Interaction of the Histone mRNA Hairpin with Stem–Loop Binding Protein (SLBP) and Regulation of the SLBP–RNA Complex by Phosphorylation and Proline Isomerization

In metazoans, the majority of histone proteins are generated from replication-dependent histone mRNAs. These mRNAs are unique in that they are not polyadenylated but have a stem–loop structure in their 3′ untranslated region. An early event in 3′ end formation of histone mRNAs is the binding of stem–loop binding protein (SLBP) to the stem–loop structure. Here we provide insight into the mechanism by which SLBP contacts the histone mRNA. There are two binding sites in the SLBP RNA binding domain for the histone mRNA hairpin. The first binding site (Glu129–Val158) consists of a helix–turn–helix motif that likely recognizes the unpaired uridines in the loop of the histone hairpin and, upon binding, destabilizes the first G-C base pair at the base of the stem. The second binding site lies between residues Arg180 and Pro200, which appears to recognize the second G-C base pair from the base of the stem and possibly regions flanking the stem–loop structure. We show that the SLBP–histone mRNA complex is regulated by threonine phosphorylation and proline isomerization in a conserved TPNK sequence that lies between the two binding sites. Threonine phosphorylation increases the affinity of SLBP for histone mRNA by slowing the off rate for complex dissociation, whereas the adjacent proline acts as a critical hinge that may orient the second binding site for formation of a stable SLBP–histone mRNA complex. The nuclear magnetic resonance and kinetic studies presented here provide a framework for understanding how SLBP recognizes histone mRNA and highlight possible structural roles of phosphorylation and proline isomerization in RNA binding proteins in remodeling ribonucleoprotein complexes

Natural isoaspartyl protein modification of ZAP70 alters T cell responses in lupus

Protein posttranslational modifications (PTMs) arise in a number of normal cellular biological pathways and in response to pathology caused by inflammation and/or infection. Indeed, a number of PTMs have been identified and linked to specific autoimmune responses and metabolic pathways. One particular PTM, termed isoaspartyl (isoAsp or isoD) modification, is among the most common spontaneous PTM occurring at physiological pH and temperature. Herein, we demonstrate that isoAsp modifications arise within the ZAP70 protein tyrosine kinase upon T-cell antigen receptor (TCR) engagement. The enzyme protein L-isoaspartate O-methyltransferase (PCMT1, or PIMT, EC 2.1.1.77) evolved to repair isoaspartyl modifications in cells. In this regard, we observe that increased levels of isoAsp modification that arise under oxidative stress are correlated with reduced PIMT activity in patients with systemic lupus erythematosus (SLE). PIMT deficiency leads to T cell hyper-proliferation and hyper-phosphorylation through ZAP70 signaling. We demonstrate that inducing the overexpression of PIMT can correct the hyper-responsive phenotype in lupus T cells. Our studies reveal a phenotypic role of isoAsp modification and phosphorylation of ZAP70 in lupus T cell autoimmunity and provide a potential therapeutic target through the repair of isoAsp modification.</p

Assembly of the SLIP1–SLBP Complex on Histone mRNA Requires Heterodimerization and Sequential Binding of SLBP Followed by SLIP1

The SLIP1–SLBP complex activates translation of replication-dependent histone mRNAs. In this report, we describe how the activity of the SLIP1–SLBP complex is modulated by phosphorylation and oligomerization. Biophysical characterization of the free proteins shows that whereas SLIP1 is a homodimer that does not bind RNA, human SLBP is an intrinsically disordered protein that is phosphorylated at 23 Ser/Thr sites when expressed in a eukaryotic expression system such as baculovirus. The bacterially expressed unphosphorylated SLIP1–SLBP complex forms a 2:2 high-affinity (<i>K</i>_D < 0.9 nM) heterotetramer that is also incapable of binding histone mRNA. In contrast, phosphorylated SLBP from baculovirus has a weak affinity (<i>K</i>_D ∼3 μM) for SLIP1. Sequential binding of phosphorylated SLBP to the histone mRNA stem–loop motif followed by association with SLIP1 is required to form an “active” ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to the SLIP1–SLBP heterodimer. Using alanine scanning mutagenesis, we demonstrate that the binding site on SLIP1 for SLBP lies close to the dimer interface. A single-point mutant near the SLIP1 homodimer interface abolished interaction with SLBP in vitro and reduced the abundance of histone mRNA in vivo. On the basis of these biophysical studies, we propose that oligomerization and SLBP phosphorylation may regulate the SLBP–SLIP1 complex in vivo. SLIP1 may act to sequester SLBP in vivo, protecting it from proteolytic degradation as an inactive heterotetramer, or alternatively, formation of the SLIP1–SLBP heterotetramer may facilitate removal of SLBP from the histone mRNA prior to histone mRNA degradation

