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mTOR signaling in spermatogenesis and male infertility
Published in Rajender Singh, Molecular Signaling in Spermatogenesis and Male Infertility, 2019
Several factors lead to the activation of mTORC1 signaling, such as cellular energy status, nutrients and growth factors, which involve PI3 K and Akt as upstream regulators and ribosomal protein S6 kinase (also known as p70 S6 K) as an activator of downstream effector molecule rpS6 (ribosomal protein S6) (30). Some studies have reported a crucial role of mTORC1 in spermatogenesis by promoting the restructuring of BTB and making it leaky (32). rps6 is highly expressed at all stages of the epithelial cycle during spermatogenesis in adult rat testes, and its expression is found to be consistent with its localization at the BTB. Phospho-rps6 (activated form of rps6) is highly expressed at the BTB and co-localized with BTB proteins ZO-1, N-cadherin and Arp3. Phosphorylation of rps6 was confined at the time of BTB restructuring to facilitate the transit of preleptotene spermatocytes and was restricted to late stages VIII–IX of the epithelial cycle. This stage-specific upregulation of activated rps6 (p-rps6) at the BTB suggests that p-rps6 induces the opening of BTB to transit preleptotene spermatocytes into the adluminal compartment. Knockdown of rpS6 by RNAi (using either rpS6-specific siRNA or shRNA), in vitro or in vivo, promoted the formation of BTB by making it tighter. Conversely, rpS6 phosphomimetic mutant p-rpS6 overexpression in cultured Sertoli cells with an established TJ barrier (mimicking the BTB in vivo) made the BTB leaky. The possible mechanisms explained were that the mutant p-rpS6 in Sertoli cells downregulates Akt1/2, which induces matrix metalloproteinase 9 (MMP-9) production by the Sertoli cells causing proteolysis of TJ and/or basal adhesion protein complexes, making the BTB leaky (32). Another mechanism explained (33,31) was through upregulation of p-rps6 causing downregulation of p-Akt1/2, which in turn increased association of Arp3 and its upstream activator N-WASP (neuronal Wiskott-Aldrich syndrome protein), resulting in actin nucleation at the barbed ends of a linear actin microfilament (i.e., branched actin polymerization). This reorganizes the actin filaments from a bundled to a branched/un-bundled network, thereby destabilizing cell adhesion protein complexes at the BTB (e.g., occluding, ZO-1, N-cadherin, α-catenin which utilize F-actin for attachment at the basal ES/BTB), making the BTB leaky (31,33). These studies are further supported by the study of Wen et al. on Sertoli cells with an established TJ-permeability barrier that mimicked the BTB. On treating these cells with Rapamycin, the mTORC1 complex function was blocked causing the barrier to become tighter (30). These studies highlighted that the mTORC1 signaling exerts its disruptive effects on BTB dynamics during spermatogenesis (Figure 14.2).
The Ethanolic Extract of Trichosanthes Kirilowii Root Exerts anti-Cancer Effects in Human Non-Small Cell Lung Cancer Cells Resistant to EGFR TKI
Published in Nutrition and Cancer, 2022
In order to investigate whether STAT3 mediated the anticancer effects of ETK, we transfected constitutively active STAT3 in H1975 cells. We hypothesized that constitutive activation of STAT3 by Y705D mutations compromises the anti-proliferative effect of ETK. As shown in Figure 5A, STAT3 was strongly phosphorylated by transfection of constitutively activated STAT3 (Figure 5A). In addition, as expected, STAT3-activated H1975 cells were highly resistant to ETK treatment compared with empty vector (EV)-transfected cells. Following treatment with 25, 50, and 100 μg/mL ETK, the cell viability of EV-transfected cells was %, 51.24%, and 43.77%, respectively, while that of STAT3-activated cells was %, 85.27%, and 64.81%, respectively (Figure 5B). The percentage of apoptotic cells following ETK treatment also decreased by transfection of phospho-mimetic STAT3 Y705D (Figure 5C). Collectively, our observations clearly show that ETK-induced apoptosis was reversed by STAT3 activation, suggesting that ETK exerted anticancer effect on EGFR TKI-resistant NSCLC cells via inactivation of STAT3.
A review of protein-protein interaction and signaling pathway of Vimentin in cell regulation, morphology and cell differentiation in normal cells
Published in Journal of Receptors and Signal Transduction, 2022
Danial Hashemi Karoii, Hossein Azizi
While the molecular mechanisms underlying these interactions have not been clearly illustrated, examples of integrin and 14-3-3 interactions with their own specific phosphorylation motifs on vimentin suggest that vimentin’s dynamic and very complex phosphorylation pattern is a likely mechanism behind the regulation of many of the newly identified partners. While the activities of numerous phosphorylation motifs have already been discussed, a significant portion of the N and C-terminal phosphorylation sites remain unidentified. Since these phosphorylation sites are usually highly conserved, it is to be assumed that essential interaction mechanisms and regulatory roles have yet to be found. As a result, functional investigation of phosphomimetic mutations at these locations will pave the way for a better understanding of vimentin’s regulatory roles.
SAMHD1: Recurring roles in cell cycle, viral restriction, cancer, and innate immunity
Published in Autoimmunity, 2018
Christopher H. Mauney, Thomas Hollis
Studies attempting to shed light on these discrepancies reveal that P-T592 results in altered kinetics of tetramer association and dissociation and expedited regulatory nucleotide release [77,129]. P-T592 is also less likely to form the activated tetramer at low concentration of activating nucleotides [78,81]. Structural data support these findings by identifying protein conformational shifts induced by a phosphomimetic mutant (T592E) that destabilizes the tetramer interface [127]. Taken in sum, these data appear to indicate that phosphorylation at T592 is a mechanism for calibrating SAMHD1 activity through altering the thermodynamics of subunit association. The discrepant results may stem from variations in culture conditions or assay methods that lack the sensitivity to accurately capture the true effect of phosphorylation. Fine tuning SAMHD1 activity within the cell by phosphorylation, as opposed to a binary on or off state, may be important as a complementary method of regulation in order to maintain dNTP pools within the narrow window conducive to genomic integrity.