Glu342) and MO25α (Glu165, Pro203 and Phe204) are observed to play a significant role in stabilizing the (LKB1-ATP)-(STRADα-ATP)-MO25α complex. Overall, the present work highlighting the structural dynamics of LKB1 by the binding of allosteric co-activators is expected to provide a basic understanding on drug design specific to PJS syndrome.

Minghui Zou - One of the best experts on this subject based on the ideXlab platform.

  • lipopolysaccharides promote s nitrosylation and proteasomal degradation of liver kinase b1 lkb1 in macrophages in vivo
    Journal of Biological Chemistry, 2015
    Co-Authors: Zhaoyu Liu, Xiaoyan Dai, Huaiping Zhu, Miao Zhang, Minghui Zou
    Abstract:

    LKB1 (liver kinase B1) plays important roles in tumor suppression, energy metabolism, and, recently, in innate immune responses. However, how LKB1 is regulated under physiological or pathological conditions is still unclear. Here, we report that LKB1 protein (but not mRNA) was decreased in both LPS-treated RAW 264.7 cells and peritoneal macrophages isolated from LPS-challenged mice. Additional LPS treatment promoted protein ubiquitination and degradation of LKB1. Pharmacological inhibition or gene silencing of inducible NOS abrogated LPS-induced LKB1 degradation, whereas exposure of RAW 264.7 cells to S-nitroso-l-glutathione, a NO donor, triggered LKB1 S-nitrosylation. Consistently, mutation of one cysteine (C430S) in LKB1 prevented LPS-induced S-nitrosylation, ubiquitination, and degradation. Moreover, S-nitrosylation and ubiquitination of LKB1 were confirmed in macrophages from LPS-challenged mice in vivo. Co-administration of the inducible NOS inhibitor S-methylisothiourea or the proteasome inhibitor MG132 prevented LPS-induced LKB1 degradation and improved the survival rate. Finally, mice lacking LKB1 in macrophages had significantly lower survival rates in response to LPS challenge compared with wild-type mice. Thus, we concluded that LKB1 is degraded by LPS treatment via S-nitrosylation-dependent proteasome pathways, and this had a protective role in LPS-induced septic shock.

  • phosphorylation of serine 399 in lkb1 protein short form by protein kinase cζ is required for its nucleocytoplasmic transport and consequent amp activated protein kinase ampk activation
    Journal of Biological Chemistry, 2013
    Co-Authors: Huaiping Zhu, Miao Zhang, Cate Moriasi, Yu Zhao, Minghui Zou
    Abstract:

    Two splice variants of LKB1 exist: LKB1 long form (LKB1(L)) and LKB1 short form (LKB1(S)). In a previous study, we demonstrated that phosphorylation of Ser-428/431 (in LKB1(L)) by protein kinase Cζ (PKCζ) was essential for LKB1-mediated activation of AMP-activated protein kinase (AMPK) in response to oxidants or metformin. Paradoxically, LKB1S also activates AMPK although it lacks Ser-428/431. Thus, we hypothesized that LKB1(S) contained additional phosphorylation sites important in AMPK activation. Truncation analysis and site-directed mutagenesis were used to identify putative PKCζ phosphorylation sites in LKB1(S). Substitution of Ser-399 to alanine did not alter the activity of LKB1(S), but abolished peroxynitrite- and metformin-induced activation of AMPK. Furthermore, the phosphomimetic mutation (S399D) increased the phosphorylation of AMPK and its downstream target phospho-acetyl-coenzyme A carboxylase (ACC). PKCζ-dependent phosphorylation of Ser-399 triggered nucleocytoplasmic translocation of LKB1(S) in response to metformin or peroxynitrite treatment. This effect was ablated by pharmacological and genetic inhibition of PKCζ, by inhibition of CRM1 activity and by substituting Ser-399 with alanine (S399A). Overexpression of PKCζ up-regulated metformin-mediated phosphorylation of both AMPK (Thr-172) and ACC (Ser-79), but the effect was ablated in the S399A mutant. We conclude that, similar to Ser-428/431 (in LKB1(L)), Ser-399 (in LKB1(S)) is a PKCζ-dependent phosphorylation site essential for nucleocytoplasmic export of LKB1(S) and consequent AMPK activation.

