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Summary [ABSTRACT FROM AUTHOR]

Fe2+ to Fe3+. The electronic structure of the different copper centers in this family of enzymes has been extensively studied and discussed for years with a particular focus on the exchange coupling regime in the trinuclear cluster (TNC). Using NMR spectroscopy we have quantified the exchange coupling constant in the type 3 center in a fully metalated oxidase; this value in Fet3p is significantly higher than that reported for proteins containing isolated type 3 centers as tyrosinase. We also provide evidence of exchange coupling between the type 2 and the type 3 Cu2+ ions, which supports the crystallographic evidence of dioxygen binding to the TNC. This work provides the foundation for the application of NMR to these complex systems. [ABSTRACT FROM AUTHOR]

Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) that contains 3 cupredoxin-like β-barrel domains and 4 copper ions located in 3 distinct metal sites (T1 in domain 3, T2, and the binuclear T3 at the interface between domains 1 and 3). To better understand how protein structure and stability is defined by cofactor coordination in MCO proteins, we assessed thermal unfolding of apo and metallated forms of Fet3p by using spectroscopic and calorimetric methods in vitro (pH 7). We find that unfolding reactions of apo and different holo forms of Fet3p are irreversible reactions that depend on the scan rate. The domains in apo-Fet3p unfold sequentially [thermal midpoint (T[subm]) of 45 °C, 62 °C, and 72 °C; 1 K/minI. Addition of T3 imposes strain in the apo structure that results in coupled domain unfolding and low stability (T[subm] of 50 °C; 1 K/mm). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by ≈5 °C but unfolding remains coupled in 1 step. Introduction of T1, producing fully-loaded holo-Fet3p (or in the absence of T2), results in stabilization of domain 3, which uncouples unfolding of the domains; unfolding of domain 2 occurs first along with Cu-site perturbations (T[subm] 50 55 °C; 1 K/mm), followed by unfolding of domains 1 and 3 (≈6570 °C; 1 K/mm). Our results suggest that there is a metal-induced tradeoff between overall protein stability and metal coordination in members of the MCO family. [ABSTRACT FROM AUTHOR]

Previous studies have shown that transcription factors IIB (TFIIB), IIF (TFIIF), and RNA polymerase II (RNAPII) play important roles in determining the position of mRNA 5'-ends in the yeast Saccharomyces cerevisiae. Yeast strains containing a deletion of the small, nonessential Rpb9 subunit of RNAPII exhibit an upstream shift in the positions of mRNA 5'-ends, whereas mutation of the large subunit of yeast TFIIF (Tfg1) can suppress downstream shifts that are conferred by mutations in TFIIB. In this study, we report an approach for the production of functional recombinant yeast boloTFIIF (Tfg1-Tfg2 complex) and use of the recombinant protein in both reconstitute 1 transcription assays and gel mobility shifts in order to investigate the biochemical alterations associated with the ΔRpb9 polymerase. The results demonstrated that upstream shifts in the positions of mRNA 5'-ends could be conferred by the ΔRpb9 RNAPII in transcription reactions reconstituted with highly purified yeast general transcription factors and, importantly, that these shifts are associated with an impaired interaction between the ΔRpb9 polymerase and TFIIF. Potential mechanisms by which an altered interaction between the ΔRpb9 RNAPII and TFIIF confers an upstream shift in the positions of mRNA 5'-ends are discussed. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) elevates Abeta (Aβ) peptides in the brain and cerebral spinal fluid. Aβ peptides are amphipathic molecules that can modulate membrane mechanics. Because the mechanosensitive cation channel PIEZO1 is gated by membrane tension and curvature, it prompted us to test the effects of Aβ on PIEZO1. Using precision fluid shear stress as a stimulus, we found that Aβ monomers inhibit PIEZO1 at femtomolar to picomolar concentrations. The Aβ oligomers proved much less potent. The effect of Aβs on Piezo gating did not involve peptide-protein interactions since the D and L enantiomers had similar effects. Incubating a fluorescent derivative of Aβ and a fluorescently tagged PIEZO1, we showed that Aβ can colocalize with PIEZO1, suggesting that they both had an affinity for particular regions of the bilayer. To better understand the PIEZO1 inhibitory effects of Aβ, we examined their effect on wound healing. We observed that over-expression of PIEZO1 in HEK293 cells increased cell migration velocity ~10-fold, and both enantiomeric Aβ peptides and GsMTx4 independently inhibited migration, demonstrating involvement of PIEZO1 in cell motility. As part of the motility study we examined the correlation of PIEZO1 function with tension in the cytoskeleton using a genetically encoded fluorescent stress probe. Aβ peptides increased resting stress in F-actin, and is correlated with Aβ block of PIEZO1-mediated Ca2+ influx. Aβ inhibition of PIEZO1 in the absence of stereospecific peptide-protein interactions shows that Aβ peptides modulate both cell membrane and cytoskeletal mechanics to control PIEZO1-triggered Ca2+ influx. [ABSTRACT FROM AUTHOR]