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long-term potentiation and in the effects of cocaine on behavior and spine morphology. The <i>KALRN</i> gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multisite phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity, and reduced GEF activity

Inhibitor of the Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's Disease

<div><p>STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC₅₀ of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and <i>in vivo</i> in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels.</p></div

<i>Mycobacterium tuberculosis</i> universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth

<div><p>We have previously shown that the <i>Mycobacterium tuberculosis</i> universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623_WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623_T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an <i>M</i>. <i>tuberculosis Rv2623</i> null mutant (Δ<i>Rv2623</i>) displays enhanced expression of phosphatidyl-<i>myo</i>-inositol mannosides (PIMs), while the Δ<i>Rv1747</i> mutant expresses decreased levels of PIMs. Animal studies have previously shown that Δ<i>Rv2623</i> is hypervirulent, while Δ<i>Rv1747</i> is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing <i>in vivo</i> growth phenotypes of Δ<i>Rv2623</i> and Δ<i>Rv1747</i>, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of <i>M</i>. <i>tuberculosis in vivo</i>.</p></div

Compound 3 fractionation and initial characterization.

<p>(A) Commercially purchased Compound <b>3</b> was dissolved in methanol at 10 mg/mL, and 300 µL portions were injected onto a Zorbax (Agilent) 5 µm 300SB-C18 column (0.94×25 cm, 3 mL/min 75% methanol/25% pH 4.0 0.1 M ammonium acetate). Thirty-five fractions (3 mL each) were collected, evaporated, and reconstituted in 100 µL of DMSO. Fractions were tested with pNPP assays to determine inhibition of STEP activity by using 0.1 µL of each fraction and 100 nM of STEP protein in 96-well plates. DMSO alone was used as a control. Shown in the insert is a representative chromatogram (UV absorbance detection, 350 nm). Peaks A, B, and C indicate early unretained material, Compound 3, and the unknown compound. (B) Structure of S₈, the benzopentathiepin core, and 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153). (C and D) Dose–response curves for S₈ and TC-2153. (C) The IC₅₀ for S₈ was determined to be 17.2±0.4 nM (mean ± s.e.m., <i>n</i> = 4). (D) The IC₅₀ for TC-2153 was determined to be 24.6±0.8 nM (mean ± s.e.m., <i>n</i> = 4).</p

Schematic of the regulation of Rv1747 putative PIM transport by Rv2623.

<p>In response to certain signals encountered in the host <b>(1)</b>, the threonine residue of Rv2623 of <i>M</i>. <i>tuberculosis</i> (a universal stress protein) at position 237 (red “T” in purple sphere) is phosphorylated <b>(2).</b> This results in the formation of a conserved phosphothreonine (pT237)-containing motif that enables the engagement of Rv2623 with the FHA I domain of Rv1747 <b>(3).</b> This interaction negatively modulates the function of the putative transporter Rv1747, turning it off <b>(4)</b>. In the absence of the signals operative in step <b>(1)</b>, or in the presence of additional signals <b>(5)</b>, dephosphorylation of the phosphorylated Rv2623 occurs <b>(6)</b>, leading to disengagement of Rv2623 from Rv1747 FHA I <b>(7)</b>. This disengagement releases the inhibitory effect of the phosphorylated Rv2623, allowing Rv1747 to transport the putative substrates PIMs <b>(8)</b>. Whether PIMs are the substrates for Rv1747 remains to be proven. The signals that induce the phosphorylation of Rv2623 are presently unclear–potential candidates include hypoxia and nitrosative stress, as well as nutritional restriction. The nature of the kinase that phosphorylates Rv2623 <i>in vivo</i> is also unknown. The Rv1747 is depicted in its monomeric form except for its transmembrane domain (TMD) for clarity. Rv2623 tethered to an orange circle with a red P represents the phosphorylated form. Red “T” in purple sphere: T237. Small red spheres represent the substrates transported by the Rv1747 transporter.</p

Superposition of <i>S</i>. <i>cerevisiae</i> Rad53 FHA domain with Rv1747 FHA I.