  • protein kinase cζ dependent lkb1 serine 428 phosphorylation increases lkb1 nucleus export and apoptosis in endothelial cells
    Journal of Biological Chemistry, 2008
    Co-Authors: Ping Song, Zhonglin Xie, Yunzhou Dong, Minghui Zou
    Abstract:

    LKB1 is a serine-threonine protein kinase that, when inhibited, may result in unregulated cell growth and tumor formation. However, how LKB1 is regulated remains poorly understood. The aim of the present study was to define the upstream signaling events responsible for peroxynitrite (ONOO-)-induced LKB1 activation. Exposure of cultured human umbilical vein endothelial cells to a low concentration of ONOO- (5 μm) significantly increased the phosphorylation of LKB1 at Ser428 and protein kinase Cζ (PKCζ) at Thr410. These effects were accompanied by increased activity of the lipid phosphatase PTEN, decreased activity and phosphorylation (Ser473) of Akt, and induction of apoptosis. ONOO- enhanced Akt-Ser473 phosphorylation in LKB1-deficient HeLa S3 cells or in HeLa S3 cells transfected with kinase-dead LKB1. Conversely, ONOO- inhibited Akt Ser473 phosphorylation when wild type LKB1 were reintroduced in HeLa S3 cells. Further analysis revealed that PKCζ directly phosphorylated LKB1 at Ser428 in vitro and in intact cells, resulting in increased PTEN phosphorylation at Ser380/Thr382/383. Finally, ONOO- enhanced PKCζ nuclear import and LKB1 nuclear export. We conclude that PKCζ mediates LKB1-dependent Akt inhibition in response to ONOO-, resulting in endothelial apoptosis.

Nahoko Komatsu - One of the best experts on this subject based on the ideXlab platform.

  • human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients
    Experimental Dermatology, 2007
    Co-Authors: Nahoko Komatsu, Eleftherios P Diamandis, Kiyofumi Saijoh, Fumiaki Shirasaki, Kazuhiko Takehara, Saba Khan
    Abstract:

    :  Human tissue kallikreins are a family of 15 trypsin- or chymotrypsin-like secreted serine proteases (KLK1–KLK15). Many KLKs have been identified in normal stratum corneum (SC) and sweat, and are candidate desquamation-related proteases. We report quantification by enzyme-linked immunosorbent assay (ELISA) of KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of atopic dermatitis (AD) patients by ELISA, and examine their variation with clinical phenotype, correlation with blood levels of eosinophils, lactate dehydrogenase (LDH) and immunoglobulin E. The overall SC serine protease activities were also measured. In the SC of AD, all KLKs, except KLK11, were significantly elevated. The elevation of chymotrypsin-like KLK7 was predominant, compared with trypsin-like KLKs. The SC overall plasmin- and furin-like activities were significantly elevated, while trypsin- and chymotrypsin-like activities did not differ significantly. In the serum of AD patients, KLK8 was significantly elevated and KLK5 and KLK11 were significantly decreased. However, their serum levels were not modified by corticosteroid topical agents. The alterations of KLK levels in the SC of AD were more pronounced than those in the serum. KLK7 in the serum was significantly correlated with eosinophil counts in the blood of AD patients, while KLK5, KLK8 and KLK11 were significantly correlated with LDH in the serum. In conclusion, we report abnormal kallikrein levels in the SC and the serum of AD patients. KLKs might be involved in skin manifestation and/or focal/systemic inflammatory reactions in AD. Our data may contribute to a better understanding of the pathogenesis of AD.

