32 research outputs found
Selective Phosphorylation Inhibitor of Delta Protein Kinase CāPyruvate Dehydrogenase Kinase ProteināProtein Interactions: Application for Myocardial Injury <i>in Vivo</i>
Protein
kinases regulate numerous cellular processes, including
cell growth, metabolism, and cell death. Because the primary sequence
and the three-dimensional structure of many kinases are highly similar,
the development of selective inhibitors for only one kinase is challenging.
Furthermore, many protein kinases are pleiotropic, mediating diverse
and sometimes even opposing functions by phosphorylating multiple
protein substrates. Here, we set out to develop an inhibitor of a
selective protein kinase phosphorylation of only one of its substrates.
Focusing on the pleiotropic delta protein kinase C (Ī“PKC), we
used a rational approach to identify a distal docking site on Ī“PKC
for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned
that an inhibitor of PDKās docking should selectively inhibit
the phosphorylation of only PDK without affecting phosphorylation
of the other Ī“PKC substrates. Our approach identified a selective
inhibitor of PDK docking to Ī“PKC with an <i>in vitro</i> <i>K</i><sub>d</sub> of ā¼50 nM and reducing cardiac
injury IC<sub>50</sub> of ā¼5 nM. This inhibitor, which did
not affect the phosphorylation of other Ī“PKC substrates even
at 1 Ī¼M, demonstrated that PDK phosphorylation alone is critical
for Ī“PKC-mediated injury by heart attack. The approach we describe
is likely applicable for the identification of other substrate-specific
kinase inhibitors
Additional file 1: of Aldehyde dehydrogenase 2 activation and coevolution of its ĪµPKC-mediated phosphorylation sites
Phylogenetic Tree of the 20 species for ALDH2 and ĪµPKC coevolution comparison. Species in green letters are those with a homology of ĪµPKC. Species in red letters are those without a homology of ĪµPKC. (PDF 73Ā kb
Additional file 2: of Aldehyde dehydrogenase 2 activation and coevolution of its ĪµPKC-mediated phosphorylation sites
The table shows the 10 species with ĪµPKC (left panel), the 10 species without ĪµPKC (right panel) and their amino acid residues at the three human ALDH2 phosphorylation sites, T185, S279 and T412. (PDF 313Ā kb
Impaired protein quality control in left ventricular remodeling and heart failure in humans.
<p><b>A</b>. ATP-dependent and -independent proteasomal activity, <b>B</b>. oxidized protein levels (determined by Western blot) and <b>C</b>. polyubiquitinated protein levels (determined by Western blot) in biopsied hearts from aortic stenosis-induced left ventricular remodeling (LVR) patients (green bars), ischemic cardiomyopathy-induced heart failure patients (red bars) and autopsied non-failing human hearts (white bars). <b>D</b>. Negative correlation between proteasomal function and oxidized protein accumulation in failing (aortic stenosis-LVR and ischemic-HF) and non-failing heart samples. <b>E</b>. Total PKC levels in failing hearts compared to non-failing hearts and <b>F</b>. Representative blots of PKCĪ²II and PKCĪ± proteins in total and Triton-soluble fraction (particulate fraction) in biopsied hearts from aortic stenosis-induced left ventricular remodeling patients (nā=ā6, green bars) and ischemic cardiomyopathy-induced heart failure patients (nā=ā3, red bars) compared to autopsied non-failing human hearts (nā=ā6, trace). Total and Triton-soluble fractions were normalized against GAPDH and GĪ±o, respectively. Error bars indicate SEM. *, p<0.05 compared to control (non-failing heart). Ā§, p<0.05 compared to aortic stenosis-LVR patients.</p
Sustained PKCĪ²II inhibition re-establishes protein quality control and improves cardiac function in myocardial infarction-induced model of heart failure.
<p><b>A</b>. Schematic panel of PKCĪ²II treatment in the post-MI heart failure model, representative blots of PKCĪ²II total level and translocation to particulate fraction, and PKCĪ²II activity from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, Ī²IIV5-3-treated and control (sham) rats (nā=ā3 per group). <b>B</b>. 20S proteasome subunits (Ī±5/7, Ī²1, Ī²5 and Ī²7) were precipitated from left ventricle tissue from 22-week-old myocardial infarction-induced heart failure (10 wks after MI surgery) TAT-treated, Ī²IIV5-3-treated and control (sham) rats (nā=ā3 per group), and then probed with PKCĪ²II, PKCĪµ and anti-serine and threonine phosphorylation antibodies. Equal sample loading was verified using Ī±5/7, Ī²1, Ī²5 and Ī²7 proteasome subunits antibody. <b>C</b>. ATP-dependent and -independent cardiac proteasomal activity. <b>D</b>. Representative blots of proteasome 20S, Ī±-Ī²-crystallin, HSP27, caspase-3, cleaved caspase-3 and GAPDH in heart samples from 22 week-old rats (10 wks after MI surgery) (nā=ā6 per group). Data quantification and statistical details are in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033175#pone-0033175-g004" target="_blank">Fig. 4</a>. <b>E</b>. Oxidized protein levels, <b>F</b>. polyubiquitinated protein levels and <b>G</b>. soluble oligomer accumulation in heart samples from control (sham, white bars), TAT-treated (gray bars) and Ī²IIV5-3-treated (green bars) heart failure rats as determined by Western blot (E, F) and slot-blot analysis (G). <b>H</b>. Average fractional shortening data from each group at 16 weeks and 22 weeks. All biochemical analyses were performed in the ventricular remote area. Error bars indicate SEM. *, p<0.05 compared to control (sham) rats. Ā§, p<0.05 compared to Ī²IIV5-3-treated heart failure rats.</p
Scheme depicting a possible mechanism of PKCĪ²II-mediated PQC disruption during heart failure establishment.
<p>Scheme depicting a possible mechanism of PKCĪ²II-mediated PQC disruption during heart failure establishment.</p
Development of Selective Inhibitors for Aldehyde Dehydrogenases Based on Substituted Indole-2,3-diones
Aldehyde
dehydrogenases (ALDH) participate in multiple metabolic
pathways and have been indicated to play a role in several cancerous
disease states. Our laboratory is interested in developing novel and
selective ALDH inhibitors. We looked to further work recently published
by developing a class of isoenzyme-selective inhibitors using similar
indole-2,3-diones that exhibit differential inhibition of ALDH1A1,
ALDH2, and ALDH3A1. Kinetic and X-ray crystallography data suggest
that these inhibitors are competitive against aldehyde binding, forming
direct interactions with active-site cysteine residues. The selectivity
is precise in that these compounds appear to interact directly with
the catalytic nucleophile, Cys243, in ALDH3A1 but not in ALDH2. In
ALDH2, the 3-keto group is surrounded by the adjacent Cys301/303.
Surprisingly, the orientation of the interaction changes depending
on the nature of the substitutions on the basic indole ring structure
and correlates well with the observed structureāactivity relationships
for each ALDH isoenzyme
PKCĪ²II activation inhibits proteasomal activity <i>in vitro</i> and disrupts protein quality control in cultured neonatal cardiomyocytes.
<p><b>A</b>. Proteolytic activity (upper panel) and phosphorylation of purified proteasome 20S (middle and lower panels) by different recombinant PKC isozymes (nā=ā3 per group). Purified 20S proteasome (1 ug) was individually incubated with recombinant PKCĪ±, PKCĪ²I, PKCĪ²II or PKCĪµ (50 ng) at 37Ā°C for 30 minutes. Proteasome phosphorylation was evaluated using serine/threonine phosphorylation antibody (1ā¶1000) and [Ī³<sup>32</sup>P] ATP incorporation. Histone phosphorylation was used to check the effectiveness of different PKC isozymes. Error bars indicate SEM. *, p<0.05 compared to other groups. <b>B</b>. Oxidized protein levels (upper panel) and ATP-dependent proteasomal activity (lower panel) in cultured neonatal cardiomyocytes. Cells were stimulated with PMA (a non-specific PKC activator) and the effect of PKC isozyme-specific peptide inhibitors (Ī±V5-3, an Ī±PKC-specific inhibitor; Ī²IV5-3, a Ī²IPKC-specific inhibitor; Ī²IIV5-3, a Ī²IIPKC-specific inhibitor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033175#pone.0033175-Stebbins1" target="_blank">[16]</a>; and ĪµV1-2, an ĪµPKC-specific inhibitor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033175#pone.0033175-Gray1" target="_blank">[46]</a>; or epoxomicin were determined. Error bars indicate SEM. *, p<0.05 compared to control-TAT and Ī²IIV5-3-treated groups. Representative blots of oxidized proteins and GAPDH. Oxidized proteins were normalized against GAPDH. <b>C</b>. Oxidized protein levels (upper panel) and ATP-dependent proteasomal activity (lower panel) in cultured neonatal cardiomyocytes. Cultured neonatal cardiomyocytes were PKCĪ² or PKCĪ± down-regulated (siRNA) plus/minus Ī²IIV5-3 and challenged with phorbol ester (PMA). NC, negative control for siRNA. Representative blots of PKCĪ²II and PKCĪ± showed siRNA effectiveness. Oxidized proteins were normalized against GAPDH. Error bars indicate SEM. *, p<0.05 compared to control (non-treated cells), PKCĪ² down-regulated treated cells and PKCĪ± down-regulated treated cells plus Ī²IIV5-3.</p
Protein quality control disruption and PKCĪ²II activation during progression of heart failure in a post-myocardial infarction (MI) model of HF in rats.
