Frontiers in chemical biology最新文献

Polyoxometalates (POMs) are metal-oxygen clusters composed of {MO₆} octahedra that have attracted considerable attention due to their remarkable antiviral, antibacterial and antitumor activities. Despite their potential, the molecular mechanisms underlying their cellular toxicity remain poorly understood. This study investigates how Anderson-Evans type polyoxotungstates (POTs) and polyoxomolybdates (POMos) interact with biological membranes by examining their effects on the zeta (ζ) - potential of the lipid bilayer and the size of small unilamellar liposomes of different phospholipid compositions. POTs affected the ζ-potential of neutral (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) and slightly negatively charged (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPC:DOPE) membranes in the order [MnW₆O₂₄]^8- > [Ni(OH)₆W₆O₁₈]^4- > [TeW₆O₂₄]^6-. The addition of negatively charged cardiolipin (CL) to DOPC reduced the interaction of POTs with the membrane. An opposite effect was observed for POMos, which changed the ζ-potential of neutral and slightly negatively charged membranes in the order [Al(OH)₆Mo₆O₁₈]^3- > [Cr(OH)₆Mo₆O₁₈]^3- >> [Ni(OH)₆Mo₆O₁₈]^4-. The addition of POMos increased the size of the liposomes in reverse order. The binding of [Al(OH)₆Mo₆O₁₈]^3- to the PE-containing phospholipid membranes and the effect of ionic strength on the interaction of [Cr(OH)₆Mo₆O₁₈]^3- with DOPC:CL liposomes could be inhibited by potassium fluoride (KF). Interestingly, KF did not inhibit the interaction of other POMos with membranes as indicated by ζ-potential measurements. These results suggest that the interaction of Anderson-Evans type POMs with phospholipid membranes is influenced more by their addenda and central ions than by their total charge. By unravelling the structure-activity relationships for the different POMs, we contribute to the design of biologically active POMs for therapeutic use.

Introduction: Dual specific phosphatases (DUSPs) are mitogen-activated protein kinase (MAPK) regulators, which also serve as drug targets for treating various vascular diseases. Previously, we have presented mechanistic characterizations of DUSP5 and its interaction with pERK, proposing a dual active site.

Methods: Herein, we characterize the interactions between the DUSP5 phosphatase domain and the pT-E-pY activation loop of ERK2, with specific active site assignments. We also report the full NMR chemical shift assignments of DUSP5 that now enable chemical shift perturbation and dynamics studies.

Results and discussion: Both phosphates of the pT-E-pY tripeptide are dephosphorylated, based on ³¹P NMR; but, steady state kinetic studies of the tripeptide both as a substrate and as an inhibitor indicate a preference for binding and dephosphorylation of the phospho-tyrosine before the phospho-threonine. Catalytic efficiency (k_cat/K_m) is 3.7 M^-1S^-1 for T-E-pY vs 1.3 M^-1S^-1 for pT-E-Y, although the diphosphorylated peptide (pT-E-pY) is a better substrate than both, with k_cat/K_m = 18.2 M^-1S^-1. Steady state inhibition studies with the pNPP substrate yields K_is values for the peptide inhibitors of: 15.82 mM (pT-E-Y), 4.932 mM (T-E-pY), 1.672 mM (pT-E-pY). Steady state inhibition studies with pNPP substrate and with vanadate or phosphate inhibitors indicated competitive inhibition with Kis values of 0.0006122 mM (sodium vanadate) and 17.32 mM (sodium phosphate), similar to other Protein Tyrosine Phosphatases with an active site cysteine nucleophile that go through a five-coordinate high energy transition state or intermediate. Molecular dynamics (MD) studies confirm preferential binding of the diphosphorylated peptide, but with preference for binding the pY over the pT reside in the catalytic site proximal to the Cys263 nucleophile. Based on MD, the monophosphorylated peptide binds tighter if phosphorylated on the Tyr vs the Thr. And, if the starting pose of the docked diphosphorylated peptide has pT in the catalytic site, it will adjust to have the pY in the catalytic site, suggesting a dynamic shifting of the peptide orientation. 2D ¹H-¹⁵N HSQC chemical shift perturbation studies confirm that DUSP5 with tripeptide bound is in a dynamic state, with extensive exchange broadening observed-especially of catalytic site residues. The availability of NMR chemical shift assignments enables additional future studies of DUSP5 binding to the ERK2 diphosphorylated activation loop.

Summary: These studies indicate a preference for pY before pT binding, but with ability to bind and dephosphorylate both residues, and with a dynamic active site pocket that accommodates multiple tripeptide orientations.

Inflammation-related disorders, such as autoimmune diseases and cancer, impose a significant global health burden. Zinc finger proteins (ZFs) are ubiquitous metalloproteins which regulate inflammation and many biological signaling pathways related to growth, development, and immune function. Numerous ZFs are involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway, associating them with inflammation-related diseases that feature chronically elevated pro-inflammatory cytokines. This review highlights the predominance of ZFs in NFκB-related signaling and summarizes the breadth of functions that these proteins perform. The cysteine-specific post-translational modification (PTM) of persulfidation is also discussed in the context of these cysteine-rich ZFs, including what is known from the few available reports on the functional implications of ZF persulfidation. Persulfidation, mediated by endogenously produced hydrogen sulfide (H₂S), has a recently established role in signaling inflammation. This work will summarize the known connections between ZFs and persulfidation and has the potential to inform on the development of related therapies.