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In agriculture, Trehalase is considered the main target of the biological fungicide validamycin A, and the toxicology mechanism of validamycin A is unknown. 14-3-3 proteins, highly conserved proteins, participate in diverse cellular processes, including enzyme activation, protein localization, and acting as a molecular chaperone. In Saccharomyces cerevisiae, the 14-3-3 protein Bmh1could interact with Nth1 to respond to specific external stimuli. Here, we characterized FgNth, FgBmh1, and FgBmh2 in Fusarium graminearum. ΔFgNth, ΔFgBmh1, and ΔFgBmh2 displayed great growth defects and their peripheral tips hyphae generated more branches when compared with wild-type (WT) PH-1. When exposed to validamycin A as well as high osmotic and high temperature stresses, ΔFgNth, ΔFgBmh1, and ΔFgBmh2 showed more tolerance than WT. Both ΔFgNth and ΔFgBmh1 displayed reduced deoxynivalenol production but opposite for ΔFgBmh2, and all three deletion mutants showed reduced virulence on wheat coleoptiles. In addition, coimmunoprecipitation (Co-IP) experiments suggested that FgBmh1 and FgBmh2 both interact with FgNth, but no interaction was detected between FgBmh1 and FgBmh2 in our experiments. Further, validamycin A enhances the interaction between FgBmh1 and FgNth in a positive correlation under concentrations of 1 to 100 μg/ml. In addition, both high osmotic and high temperature stresses promote the interaction between FgBmh1 and FgNth. Co-IP assay also showed that neither FgBmh1 nor FgBmh2 could interact with FgPbs2, a MAPKK kinase in the high-osmolarity glycerol pathway. However, FgBmh2 but not FgBmh1 binds to the heat shock protein FgHsp70 in F. graminearum. Taken together, our results demonstrate that FgNth and FgBmh proteins are involved in growth and responses to external stresses and virulence; and validamycin enhanced the interaction between FgNth and FgBmh1in F. graminearum.

Potassium (K+) is the predominant intracellular cation in the body. The intracellular [K+] is 140–150 mEq/L; in blood it is 3.5–5 mEq/L. Serum contains a slightly higher concentration of K+ than plasma because K+ is released from red blood cells during clot formation. Maintenance of a high cellular concentration of K+ is necessary for several cellular functions, including growth, nucleic acid and protein synthesis, and regulation of cell volume, as well as pH and enzyme activation. In addition, a high intracellular concentration of K+ is essential to the maintenance of the resting membrane potential for cellular excitability and contraction. The high intracellular K+ concentration is maintained by the Na/K-ATPase located in the cell membranes of all animal cells. The activity of this enzyme is influenced by a variety of hormones. The kidney is the primary route for K+ excretion. In general, K+ excretion in the urine, called kaliuresis, parallels dietary intake. The other route for K+ excretion is the colon. Under conditions of decreased renal function, K+ excretion by the colon is enhanced.

Syntheses of fluorescent substrate and product for arginyltransferase, N-aspartyl-N'-dansylamido-1,4-butanediamine (Asp-4DNS), and N-arginylaspartyl-N'-dansylamido-1,4-butanediamine (ArgAsp-4DNS), respectively, including their precursor 4-dansylamidobutylamine (4DNS), are described. Then, HPLC conditions are summarized for a baseline separation of the three compounds in 10 min. The present method, which permits the simultaneous determination of Asp-4DNS, 4DNS, and ArgAsp-4DNS (in eluting order), is advantageous in measuring arginyltransferase activity and detecting the unfavorable enzyme(s) in 105,000 × g supernatant of tissues to ensure accurate determination.

Following our early discovery of arginylation in 1963, we have performed several studies to correlate its activity with essential biological processes. We employed cell- and tissue-based assays to detect both the level of acceptor proteins and the level of ATE1 activity under different conditions. Remarkably, in these assays, we found a close correlation between arginylation and aging, a discovery that we believe has longer-term implications in uncovering the importance of ATE1 in normal biology and disease therapies. Here we describe the original methods we used to measure ATE1 activity in tissues and correlate it with key biological events.

Subcellular organelle targeted imaging and therapy are of enormous interest in cancer theranostics. However, the lack of tumor-selective organelle targeting has compromised their efficacy and safety. In this work, we found that the near-infrared (NIR) fluorophore hemicyanine (CyNH2) can selectively target mitochondria with strong cytotoxicity through decreasing the mitochondrial membrane potential and increasing the intracellular reactive oxygen species (ROS) levels. A macrotheranostic probe (denoted as PLCy) based on conjugating CyNH2 with an acetylated lysine group was developed with masked fluorescence and cytotoxicity, which could both be unmasked through sequential activation by cancer cells overexpressing histone deacetylases (HDACs) and cathepsin L (CTSL) enzymes for selective cancer cell mitochondria-targeted imaging and therapy. In vitro and in vivo studies confirmed that the specific fluorescence turn-on and toxicity were restored in cancer cells and efficiently inhibited tumor growth. This macrotheranostic probe with sequential enzyme activation and mitochondrial targeting is expected to have promising applications in imaging-guided cancer therapy with high specificity and efficiency. Statement of significance To improve the targeting efficiency and enhance the anti-cancer activities of macrotheranostic probe. We designed macrotheranostic probe PLCy that can be activated via sequential enzymes for selective tumor mitochondria targeting. More importantly, the activated CyNH2 can decrease the mitochondrial membrane potential and elevate the reactive oxygen species level in cancer cells without light irradiation, which can further induce apoptosis of tumor cells for chemotherapy. Therefore, the use of sequential enzyme activation and mitochondria targeting strategies in the context of enzymatic activation may provide a general strategy for organelle-targeted imaging and therapy with high specificity and efficiency.

