Periostin is essential for cardiac healingafter acute myocardial infarction

Acute myocardial infarction (AMI) is a common and lethal heart disease, and the recruitment of fibroblastic cells to the infarct region is essential for the cardiac healing process. Although stiffness of the extracellular matrix in the infarct myocardium is associated with cardiac healing, the molecular mechanism of cardiac healing is not fully understood. We show that periostin, which is a matricellular protein, is important for the cardiac healing process after AMI. The expression of periostin protein was abundant in the infarct border of human and mouse hearts with AMI. We generated periostin−/− mice and found no morphologically abnormal cardiomyocyte phenotypes; however, after AMI, cardiac healing was impaired in these mice, resulting in cardiac rupture as a consequence of reduced myocardial stiffness caused by a reduced number of α smooth muscle actin–positive cells, impaired collagen fibril formation, and decreased phosphorylation of FAK. These phenotypes were rescued by gene transfer of a spliced form of periostin. Moreover, the inhibition of FAK or αv-integrin, which blocked the periostin-promoted cell migration, revealed that αv-integrin, FAK, and Akt are involved in periostin signaling. Our novel findings show the effects of periostin on recruitment of activated fibroblasts through FAK-integrin signaling and on their collagen fibril formation specific to healing after AMI

Delayed Re-Epithelialization in Periostin-Deficient Mice during Cutaneous Wound Healing

BACKGROUND: Matricellular proteins, including periostin, are important for tissue regeneration. METHODS AND FINDINGS: Presently we investigated the function of periostin in cutaneous wound healing by using periostin-deficient ⁻/⁻ mice. Periostin mRNA was expressed in both the epidermis and hair follicles, and periostin protein was located at the basement membrane in the hair follicles together with fibronectin and laminin γ2. Periostin was associated with laminin γ2, and this association enhanced the proteolytic cleavage of the laminin γ2 long form to produce its short form. To address the role of periostin in wound healing, we employed a wound healing model using WT and periostin⁻/⁻ mice and the scratch wound assay in vitro. We found that the wound closure was delayed in the periostin⁻/⁻ mice coupled with a delay in re-epithelialization and with reduced proliferation of keratinocytes. Furthermore, keratinocyte proliferation was enhanced in periostin-overexpressing HaCaT cells along with up-regulation of phosphorylated NF-κB. CONCLUSION: These results indicate that periostin was essential for keratinocyte proliferation for re-epithelialization during cutaneous wound healing

Druggable Transient Pockets in Protein Kinases

Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of “cryptic inhibitor-binding sites.” These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors

DYRK1B mutations associated with metabolic syndrome impair the chaperone-dependent maturation of the kinase domain

Two missense mutations of the DYRK1B gene have recently been found to co-segregate with a rare autosomal-dominant form of metabolic syndrome. This gene encodes a member of the DYRK family of protein kinases, which depend on tyrosine autophosphorylation to acquire the catalytically active conformation. The mutations (H90P and R102C) affect a structural element named DYRK homology (DH) box and did not directly interfere with the conformation of the catalytic domain in a structural model of DYRK1B. Cellular assays showed that the mutations did not alter the specific activity of mature kinase molecules. However, a significant part of the mutant DYRK1B protein accumulated in detergent-insoluble cytoplasmic aggregates and was underphosphorylated on tyrosine. The mutant DYRK1B variants were more vulnerable to the HSP90 inhibitor ganetespib and showed enhanced binding to the co-chaperone CDC37 as compared to wild type DYRK1B. These results support the hypothesis that the mutations in the DH box interfere with the maturation of DYRK1B by tyrosine autophosphorylation and compromise the conformational stability of the catalytic domain, which renders the kinase susceptible to misfolding

Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ

The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimers disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases