22 research outputs found
Grafting Miniature DNA Binding Proteins
AbstractMiniature proteins serve as leads for biological and medicinal applications by positioning all amino acids necessary for biomolecular recognition on a compact protein structure. Protein grafting was recently used to create miniature helical proteins with high DNA binding affinity and specificity
Limited proteolysis of human histone deacetylase 1
BACKGROUND: Histone deacetylase (HDAC) proteins are associated with cell proliferation, differentiation, apoptosis, and cancer. Specifically, HDAC1 is linked with cell growth, a hallmark of cancer formation. HDAC1 is a phosphoprotein and phosphorylation at S421 and S423 promotes HDAC1 enzymatic activity and protein association. While single and double point mutants of HDAC1 at S421 and S423 appear functionally similar, the evidence suggests that HDAC1 is phosphorylated simultaneously at both S421 and S423 in vivo. Additional experiments are necessary to probe the role of double phosphorylation of HDAC1 at S421 and S423. RESULTS: To characterize HDAC1 phosphorylation at S421 and S423, limited proteolysis of HDAC1 was performed for the first time. HDAC1 degraded without production of discrete fragments. By performing concentration-dependent proteolysis, HDAC1 double point mutants with disrupted phosphorylation at S421 and S423 displayed different trypsin sensitivities compared to wild type HDAC1. Unexpectedly, HDAC1 single point mutants with disrupted phosphorylation at either S421 or S423 demonstrated protease sensitivity similar to the wild type HDAC1. CONCLUSION: Concentration-dependent proteolysis experiments provide evidence that phosphorylation of S421 and S423 individually contribute to HDAC1 function. In addition, the limited proteolysis experiments support a model where associated proteins promote HDAC1 enzymatic activity, reinforcing the importance of protein interactions in HDAC1 structure and function. Finally, because HDAC1 does not display distinct regions of protease sensitivity, the proteolysis studies suggest that HDAC1 comprises inter-related structural regions
Phosphorylation of cell cycle and apoptosis regulatory protein-1 by stress activated protein kinase P38γ is a novel mechanism of apoptosis signaling by genotoxic chemotherapy
CARP-1, a perinuclear phospho-protein, regulates cell survival and apoptosis signaling induced by genotoxic drugs. However, kinase(s) phosphorylating CARP-1 and down-stream signal transduction events remain unclear. Here we find that CARP-1 Serine (S)626 and Threonine (T)627 substitution to Alanines (AA) inhibits genotoxic drug-induced apoptosis. CARP-1 T627 is followed by a Proline (P), and this TP motif is conserved in vertebrates. Based on these findings, we generated affinity-purified, anti-phospho-CARP-1 T627 rabbit polyclonal antibodies, and utilized them to elucidate chemotherapy-activated, CARP-1-dependent cell growth signaling mechanisms. Our kinase profiling studies revealed that MAPKs/SAPKs phosphorylated CARP-1 T627. We then UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614–638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed SAPK p38γ interaction with CARP-1 (614–638) peptide. Our studies further established that SAPK p38γ, but not other MAPKs, phosphorylates CARP-1 T627 in cancer cells treated with genotoxic drugs. Loss of p38γ abrogates CARP-1 T627 phosphorylation, and results in enhanced survival of breast cancer cells by genotoxic drugs. CARP-1 T627 phosphorylation was also noted in breast tumors from patients treated with radiation or endocrine therapies. We conclude that genotoxic drugs activate p38γ-dependent CARP-1 T627 phosphorylation to inhibit cell growth
LSD1 Substrate Binding and Gene Expression Are Affected by HDAC1-Mediated Deacetylation
Lysine
Specific Demethylase 1 (LSD1) catalyzes the demethylation
of histone 3 to regulate gene expression. With a fundamental role
in gene regulation, LSD1 is involved in multiple cellular processes,
including embryonic development, cell proliferation, and metastasis.
Significantly, LSD1 is overexpressed in multiple cancers and has emerged
as a potential anticancer drug target. LSD1 is typically found in
association with another epigenetic enzyme, histone deacetylase (HDAC).
HDAC and LSD1 inhibitor compounds have been tested as combination
anticancer agents. However, the functional link between LSD1 and HDAC
has yet to be understood in detail. Here, we used a substrate trapping
strategy to identify cellular substrates of HDAC1. Using inactive
HDAC1 mutants, we identified LSD1 as an HDAC1 substrate. HDAC1 mediated
deacetylation of LSD1 at K374 in the substrate binding lobe, which
affected the histone 3 binding and gene expression activity of LSD1.
The mechanistic link between HDAC1 and LSD1 established here suggests
that HDAC inhibitors influence LSD1 activity, which will ultimately
guide drug design targeting epigenetic enzymes
K‑CLASP: A Tool to Identify Phosphosite Specific Kinases and Interacting Proteins
Few
methods are available to discover the cellular kinase that
phosphorylates a specific amino acid, or phosphosite, on a protein.
