Meredith A. Borman

Identification of the endogenous smooth muscle myosin phosphatase-associated kinase

Ca2+ sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by “mixed-peptide” Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca2+ sensitizing kinase cascade.

Comparative Effectiveness of Immunosuppressants and Biologics for Inducing and Maintaining Remission in Crohn's Disease: A Network Meta-analysis

BACKGROUND & AIMS There is controversy regarding the best treatment for patients with Crohns disease because of the lack of direct comparative trials. We compared therapies for induction and maintenance of remission in patients with Crohns disease, based on direct and indirect evidence. METHODS We performed systematic reviews of MEDLINE, EMBASE, and Cochrane Central databases, through June 2014. We identified randomized controlled trials (N = 39) comparing methotrexate, azathioprine/6-mercaptopurine, infliximab, adalimumab, certolizumab, vedolizumab, or combined therapies with placebo or an active agent for induction and maintenance of remission in adult patients with Crohns disease. Pairwise treatment effects were estimated through a Bayesian random-effects network meta-analysis and reported as odds ratios (OR) with a 95% credible interval (CrI). RESULTS Infliximab, the combination of infliximab and azathioprine (infliximab + azathioprine), adalimumab, and vedolizumab were superior to placebo for induction of remission. In pair-wise comparisons of anti-tumor necrosis factor agents, infliximab + azathioprine (OR, 3.1; 95% CrI, 1.4-7.7) and adalimumab (OR, 2.1; 95% CrI, 1.0-4.6) were superior to certolizumab for induction of remission. All treatments were superior to placebo for maintaining remission, except for the combination of infliximab and methotrexate. Adalimumab, infliximab, and infliximab + azathioprine were superior to azathioprine/6-mercaptopurine: adalimumab (OR, 2.9; 95% CrI, 1.6-5.1), infliximab (OR, 1.6; 95% CrI, 1.0-2.5), infliximab + azathioprine (OR, 3.0; 95% CrI, 1.7-5.5) for maintenance of remission. Adalimumab and infliximab + azathioprine were superior to certolizumab: adalimumab (OR, 2.5; 95% CrI, 1.4-4.6) and infliximab + azathioprine (OR, 2.6; 95% CrI, 1.3-6.0). Adalimumab was superior to vedolizumab (OR, 2.4; 95% CrI, 1.2-4.6). CONCLUSIONS Based on a network meta-analysis, adalimumab and infliximab + azathioprine are the most effective therapies for induction and maintenance of remission of Crohns disease.

Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase

Phosphorylation of CPI‐17 and PHI‐1 by the MYPT1‐associated kinase (M110 kinase) was investigated. M110 kinase is a recently identified serine/threonine kinase with a catalytic domain that is homologous to that of ZIP kinase (ZIPK. GST‐rN‐ZIPK, a constitutively active GST fusion fragment, phosphorylates CPI‐17 (but not PHI‐1) to a stoichiometry of 1.7 mol/mol. Phosphoamino acid analysis revealed phosphorylation of both Ser and Thr residues. Phosphorylation sites in CPI‐17 were identified as Thr 38 and Ser 12 using Edman sequencing with 32P release and a point mutant of Thr 38.

Integrin-linked kinase is responsible for Ca2+-independent myosin diphosphorylation and contraction of vascular smooth muscle

Smooth muscle contraction is activated by phosphorylation at Ser-19 of LC20 (the 20 kDa light chains of myosin II) by Ca2+/calmodulin-dependent MLCK (myosin light-chain kinase). Diphosphorylation of LC20 at Ser-19 and Thr-18 is observed in smooth muscle tissues and cultured cells in response to various contractile stimuli, and in pathological circumstances associated with hypercontractility. MLCP (myosin light-chain phosphatase) inhibition can lead to LC20 diphosphorylation and Ca2+-independent contraction, which is not attributable to MLCK. Two kinases have emerged as candidates for Ca2+-independent LC20 diphosphorylation: ILK (integrin-linked kinase) and ZIPK (zipper-interacting protein kinase). Triton X-100-skinned rat caudal arterial smooth muscle was used to investigate the relative importance of ILK and ZIPK in Ca2+-independent, microcystin (phosphatase inhibitor)-induced LC20 diphosphorylation and contraction. Western blotting and in-gel kinase assays revealed that both kinases were retained in this preparation. Ca2+-independent contraction of calmodulin-depleted tissue in response to microcystin was resistant to MLCK inhibitors [AV25 (a 25-amino-acid peptide derived from the autoinhibitory domain of MLCK), ML-7, ML-9 and wortmannin], protein kinase C inhibitor (GF109203X) and Rho-associated kinase inhibitors (Y-27632 and H-1152), but blocked by the non-selective kinase inhibitor staurosporine. ZIPK was inhibited by AV25 (IC50 0.63+/-0.05 microM), whereas ILK was insensitive to AV25 (at concentrations as high as 100 microM). AV25 had no effect on Ca2+-independent, microcystin-induced LC20 mono- or di-phosphorylation, with a modest effect on force. We conclude that direct inhibition of MLCP in the absence of Ca2+ unmasks ILK activity, which phosphorylates LC20 at Ser-19 and Thr-18 to induce contraction. ILK is probably the kinase responsible for myosin diphosphorylation in vascular smooth muscle cells and tissues.