  • aberrant human tissue kallikrein levels in the stratum corneum and serum of patients with psoriasis dependence on phenotype severity and therapy
    British Journal of Dermatology, 2007
    Co-Authors: Nahoko Komatsu, Eleftherios P Diamandis, Kiyofumi Saijoh, Cynthia Kuk, Fumiaki Shirasaki, Kazuhiko Takehara
    Abstract:

    Summary Background  Human tissue kallikreins (KLKs) are a family of 15 trypsin-like or chymotrypsin-like secreted serine proteases (KLK1–KLK15). Multiple KLKs have been quantitatively identified in normal stratum corneum (SC) and sweat as candidate desquamation-related proteases. Objectives  To quantify KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of patients with psoriasis, and their variation between lesional and nonlesional areas and with phenotype, therapy and severity. The overall SC serine protease activities were also measured. Methods  Enzyme-linked immunosorbent assays and enzymatic assays were used. Results  The lesional SC of psoriasis generally contained significantly higher levels of all KLKs. KLK6, KLK10 and KLK13 levels were significantly elevated even in the nonlesional SC. The overall trypsin-like, plasmin-like and furin-like activities were significantly elevated in the lesional SC. Plasmin-like activity was significantly elevated also in the nonlesional SC. The SC chymotrypsin-like activity was only slightly elevated in psoriasis. KLK7 serum levels did not differ between normal volunteers and patients with psoriasis. Serum KLK6, KLK8, KLK10 and KLK13 levels in patients with untreated psoriasis significantly correlated with Psoriasis Area and Severity Index score. Serum KLK5 and KLK11 levels decreased in patients with psoriasis after therapy, especially with etretinate. Patients with erythrodermic psoriasis exhibited significantly higher serum KLK levels than normal subjects or patients with psoriasis vulgaris or arthropathic psoriasis. Conclusions  We found aberrant KLK levels in the SC and serum of patients with psoriasis and suggest that KLKs might be involved in the pathogenesis of this disease.

  • expression and localization of tissue kallikrein mrnas in human epidermis and appendages
    Journal of Investigative Dermatology, 2003
    Co-Authors: Nahoko Komatsu, Kazuhiko Takehara, Minoru Takata, Norio Otsuki, Tadashi Toyama, Rie Ohka, Kiyofumi Saijoh
    Abstract:

    Tissue kallikreins are a group of serine proteases that are found in many organs and biologic fluids. Tissue kallikrein genes (KLKs) are found on chromosome 19q13.3–4 as a gene cluster encoding 15 different serine proteases. In skin, two tissue kallikrein proteins, hK5 and hK7, are expressed in the stratum corneum and are known to be involved in desquamation of corneocytes. The possible involvement of other kallikrein proteins has not been clarified, however, nor has the significance of each member in the serine protease activity of skin been delineated. In the study described here, we examined expression and localization of KLK mRNA in normal human skin by means of RT-PCR and in situ hybridization. Quantitative RT-PCR analysis showed abundant expression of KLK1 and KLK11 mRNA, moderate expression of KLK4, KLK5, KLK6, KLK7, and KLK13 mRNA, and low expression of KLK8 mRNA in normal human skin. For KLK4, KLK8, and KLK13 mRNA, splice variants were identified to be their major mRNA species. Two variants for KLK13 mRNA were novel. The amount of the serine protease inhibitor Kazal-type 5 (SPINK5) mRNA was comparable to KLK1 and KLK11 mRNA. In situ hybridization revealed intense expression of all KLK mRNA studied except KLK12 mRNA in the stratum granulosum of normal epidermis, where SPINK5 mRNA coexisted. Excluding KLK13 mRNA, they are also expressed in hair sheath, eccrine sweat glands, and sebaceous glands. Coexpression of various KLK and SPINK5 mRNA suggests that their proteins are the candidates to balance and maintain serine protease activities in both the skin and appendages.