<p><b>(A)</b> PyMol (<a href="http://www.pymol.org/" target="_blank">www.pymol.org</a>) depiction of the surface of the <i>S</i>. <i>cerevisiae</i> Rad53 FHA based on solved structures: R70, S85, and N107, the three residues in the conserved FHA domain region that have been shown to play significant roles in mediating interaction with the phosphothreonine (pT)-containing peptide motif (<u>pT</u>EA<u>D</u>) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref032" target="_blank">32</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref033" target="_blank">33</a>], are located on the surface of the yeast Rad53 FHA domain. <b>(B)</b> Ribbon diagram (<a href="http://www.pymol.org/" target="_blank">www.pymol.org</a>) of Rad53 FHA domain demonstrating interaction between R70, S85, and N107 of Rad53 with the pTEAD residues of its interacting partner’s conserved FHA-binding motif [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref032" target="_blank">32</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref033" target="_blank">33</a>]. <b>(A&B)</b> H88 and G69 residues of the Rad53 FHA domain, which play a role in stabilizing the interaction between the Rad53 FHA domain and its interacting partner [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref032" target="_blank">32</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref033" target="_blank">33</a>], are non-surface located. <b>(C)</b> The homology model of Rv1747 FHA I domain was generated via the M4T server ver 3.0 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref034" target="_blank">34</a>] based on comparative modeling using a combination of 2 templates (PDB codes 2LC1 and 1UHT). The homology model of the Rv1747 FHA I domain was then superimposed onto the Rad53 FHA domain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref032" target="_blank">32</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref033" target="_blank">33</a>] using Pymol (<a href="http://www.pymol.org/" target="_blank">www.pymol.org</a>): Note the superposition of conversed amino acids shown in Rad53 (N107, H88, S85, G69, R70) to play important roles in recognizing the pT-containing motif (pTEAD) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref024" target="_blank">24</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref032" target="_blank">32</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref033" target="_blank">33</a>] with the corresponding residues of Rv1747 FHA I domain (N69, H50, S47, G32, R33). <b>(D)</b> Alignment of various FHA domains with the prototypic Rad53 FHA1 and FHA2 has revealed near complete match of spacing between the conserved residues of Rad53 FHA1, known to participate in the interaction with the phosphorylated FHA domain-binding motif of its interacting partner (G69, R70, S85, H88, N107), with those of the <i>M</i>. <i>tuberculosis</i> Rv1747 FHA I domain (G32, R33, S47, H50. N69), except for one amino acid difference between Rad53 R70/S85 and the Rv1747 R33/S47 spacing. KAPP: kinase associated protein phosphatase of <i>Arabidopsis thaliana</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref072" target="_blank">72</a>]; KIAA: also known as KIAA0710/NFBD1 (nuclear factor BRCT domain 1) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref073" target="_blank">73</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006515#ppat.1006515.ref074" target="_blank">74</a>]. Note the highly conserved N, S, R (green asterisks: surface location) and H and G (Grey asterisks: non-surface location) residues.</p

TC-2153 targets the active site cysteine of STEP.

<p>(A) STEP activity was measured with pNPP and IC₅₀s were 24.6±0.8 nM and 8.79±0.43 µM in the absence and presence of 1 mM GSH (mean ± s.e.m., <i>n</i> = 2). (B) STEP (200 nM) and TC-2153 (1 µM) (or DMSO control) were incubated for 60 min to inhibit enzymatic activity prior to dialysis. Aliquots were tested against pNPP (mean ± s.e.m., <i>n</i> = 4). (C) The progress curve method was used to determine the second-order rate constant: <i>k</i>_inact/<i>K</i>_i = 153,000±15,000 M⁻¹s⁻¹ (mean ± s.e.m., <i>n</i> = 4). (D) STEP (200 nM) and TC-2153 (5 µM) were incubated for 10 min and then incubated with GSH or DTT (1 mM each) or water (no reductant) for 0, 15, 30, or 60 min, and the enzymatic activity of STEP was measured using the pNPP assay (mean ± s.e.m., <i>n</i> = 4). (E) Detection of trisulfide bridge formation between C⁴⁶⁵ and C⁴⁷². The peptide sequence in (1) illustrates the trisulfide bridge along with the b and y-ion assignments detected in the MS/MS fragmentations spectrum (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001923#pbio.1001923.s006" target="_blank">Figure S6</a>). (2) compares the 3D elution profile of the trisulfide peptide (mass  = 2,746.242 Da). The trisulfide bridge (modified) peptide is only detected in the WT STEP in the presence of TC-2153. The corresponding disulfide (non-modified) peptide (mass  = 2,714.254 Da) was detected in WT STEP (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001923#pbio.1001923.s006" target="_blank">Figure S6</a>).</p