Smooth Muscle Myosin Phosphatase-associated Kinase Induces Ca2+ Sensitization via Myosin Phosphatase Inhibition

Smooth muscle calcium sensitization reflects an inhibition of myosin light chain phosphatase (SMPP-1m) activity; however, the underlying mechanisms are not well understood. SMPP-1m activity can be modulated through phosphorylation of the myosin targeting subunit (MYPT1) by the endogenous myosin phosphatase-associated kinase, MYPT1 kinase (MacDonald, J. A., Borman, M. A., Muranyi, A., Somlyo, A. V., Hartshorne, D. J., and Haystead, T. A. (2001)Proc. Natl. Acad. Sci. U. S. A. 98, 2419–2424). Recombinant chicken gizzard MYPT1 (M130) was phosphorylated in vitro by a recombinant MYPT1 kinase, and the sites of phosphorylation were identified as Thr654, Ser808, and Thr675. Introduction of recombinant MYPT1 kinase elicited a calcium-independent contraction in β-escin-permeabilized rabbit ileal smooth muscle. Using an antibody that specifically recognizes MYPT1 phosphorylated at Thr654(M130 numbering), we determined that this calcium-independent contraction was correlated with an increase in MYPT1 phosphorylation. These results indicate that SMPP-1m phosphorylation by MYPT1 kinase is a mechanism of smooth muscle calcium sensitization.

Modulation of smooth muscle contractility by CHASM, a novel member of the smoothelin family of proteins.

Cyclic nucleotides acting through their associated protein kinases, the cGMP‐ and cAMP‐dependent protein kinases, can relax smooth muscles without a change in free intracellular calcium concentration ([Ca2+] i ), a phenomenon referred to as Ca2+ desensitization. The molecular mechanisms by which these kinases bring about Ca2+ desensitization are unknown and an understanding of this phenomenon may lead to better therapies for treating diseases involving defects in the contractile response of smooth muscles such as hypertension, bronchospasm, sexual dysfunction, gastrointestinal disorders and glaucoma. Utilizing a combination of real‐time proteomics and smooth muscle physiology, we characterized a distinct subset of protein targets for cGMP‐dependent protein kinase in smooth muscle. Among those phosphoproteins identified was calponin homology‐associated smooth muscle (CHASM), a novel protein that contains a calponin homology domain and shares sequence similarity with the smoothelin family of smooth muscle specific proteins. Recombinant CHASM was found to evoke relaxation in a concentration dependent manner when added to permeabilized smooth muscle. A co‐sedimentation assay with actin demonstrated that CHASM does not possess actin binding activity. Our findings indicate that CHASM is a novel member of the smoothelin protein family that elicits Ca2+ desensitization in smooth muscle.

Solution Structure of the Calponin Homology (CH) Domain from the Smoothelin-like 1 Protein A UNIQUE APOCALMODULIN-BINDING MODE AND THE POSSIBLE ROLE OF THE C-TERMINAL TYPE-2 CH-DOMAIN IN SMOOTH MUSCLE RELAXATION

The SMTNL1 protein contains a single type-2 calponin homology (CH) domain at its C terminus that shares sequence identity with the smoothelin family of smooth muscle-specific proteins. In contrast to the smoothelins, SMTNL1 does not associate with F-actin in vitro, and its specific role in smooth muscle remains unclear. In addition, the biological function of the C-terminal CH-domains found in the smoothelin proteins is also poorly understood. In this work, we have therefore determined the solution structure of the CH-domain of mouse SMTNL1 (SMTNL1-CH; residues 346-459). The secondary structure and the overall fold for the C-terminal type-2 CH-domain is very similar to that of other CH-domains. However, two clusters of basic residues form a unique surface structure that is characteristic of SMTNL1-CH. Moreover, the protein has an extended C-terminal α-helix, which contains a calmodulin (CaM)-binding IQ-motif, that is also a distinct feature of the smoothelins. We have characterized the binding of apo-CaM to SMTNL1-CH through its IQ-motif by isothermal titration calorimetry and NMR chemical shift perturbation studies. In addition, we have used the HADDOCK protein-protein docking approach to construct a model for the complex of apo-CaM and SMTNL1-CH. The model revealed a close interaction of SMTNL1-CH with the two Ca2+ binding loop regions of the C-terminal domain of apo-CaM; this mode of apo-CaM binding is distinct from previously reported interactions of apo-CaM with IQ-motifs. Finally, we comment on the putative role of the CH-domain in the biological function of SMTNL1.

Ca2+-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide.

As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC20) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca2+-independent, microcystin- and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca2+-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca2+-independent LC20 kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK((1-320))-D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC(20) and MYPT1 in situ. AV25 or molecules based on its structure could be used in therapeutic situations to induce contractility in diseases of the gastrointestinal tract associated with hypomotility.

Anti-TNF-induced autoimmune hepatitis

Fig. 1. Liver biopsies demonstrating typical features of AIH. Liver biopsy from the first patient, after four months treatment with adalimumab, shows expansion of portal areas due to predominant plasma cell infiltrate with prominent interface inflammatory activity and bridging necrosis (A and B). Liver biopsy from the second patient, after twelve months treatment with infliximab, shows massive hepatic necrosis with almost complete destruction of hepatocytes within the sampled tissue, portal triad expansion with connective tissue, bile duct proliferation and predominant lymphoplasmacytic cell infiltrate (C and D). To the Editor: We read with interest the report by Weiler-Normann et al. [1] evaluating the safety and efficacy of infliximab as a rescue therapy in difficult-to-treat autoimmune hepatitis (AIH). TNF-targeted therapies are widely used for treating a rapidly growing number of autoimmune diseases. However despite their effectiveness in many of these diseases, there have been increasing reports of autoimmune processes developing de novo after their use, including AIH. This is highlighted by our experience with two cases of severe anti-TNF therapy-induced AIH. The first patient, a 60-year-old woman with a 20-year history of rheumatoid arthritis, treated with prednisone 5 mg/day, with remote exposure to methotrexate (MTX). She presented with elevated liver enzymes four months after treatment with adalimumab, which had been normal prior to anti-TNF therapy. Her liver enzymes were grossly abnormal with ALT 923 IU/L (normal <40 IU/L), AST 1146 IU/L (normal <32 IU/L), bilirubin 76 lmol/L (normal <20 lmol/L), INR 1.1 (normal 0.9–1.1), and albumin 36 g/L (normal 33–48 g/L). Anti-nuclear antibody (ANA) was positive, documented prior to initiation of anti-TNF therapy, anti-smooth muscle antibody (ASMA) negative, and IgG level normal at 15.52 g/L (normal 6.80–18.00 g/L). ds-DNA antibodies were highly positive. Despite discontinuing adalimumab, over the following month she developed features of progressive liver failure with INR 1.8, albumin 16 g/L, bilirubin 271 lmol/L, pruritis, and progressive lower extremity edema. There was no hepatic encephalopathy. Fibroscan showed a liver stiffness of 33.3 kPa (IQR 4.8 kPa; 100% success). Liver biopsy showed features consistent with AIH, including expansion of portal areas with dense plasma cell infiltrates, prominent interface activity, and bridging necrosis (Fig. 1A and B). Prednisone 40 mg/day was started in combination with azathioprine 50 mg/day. Liver enzymes completely normalized within 8 weeks of treatment. Serial fibroscans showed a striking improvement in liver stiffness, with her most recent liver stiffness measure of 5.4 kPa (IQR 0.9 kPa; 77% success). She remains in biochemical remission and is currently being reconsidered for alternative anti-TNF therapy for management of her rheumatoid arthritis. The second patient is a 48-year-old woman with an 18 year history of rheumatoid arthritis. She was treated with MTX and naproxen, both of which were discontinued in November 2010 due to elevated ALT (158 IU/L). Liver enzymes completely normalized following drug withdrawal (ALT 19 IU/L; June 2011). In July 2011 she was started on infliximab (IFX) 5 mg/kg and after 12 months of therapy she was admitted to hospital with hepatic failure with INR 1.6, albumin 22 g/L, bilirubin 408 lmol/L, ALT 505 IU/L, AST 1614 IU/L, and moderate ascites. She had no hepatic encephalopathy. ANA was positive (negative prior to treatment with IFX), ASMA negative, and IgG level elevated at 27.8 g/L. Liver biopsy showed features consistent with severe AIH including submassive hepatic necrosis with plasma cell rich inflammation and significant fibrosis (Fig. 1C and D). Fibroscan revealed a liver stiffness of 24.6 kPa (IQR 14.4; 63% success). IFX was

Tropomyosin-binding properties of the CHASM protein are dependent upon its calponin homology domain

MINT‐7966090: CHASM (uniprotkb:Q99LM3) binds (MI:0407) to Tropomyosin alpha (uniprotkb:P04268) by pull down (MI:0096)