Micropollutants (ciprofloxacin and norfloxacin) remediation from wastewater through laccase derived from spent mushroom waste: Fate, toxicity, and degradation.

Fluoroquinolone antibiotics frequently found in environmental matrices (wastewater treatment plants, hospital wastewater, industrial wastewater and surface wastewater) causes potential threat to the environment. Enzymatic treatment for degradation of antibiotics from environmental matrices is a green and sustainable approach. Focusing on this, this study aimed to degrade two frequently found fluroquinolone emergent pollutants, ciprofloxacin and norfloxacin from wastewater. The trinuclear cluster of copper ions present in laccase has the ability to effectively remove organic micropollutants (OMPs). The uniqueness of this study is that it utilizes laccase enzyme extracted from spent mushroom waste (SMW) of P. florida for degradation of ciprofloxacin and norfloxacin and to achieve highest degradation efficiency various parameters were tweaked such as pH (3-6), temperature (30 °C and 50 °C), and ABTS (0.05, 0.6, and 1 mM) concentration. The results showed that the most effective degradation of ciprofloxacin (86.12-75.94%) and norfloxacin (83.27-65.94%) was achieved in 3 h at pH 4.5, temperature 30 °C, and 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 0.05 mM concentration. Nevertheless, achieving degradation at 50 °C for both antibiotics, indicates thermostability nature of laccase (P. florida). Further, the fate of transformed products obtained from laccase mediated degradation was confirmed by liquid chromatography (LC-MS). Both the antibiotics undergo decarboxylation, depiperylyzation, dealkylation and defluorination as a result of laccase-mediated bond breakage. Anti-microbial activity of the biodegraded products was monitored by residual anti-bacterial toxicity test (E. coli and Staphylococcus aureus). The biodegraded products were found to be non-toxic and resulted in the growth of E. coli and Staphylococcus aureus, as determined by the agar-diffusion method. Moreover, the storage stability of laccase was determined for 28-day duration at varying pH (3-10) and temperature (4-50 °C). The maximum storage stability was obtained at pH 4.5 and temperature 30 °C. Therefore, utilizing SMW for the degradation of OMPs from wastewater not only benefits in degradation but also reuses SMW agro waste, shedding light on agro waste management. Thus, SMW is a one-pot solution for both OMPs biodegradation and circularity in the economy.

Regulation of RHOV signaling by interaction with SH3 domain-containing adaptor proteins and phosphorylation by PKA.

RHOV and RHOU are considered atypical Rho-family small GTPases because of the existence of N- and C-terminal extension regions, abnormal GDP/GTP cycling, and post-translational modification. Particularly, RHOV and RHOU both have a proline-rich (PR) motif in the N-terminal region. It has been reported that the PR motif of RHOU interacts with GRB2, a SH3 domain-containing adaptor protein, and regulates its activity through EGF receptor signaling. However, it is unknown whether RHOV, like RHOU, interacts with SH3 domain-containing adaptor proteins. In this study, we investigated the interactions between RHOV and SH3 domain-containing adaptor proteins, including GRB2 and NCK2. The RHOV-induced serum response factor (SRF)-dependent gene transcriptional activity was attenuated in cells co-expressing either GRB2 or NCK2 compared to cells expressing RHOV alone. From the results of experiments using various gene mutants of RHOV and GRB2, it appears that the PR motif of the N-terminal region of RHOV is the crucial binding site for the SH3 domain-containing proteins. Furthermore, we found that Ser25 in the N-terminal region of RHOV is phosphorylated by PKA and that its phosphorylation is suppressed by interaction with NCK2 but not GRB2. We have found a novel regulatory mechanism for the phosphorylation of RHOV and its interaction with SH3 domain-containing adaptor proteins.

The Rho guanine nucleotide exchange factor PLEKHG1 is activated by interaction with and phosphorylation by Src family kinase member FYN.

Rho family small GTPases (Rho) regulate various cell motility processes by spatiotemporally controlling the actin cytoskeleton. Some Rho-specific guanine nucleotide exchange factors (RhoGEFs) are regulated via tyrosine phosphorylation by Src family tyrosine kinase (SFK). We also previously reported that PLEKHG2, a RhoGEF for the GTPases Rac1 and Cdc42, is tyrosine-phosphorylated by SRC. However, the details of the mechanisms by which SFK regulates RhoGEFs are not well understood. In this study, we found for the first time that PLEKHG1, which has very high homology to the Dbl and pleckstrin homology domains of PLEKHG2, activates Cdc42 following activation by FYN, a member of the SFK family. We also show that this activation of PLEKHG1 by FYN requires interaction between these two proteins and FYN-induced tyrosine phosphorylation of PLEKHG1. We also found that the region containing the Src homology 3 and Src homology 2 domains of FYN is required for this interaction. Finally, we demonstrated that tyrosine phosphorylation of Tyr-720 and Tyr-801 in PLEKHG1 is important for the activation of PLEKHG1. These results suggest that FYN is a regulator of PLEKHG1 and may regulate cell morphology through Rho signaling via the interaction with and tyrosine phosphorylation of PLEKHG1.