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G-protein-coupled receptors (GPCRs) transduce diverse signals into the cell by coupling to one or several Gα subtypes. Of the 16 Gα subtypes in human cells, Gα 12 and Gα 13 belong to the G 12 subfamily and are reported to be functionally different. Notably, certain GPCRs display selective coupling to either Gα 12 or Gα 13 , highlighting their significance in various cellular contexts. However, the structural basis underlying this selectivity remains unclear. Here, using a Gα 12 -coupled designer receptor exclusively activated by designer drugs (DREADD; G 12 D) as a model system, we identified residues in the α5 helix and the receptor that collaboratively determine Gα 12 -vs-Gα 13 selectivity. Residue-swapping experiments showed that G 12 D distinguishes differences between Gα 12 and Gα 13 in the positions G.H5.09 and G.H5.23 in the α5 helix. Molecular dynamics simulations observed that I378 G.H5.23 in Gα 12 interacts with N103 2.39 , S169 3.53 and Y176 34.53 in G 12 D, while H364 G.H5.09 in Gα 12 interact with Q264 5.71 in G 12 D. Screening of mutations at these positions in G 12 D identified G 12 D mutants that enhanced coupling with Gα 12 and to an even greater extent with Gα 13 . Combined mutations, most notably the dual Y176 34.53 H and Q264 5.71 R mutant, further enhanced Gα 12 / 13 coupling, thereby serving as a potential Gα 12/13 -DREADD. Such novel Gα 12/13 -DREADD may be useful in future efforts to develop drugs that target Gα 12/13 signaling as well as to identify their therapeutic indications., (© 2024. The Author(s).)

Polo-like kinase 1 (Plk1) is a mitotic kinase that has multiple functions throughout the cell cycle. Catalytic activation of Plk1 is known to be regulated by phosphorylation of the kinase domain, including Thr210, and by releasing the kinase domain from its inhibitory polo-box domain. However, how Plk1 is activated to fulfill its proper roles, in time and space, is not well understood. In this study, we unintentionally found that the expression of a constitutively active form of human Plk1 is toxic to bacterial cells, such that cells contained point mutations that alleviate the kinase activity. Structural prediction revealed that these mutations are adjacent to the amino acids supporting the kinase activity. When human cells express these mutants, we found decreased levels of Plk1's substrate phosphorylation, resulting in mitotic defects. Moreover, unlike in bacterial cells, the expression of activated Plk1 mutants did not affect cell proliferation in human cells unless localized at the right place in mitosis. Our observations identified new suppressor mutations and underscored the importance of spatiotemporal regulation in Plk1, providing a basis for how we might intervene in this kinase for therapeutic purpose in human cells., (© 2023 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Cancer stem cells have the capability of self-renewal and multipotency and are, therefore, associated with tumor heterogeneity, resistance to chemoradiation therapy, and metastasis. The hypothesis that multinucleated giant cells, which often emerge following chemo- and/or radiotherapy, serve as cancer stem cells has not been fully evaluated. Although a previous study demonstrated that these cells functioned as stem cells, only low levels of Yamanaka factors were expressed, contrasting with the high expression seen from their gestated first-generation mononuclear cells. Herein, we report a case of a plasmablastic neoplasm with multinucleated giant cells that were analyzed for stemness to test the above hypothesis. The patient was a male in his 80s who had a plasmablastic neoplasm that was not easily distinguishable as plasmablastic lymphoma versus plasma cell myeloma of plasmablastic type. Lymph node biopsy showed predominant mononuclear cell proliferation with admixed multinucleated giant cells. Immunohistochemistry and in situ hybridization showed that both multinucleated and mononuclear cells had the same profile: CD138(+), light chain restriction of κ>λ, cyclin D1(+), CD68(-), EBER-ISH (+). These results suggested that both cell types were neoplastic. In accordance with the previous study, the multinucleated giant cells showed low expression of Yamanaka factors, which were highly expressed in some of the mononuclear cells. Furthermore, the multinucleated giant cells showed a much lower proliferative activity (Mib1/Ki67 index) than the mononuclear cells. Based on these results, the multinucleated giant cells were compatible with cancer stem cells. This case is expected to expand the knowledge base regarding biology of cancer stem cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Otsuka-Kamakura, Sugiura, Yamazaki, Shimizu and Hiruta.)

Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods.

Matrix metalloproteinase-20 (MMP20; enamelysin) is important for proteolytic processing of extracellular matrix (ECM) proteins during the formation of enamel and plays a critical role in proteolytic processing of amelogenin (AMEL), the most abundant enamel ECM protein. MMP20 might have played a role in the emergence of teeth, because jawless vertebrates with primordial teeth on their external skeletons may have possessed the MMP20 gene, and MMP20 and enamel ECM proteins are thought to have evolved together in a special relationship over time. Thus, an understanding of the molecular evolution of the MMP20 gene is important for elucidating the evolution of enamel and it is necessary to identify the orthologs of the MMP20 gene in non-mammals, as it has been identified in mammals. In the present study, orthologs of the MMP20 genes from a reptile (caiman) and an amphibian (African clawed toad) were cloned and characterized. Comparisons of the orthologs revealed that the MMP20 proteins were highly conserved throughout the evolution of tetrapods. Further, the caiman, toad, and mammalian MMP20 shared several unique features specific for MMP20, but not for other matrix metalloproteinases. In addition, the toad MMP20 gene was transcribed only in the upper jaw, presumably in teeth. These results suggest that MMP20 in a common ancestor of tetrapods might have been recruited for the processing of AMEL and conserved over 350 million years of evolution.