Interrogation of Carboxy-Terminus Localized GJA1 Variants Associated with Erythrokeratodermia Variabilis et Progressiva

Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell–cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.

Tomato Prosystemin is much more than a simple Systemin precursor

SummarySystemin (Sys) is an octadecapeptide which, upon wounding, is released from the carboxy terminus of its precursor, prosystemin(ProSys) to promote plant defenses. Recent findings on the disordered structure of ProSysprompted us to investigate a putative biological role of the whole precursor deprived of Sys peptide. We produced transgenic tomato plants expressing a truncated ProSys gene in which the exon coding for Sys was removed and compared their defense response with that induced by the exogenous application of the recombinant deleted ProSys[ProSys(1-178)].By combining protein structure analyses, transcriptomic analysis, gene expression profiling and bioassays with different pests we demonstrate that the truncated ProSys, that does not induce the endogenous ProSys gene, promotes defense barriers in tomato plants through a hormone independent defense pathway, likely associated with the production of oligogalacturonides (OGs). Both transgenic and plants treated with the recombinant protein showed the modulation of the expression of genes linked with defense responses and resulted protected against the lepidopteran pest Spodoptera littoralis and the fungus Botrytis cinerea. Our results suggest that the overall function of the wild type prosystemin is more complex than previously shown as it might activate at least two tomato defense pathways: the well-known Sys-dependent pathway connected with the induction of JA biosynthesis and the successive activation of a set of defense-related genes and the ProSys(1-178)-dependent pathway associated with OGs production leading to the OGs mediate plant immunity.

Inhibition of prolyl oligopeptidase by flavonoids isolated from the roots of Allexis obanensis (Baker f.) Melch

Background: Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes peptides containing proline at the carboxy terminus of proline residues. It has been associated with several neurodegenerative diseases. Therefore, it is a target in the management of these disease conditions. Methods: Allexis obanensis was taken through cold extraction, subjected to column chromatography and flavonoids isolated via high-performance liquid chromatographic technique. The flavonoids obtained were investigated for their in vitro prolyl oligopeptidase inhibitory activity. Results: The flavonoids isolated include: 4.4'''- dimethoxylophirone A [1] and 7-hydroxy-3-(3-hydroxy-4 méthoxyphenyl)-5- méthoxy-4H chromen-4-one [2]. They inhibited prolyl oligopeptidase at low IC50 concentrations of 7.201±3.021 µM and 6.223±2.002 µM respectively. Conclusion: The results obtained from this study proves the potential of these flavonoids as prolyl oligopeptidase inhibitors, by inference, their potentiality in the management of neuropsychiatric disorders.

Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo.

Identification of Critical Residues in the Carboxy Terminus of the Dopamine Transporter Involved in the G Protein βγ-Induced Dopamine Efflux

The dopamine transporter (DAT) plays a crucial role in the regulation of brain dopamine (DA) homeostasis through the re-uptake of DA back into the presynaptic terminal. In addition to re-uptake, DAT is also able to release DA through a process referred to as DAT-mediated DA efflux. This is the mechanism by which potent and highly addictive psychostimulants, such as amphetamine (AMPH) and its analogues, increase extracellular DA levels in motivational and reward areas of the brain. Recently, we discovered that G protein βγ subunits (Gβγ) binds to the DAT, and that activation of Gβγ results in DAT-mediated efflux - a similar mechanism as AMPH. Previously, we have shown that Gβγ binds directly to a stretch of 15 residues within the intracellular carboxy terminus of DAT (residues 582–596). Additionally, a TAT peptide containing residues 582 to 596 of DAT was able to block the Gβγ-induced DA efflux through DAT. Here, we use a combination of computational biology, mutagenesis, biochemical, and functional assays to identify the amino acid residues within the 582–596 sequence of the DAT carboxy terminus involved in the DAT-Gβγ interaction and Gβγ-induced DA efflux. Our in-silico protein-protein docking analysis predicted the importance of F587 and R588 residues in a network of interactions with residues in Gβγ. In addition, we observed that mutating R588 to alanine residue resulted in a mutant DAT which exhibited attenuated DA efflux induced by Gβγ activation. We demonstrate that R588, and to a lesser extent F5837, located within the carboxy terminus of DAT play a critical role in the DAT-Gβγ physical interaction and promotion of DA efflux. These results identify a potential new pharmacological target for the treatment of neuropsychiatric conditions in which DAT functionality is implicated including ADHD and substance use disorder.

TTL-Expression Modulates Epithelial Morphogenesis

Epithelial monolayer formation depends on the architecture and composition of the microtubule cytoskeleton. Microtubules control bidirectional trafficking and determine the positioning of structural cellular proteins. We studied the role of tubulin tyrosination in epithelial cell shape and motility. Tubulin tyrosine ligase (TTL), the enzyme that adds tyrosine to the carboxy terminus of detyrosinated α-tubulin, was depleted or overexpressed in 2D epithelial monolayers as well as in 3D intestinal organoids. We demonstrate qualitatively and quantitatively that in the absence of TTL the cells comprise high levels of detyrosinated tubulin, change their shape into an initial flat morphology and retardedly acquire a differentiated columnar epithelial cell shape. Enhanced adhesion and accelerated migration patterns of TTL-knockout cells combined with reverse effects in TTL-overexpressing cells indicate that the loss of TTL affects the organization of cell adhesion foci. Precipitation of detyrosinated tubulin with focal adhesion scaffold components coincides with increased quantities and persistence of focal adhesion plaques. Our results indicate that the equilibrium between microtubules enriched in detyrosinated or tyrosinated tubulin modulates epithelial tissue formation, cell morphology, and adhesion.

Publisher Correction: Characterization of three TRAPPC11 variants suggests a critical role for the extreme carboxy terminus of the protein

An amendment to this paper has been published and can be accessed via a link at the top of the paper.