Vaughansiegel1758

Olfactory receptors are G-protein coupled receptors (GPCRs) that enable olfactory epithelia to detect odorants. These GPCRs may also show constitutive activity, which play important roles in the development and responses of odorant receptor neurons. However, little is known about the molecular characteristics that support the constitutive activities in olfactory receptors. Here, we characterize a pair of olfactory receptor orthologs that show similar ligand-dependent activity but different levels of constitutive activity, and elucidate the molecular characteristics that maintain the constitutive activity. Previously, PmTAAR348, a sea lamprey (Petromyzon marinus) olfactory receptor that is activated by the male sex pheromone spermine has been reported. In this study, we identified LmTAAR348 of Northeast Chinese lamprey (Lethenteron morii) as an ortholog of PmTAAR348. When expressed in HEK293T cell lines, both receptors showed similar levels of activation when exposed to spermine. However, the constitutive activity of LmTAAR348 was 100-fold higher than that of PmTAAR348. Using site-directed mutagenesis, we screened all 13 amino acid residues (aa) that differed between the two orthologs and found that a switch in position 340 reversed the constitutive activity levels between LmTAAR348 and PmTAAR348. Mutating the remaining 12 aa did not affect the ligand-dependent or constitutive activation. Moreover, both the ligand-dependent and constitutive activation of TAAR348 are Golf (G protein ⍺ subunit olfactory type) independent. We conclude that a single aa in the C-terminal maintains the constitutive activity in a spermine receptor. Receptor tyrosine kinase EphA7 is specifically expressed in otic region in Xenopus early development. However, its role in otocyst development remains unknown. Knockdown of EphA7 by a specific morpholino oligonucleotide (MO) reduced the size of the otocyst and triggered otic epithelial cell extrusion. Interestingly, EphA7 depletion attenuated the membrane level of the tight junction protein Claudin6 (CLDN6). Utilizing the Cldn6 MO, we further confirmed that CLDN6 attenuation also led to otic epithelial cell extrusion. Our work suggested that EphA7 modulates the otic epithelial homeostasis through stabilizing the CLDN6 membrane level. Breast cancer (BC), the most frequent cancer in women worldwide, is extremely heterogeneous. For effective and precise treatment and to cope with drug resistance in BC, we need to find more therapeutic molecular targets. In this study, we found that the Proteasome 26S Subunit, Non-ATPase 12 (PSMD12) was upregulated in BC samples, its expression was heterogeneous among different cell lines, and high levels of PSMD12 were related to poor prognosis of BC patients. Notably, the expression of PSMD12 increased in the nucleus. Cytological experiments revealed that PSMD12 knockdown inhibited cell growth and migration, and a genome-wide CRISPR-Cas9 knockout (GeCKO) screen also confirmed that PSMD12 is a crucial gene for the growth of BC cells. Flow cytometry showed that cell apoptosis increased in the PSMD12 knockdown, and RNA-seq indicated that the apoptosis pathway was activated, and the TXNIP, GADD45A, GADD45B, RHOB, and CDKN1A pro-apoptotic genes were highly expressed, a result that was validated by RT-qPCR and Western blot. Furthermore, restoration of PSMD12 expression decreased the expression of pro-apoptotic genes. A tumor-bearing mice assay demonstrated that BC growth was arrested by reduced PSMD12 levels in vivo. Taken together, PSMD12, a subunit of 19S regulator of 26S proteasome, was identified as a potential prognostic and therapeutic molecular target for BC, which provides a new insight for developing anticancer drugs that promote apoptosis based on the targeting of the 26S proteasome complex. Apolipoprotein A-I (ApoA-I) mimetic peptides are potential therapeutic agents for promoting the efflux of excess cellular cholesterol, which is dependent upon the presence of an amphipathic helix. Since α-methylated Ala enhances peptide helicity, we hypothesized that incorporating other types of α-methylated amino acids into ApoA-I mimetic peptides may also increase their helicity and cholesterol efflux potential. The last helix of apoA-I, peptide 'A' (VLESFKVSFLSALEEYTKKLNT), was used to design peptides containing a single type of α-methylated amino acid substitution (Ala/Aα, Glu/Dα, Lys/Kα, Leu/Lα), as well as a peptide containing both α-methylated Lys and Leu (6α). Depending on the specific residue, the α-helical content as measured by CD-spectroscopy and calculated hydrophobic moments were sometimes higher for peptides containing other types of α-methylated amino acids than those with α-methylated Ala. In ABCA1-transfected cells, cholesterol efflux to the peptides showed the following order of potency 6α>Kα≈Lα≈Aα≫Dα≈A. In general, α-methylated peptides were resistant to proteolysis, but this varied depending on the type of protease and specific amino acid substitution. In summary, increased helicity and amphilicity due to α-methylated amino acid substitutions in ApoA-I mimetic peptides resulted in improved cholesterol efflux capacity and resistance to proteolysis, indicating that this modification may be useful in the future design of therapeutic ApoA-I mimetic peptides. Published by Elsevier Inc.Proteolytic processing of membrane proteins by A disintegrin and metalloprotease-17 (ADAM17) is a key regulatory step in many physiological and pathophysiological processes. selleck inhibitor This so-called shedding is essential for development, regeneration and immune defense. An uncontrolled ADAM17 activity promotes cancer development, chronic inflammation and autoimmune diseases. Consequently, the ADAM17 activity is tightly regulated. As a final trigger for the shedding event a phosphatidylserine (PS) flip to the outer leaflet of the cell membrane was recently described. PS interacts with the extracellular part of ADAM17, which results in the shedding event by shifting the catalytic domain towards the membrane close to the cleavage sites within ADAM17 substrates. Our data indicate that the intrinsic proteolytic activity of the catalytic domain is prerequisite for the shedding activity and constantly present. However, the accessibility for substrate cleavage sites is controlled on several levels. In this report, we demonstrate that the positioning of the catalytic domain towards the cleavage sites is a crucial part of the shedding process.