<p><b>A</b>. MI-induced HF protocol (10 weeks follow up) and example of heart morphology and echo data before (left) and 4 weeks after myocardial infarction (right) in rats. <b>B</b>. Cardiac fractional shortening, <b>C</b>. Oxidized protein levels, <b>D</b>. Soluble oligomer level and <b>E</b>. Proteasomal activity during the progression of cardiac dysfunction induced by myocardial infarction. *, p<0.05 compared to week 0 (before surgery). Ā§, p<0.05 compared to week 1 after surgery. ā”, p<0.05 compared to week 2 after surgery. <b>F</b>. Concordance between fractional shortening and proteasomal activity, and <b>G</b>. Concordance between accumulation of misfolded cardiac proteins and oxidized proteins at weeks 0, 1, 2, 4 and 10 after myocardial infarction surgery in rats, <b>H</b>. Cardiac fractional shortening, proteasomal activity, oxidized and misfolded protein levels during the progression of cardiac dysfunction induced by myocardial infarction. <b>I</b>. The levels of total PKC isozymes (white bars) and translocated active PKCs (gray bars; Triton-soluble proteins or the particulate fraction/total fraction) at 10 weeks after myocardial infarction relative to control, age-matched rats (nā=ā6). Total proteins and Triton-soluble proteins of the particulate fraction were normalized against GAPDH and GĪ±o, respectively. Representative blots are of PKCĪ²II total level and translocation to particulate fraction. All biochemical analyses were performed in the ventricular remote area. Error bars indicate s.e.m. *, p<0.05 compared to control-TAT. <b>J</b>. PKCĪ²II activity at 10 weeks after myocardial infarction relative to control age-matched rats (nā=ā3). *, p<0.05 compared to control.</p
PKCĪ²II inhibition repairs protein quality control in hypertension-induced heart failure model in rats.
<p><b>A</b>. Schematic panel of sustained PKCĪ²I, Ī²II or Īµ inhibition in hypertension-induced model of heart failure in rats. Representative blots of PKCĪ²II total level and translocation to particulate fraction. <b>B</b>. ATP-dependent and -independent cardiac proteasomal activity, <b>C</b>. Oxidized proteins (as determined by Western blot) and <b>D</b>. cardiac soluble oligomer accumulation (as determined by slot blot) in heart samples from 17 week-old normotensive rats (white bar), TAT-treated (gray bar), Ī²IV5-3-treated (gray bar), Ī²IIV5-3-treated (green bar) and ĪµV1-2-treated (gray bar) hypertensive rats. <b>E</b>. Average fractional shortening data from each group at the age of 17 weeks-old. <b>F</b>. Ī²IIV5-3 improved survival of rats with hypertension-induced heart failure. Error bars indicate SEM. *, p<0.05 compared to control (sham) rats. Ā§, p<0.05 compared to Ī²IIV5-3-treated heart failure rats. Data from Fig. bād were analyzed by one-way analysis of variance (ANOVA) with <i>post-hoc</i> testing by Tukey. Survival was analyzed by the standard Kaplan-Meier analysis with log-rank test.</p