In addition, identifying the associated proteins bound near a phosphosite
during phosphorylation would provide insights into cell biology and
signaling. Here, we report K-CLASP (Kinase Catalyzed CrossLinking
And Streptavidin Purification) as a method for both phosphosite-specific
kinase identification and the discovery of kinase interacting proteins.
K-CLASP offers a powerful tool to discover unanticipated protein–protein
interactions in phosphorylation-mediated biological events
Valproate regulates inositol synthesis by reducing expression of myo-inositol-3-phosphate synthase
Abstract Inositol depletion is a hypothesized mechanism of action of mood stabilization drugs used in the treatment of bipolar disorder. It was previously reported that the mood stabilizer valproate (VPA) increased phosphorylation of myo-inositol-3-phosphate synthases (MIPS), the rate limiting enzyme of inositol synthesis. Phosphosites were identified and examination of site-directed mutants suggested that phosphorylation leads to decreased enzymatic activity. In this study, we examined the extent of MIPS phosphorylation in response to VPA and used two interaction screens to identify protein kinases that interact with MIPS. Using an epitope tagged MIPS construct, we determined the fraction of phosphorylated MIPS to be very low (less than 2% of total), and we could not detect phosphorylation of untagged MIPS in response to VPA. In vitro analyses of phosphorylation revealed that putative protein kinases, PKC and CKII, have low specificity toward MIPS. These findings suggest that VPA likely depletes inositol via a mechanism other than MIPS phosphorylation. Consistent with this, mRNA levels of the MIPS-encoding gene INO1 and MIPS protein levels were significantly reduced during the mid-log growth phase in response to VPA treatment. These findings suggest that the mechanism whereby VPA causes inositol depletion is by reducing expression of the rate-limiting enzyme MIPS
Structural Analysis of ATP Analogues Compatible with Kinase-Catalyzed Labeling
Kinase-catalyzed protein phosphorylation is an important
biochemical
process involved in cellular functions. We recently discovered that
kinases promiscuously accept γ-modified ATP analogues as cosubstrates
and used several ATP analogues as tools for studying protein phosphorylation.
Herein, we explore the structural requirements of γ-modified
ATP analogues for kinase compatibility. To understand the influence
of linker length and composition, a series of ATP analogues was synthesized,
and the efficiency of kinase-catalyzed labeling was determined by
quantitative mass spectrometry. This study on factors influencing
kinase cosubstrate promiscuity will enable design of ATP analogues
for a variety of kinase-catalyzed labeling reactions
Structural Requirements of HDAC Inhibitors: SAHA Analogues Modified at the C2 Position Display HDAC6/8 Selectivity
Histone deacetylase
(HDAC) proteins are epigenetic regulators that
deacetylate protein substrates, leading to subsequent changes in cell
function. HDAC proteins are implicated in cancers, and several HDAC
inhibitors have been approved by the FDA as anticancer drugs, including
SAHA (suberoylanilide hydroxamic acid; Vorinostat and Zolinza). Unfortunately,
SAHA inhibits most HDAC isoforms, which limits its use as a pharmacological
tool and may lead to side effects in the clinic. In this work SAHA
analogues substituted at the C2 position were synthesized and screened
for HDAC isoform selectivity <i>in vitro</i> and in cells.
The most potent and selective compound, C2-<i>n</i>-hexyl
SAHA, displayed submicromolar potency with 49- to 300-fold selectivity
for HDAC6 and HDAC8 compared to HDAC1, -2, and -3. Docking studies
provided a structural rationale for selectivity. Modification of the
nonselective inhibitor SAHA generated HDAC6/HDAC8 dual selective inhibitors,
which can be useful lead compounds toward developing pharmacological
tools and more effective anticancer drugs
Mutagenesis Studies of the 14 Ã… Internal Cavity of Histone Deacetylase 1: Insights toward the Acetate-Escape Hypothesis and Selective Inhibitor Design
Histone
deacetylase (HDAC) proteins are promising targets for cancer
treatment, as shown by the approval of two HDAC inhibitors for the
treatment of cutaneous T-cell lymphoma. HDAC1 in particular has been
linked to cell growth and cell cycle regulation and is therefore an
attractive target for anticancer drugs. The HDAC1 active site contains
a hydrophobic 11 Ã… active-site channel, with a 14 Ã… internal
cavity at the bottom of the active site. Several computational and
biochemical studies have proposed an acetate-escape hypothesis where
the acetate byproduct of the deacetylation reaction escapes via the
14 Ã… internal cavity. Selective HDAC inhibitors that bind to
the 14 Ã… cavity have also been created. To understand the influence
of amino acids lining the HDAC1 14 Ã… cavity in acetate escape
and inhibitor binding, we used mutagenesis coupled with acetate competition
assays. The results indicate that amino acids lining the 14 Ã…
cavity are critical for catalytic activity and acetate competition,
confirming the role of the cavity in acetate escape. In addition,
these mutagenesis studies will aid in HDAC1-inhibitor design that
exploits the 14 Ã… cavity