In animal tissues, N-acyltransferase (NAT) catalyzes the first reaction in the biosynthetic pathway of bioactive N-acylethanolamines, in which an acyl chain is transferred from the sn-1 position of the donor phospholipid, such as phosphatidylcholine, to the amino group of phosphatidylethanolamine, resulting in the formation of N-acylphosphatidylethanolamine. NAT has long been known to be stimulated by Ca(2+), and hence it has been referred to as Ca(2+)-dependent NAT. On the other hand, members of the phospholipase A/acyltransferase (PLA/AT) family (also known as HRAS-like suppressor family) show Ca(2+)-independent NAT activity. In this chapter, we describe (1) partial purification of Ca(2+)-dependent NAT from rat brain, (2) purification of recombinant PLA/AT-2, and (3) NAT assay using radiolabeled substrate.

Made available in DSpace on 2022-04-28T16:56:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-03-07. Added 1 bitstream(s) on 2022-04-28T17:11:40Z : No. of bitstreams: 1 S0004-27492022005004207.pdf: 473055 bytes, checksum: 07f02d57b067cc7b79769f9d75c78653 (MD5) Após a descoberta do anti fator de crescimento en dotelial vascular no tratamento da degeneração macular relacionada à idade úmida, muitas pesquisas têm sido realizadas com o intuito de elucidar os mecanismos moleculares envolvidos na gênese da degeneração macular relacionada à idade. O fator nuclear eritroide 2 relacionado ao fator 2 destaca-se pelo fato de diversas proteínas, oriundas de sua ativação, estarem envolvidas na manutenção do equilíbrio do estado redox e consequente prevenção da doença macular degenerativa. Este artigo mostra as características do fator nuclear eritroide 2 relacionado ao fator 2 e descreve as principais enzimas antioxidantes originadas da ativação que contribuem para a preservação da visão. After the discovery of anti-vascular endothelial growth factor agents as treatment of wet age-related macular degeneration, the number of studies with the objective to understand the molecular mechanisms involved in the age-re lated macular degeneration genesis has increased. The importance of the nuclear factor e2-related factor 2 lies in its activation-derived proteins being involved in the maintenance of the redox balance and consequent prevention of degenerative macular disease. This article aims to present the characteristics of nuclear factor e2-related factor 2 and describe the main nuclear factor e2-related factor 2-activated antioxidant enzymes that contribute to the preservation of vision. Universidade Estadual Paulista “Júlio de Mesquita Filho” Hospital Angelina Caron, Departamento de Oftalmologia Centro Oftalmológico de Curitiba Universidade Estadual Paulista “Júlio de Mesquita Filho”

Reversible formation of covalent adducts between a thiol and a membrane-anchored Michael acceptor has been used to control the activation of a caged enzyme encapsulated inside vesicles. A peptide substrate and papain, caged as the mixed disulfide with methane thiol, were encapsulated inside vesicles, which contained Michael acceptors embedded in the lipid bilayer. In the absence of the Michael acceptor, addition of thiols to the external aqueous solution did not activate the enzyme to any significant extent. In the presence of the Michael acceptor, addition of benzyl thiol led to uncaging of the enzyme and hydrolysis of the peptide substrate to generate a fluorescence output signal. A charged thiol used as the input signal did not activate the enzyme. A Michael acceptor with a polar head group that cannot cross the lipid bilayer was just as effective at delivering benzyl thiol to the inner compartment of the vesicles as a non-polar Michael acceptor that can diffuse across the bilayer. The concentration dependence of the output signal suggests that the mechanism of signal transduction is based on increasing the local concentration of thiol present in the vesicles by the formation of Michael adducts. An interesting feature of this system is that enzyme activation is transient, which means that sequential addition of aliquots of thiol can be used to repeatedly generate an output signal., Reversible formation of covalent adducts between a thiol and a membrane-anchored Michael acceptor has been used to control the activation of a caged enzyme encapsulated inside vesicles.

β-Arrestins are known to play a crucial role in GPCR-mediated transmembrane signaling processes. However, there are still many unanswered questions, especially those concerning the presumed similarities and differences of β-arrestin isoforms. Here, we examined the roles of β-arrestin 1 and β-arrestin 2 at different levels of μ-opioid receptor (MOR)-regulated signaling, including MOR mobility, internalization of MORs, and adenylyl cyclase (AC) activity. For this purpose, naïve HEK293 cells or HEK293 cells stably expressing YFP-tagged MOR were transfected with appropriate siRNAs to block in a specific way the expression of β-arrestin 1 or β-arrestin 2. We did not find any significant differences in the ability of β-arrestin isoforms to influence the lateral mobility of MORs in the plasma membrane. Using FRAP and line-scan FCS, we observed that knockdown of both β-arrestins similarly increased MOR lateral mobility and diminished the ability of DAMGO and endomorphin-2, respectively, to enhance and slow down receptor diffusion kinetics. However, β-arrestin 1 and β-arrestin 2 diversely affected the process of agonist-induced MOR endocytosis and exhibited distinct modulatory effects on AC function. Knockdown of β-arrestin 1, in contrast to β-arrestin 2, more effectively suppressed forskolin-stimulated AC activity and prevented the ability of activated-MORs to inhibit the enzyme activity. Moreover, we have demonstrated for the first time that β-arrestin 1, and partially β-arrestin 2, may somehow interact with AC and that this interaction is strongly supported by the enzyme activation. These data provide new insights into the functioning of β-arrestin isoforms and their distinct roles in GPCR-mediated signaling.