PIEZO1 and PIEZO2 are mechanosensitive channels (MSCs) important for cellular function and mutations in them lead to human disorders. We examined how functional heteromers form between subunits of PIEZO1 using the mutants E2117K, E2117D, and E2117A. Homomers of E2117K do not conduct. E2117A homomers have low conductance with rapid inactivation, and those of E2117D have high conductance with slow inactivation. Pairing E2117K with E2117D or E2117A with E2117D gave rise to new channel species representing heteromers with distinct conductances. Whole-cell currents from co-expression of E2117A and E2117D fit well with a linear-combination model of homomeric channel currents suggesting that functional channels do not form from freely-diffusing, randomly-mixed monomers in-vitro. Whole-cell current from coexpressed PIEZO1/PIEZO2 also fit as a linear combination of homomer currents. High-resolution optical images of fluorescently-tagged channels support this interpretation because coexpressed subunits segregate into discrete domains. [ABSTRACT FROM AUTHOR]

PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the “split protein” is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone. [ABSTRACT FROM AUTHOR]

Fet3p is a multicopper-containing glycoprotein localized to the yeast plasma membrane that catalyzes the oxidation of Fe(ll) to Fe(lll). This ferrous iron oxidation is coupled to the reduction of O2 to H2O and is termed the ferroxidase reaction. Fet3p-produced Fe(lll) is transferred to the permease Ftr1p for import into the cytosol. The posttranslational insertion of four copper ions into Fet3p is essential for its activity, thus linking copper and iron homeostasis. The mammalian ferroxidases ceruloplasmin and hephaestin are homologs of Fet3p. Loss of the Fe(ll) oxidation catalyzed by these proteins results in a spectrum of pathological states, including death. Here, we present the structure of the Fet3p extracellular ferroxidase domain and compare it with that of human ceruloplasmin and other multicopper oxidases that are devoid of ferroxidase activity. The Fet3p structure delineates features that underlie the unique reactivity of this and homologous multicopper oxidases that support the essential trafficking of iron in diverse eukaryotic organisms. The findings are correlated with biochemical and physiological data to cross-validate the elements of Fet3p that define it as both a ferroxidase and cuprous oxidase. [ABSTRACT FROM AUTHOR]

Multicopper oxidases (MCOs) carry out the most energy efficient reduction of O2 to H2O known, i.e., with the lowest overpotential. This four-electron process requires an electron mediating type 1 (T1) Cu site and an oxygen reducing trinuclear Cu cluster (TNC), consisting of a binuclear type 3 (T3)- and a mononuclear type 2 (T2) Cu center. The rate-determining step in O2 reduction is the first two-electron transfer from one of the T3 Cu’s (T3β) and the T2 Cu, forming a bridged peroxide intermediate (PI). This reaction has been investigated in T3β Cu variants of the Fet3p, where a first shell His ligand is mutated to Glu or Gln. This converts the fast two-electron reaction of the wild-type (WT) enzyme to a slow one-electron oxidation of the TNC. Both variants initially react to form a common T3β Cu(II) intermediate that converts to the Glu or Gln bound resting state. From spectroscopic evaluation, the nonmutated His ligands coordinate linearly to the T3β Cu in the reduced TNCs in the two variants, in contrast to the trigonal arrangement observed in the WT enzyme. This structural perturbation is found to significantly alter the electronic structure of the reduced TNC, which is no longer capable of rapidly transferring two electrons to the two perpendicular half occupied π*-orbitals of O2, in contrast to the WT enzyme. This study provides new insight into the geometric and electronic structure requirements of a fully functional TNC for the rate determining two-electron reduction of O2 in the MCOs. [ABSTRACT FROM AUTHOR]

The multicopper oxidase Fet3p catalyzes the four-electron reduction of dioxygen to water, coupled to the one-electron oxidation of four equivalents of substrate. To carry out this process, the enzyme utilizes four Cu atoms: a type 1, a type 2, and a coupled binuclear, type 3 site. Substrates are oxidized at the T1 Cu, which rapidly transfers electrons, 13 Å away, to a trinuclear copper cluster composed of the T2 and T3 sites, where dioxygen is reduced to water in two sequential 2e- steps. This study focuses on two variants of Fet3p, H126Q and H483Q, that perturb the two T3 Cu's, T3α. and T3β, respectively. The variants have been isolated in both hole and type 1 depleted (T1D) forms, T1DT3αQ and T1DT3/tQ, and their trinuclear copper clusters have been characterized in their oxidized and reduced states. While the variants are only mildly perturbed relative to T1D in the resting oxidized state, in contrast to T1D they are both found to have lost a ligand in their reduced states, importantly, T1DT3αQ reacts with O2, but T1DT3βQ does not. Thus loss of a ligand at T3β, but not at T3α, turns off O2 reactivity, indicating that T3β and T2 are required for the 2e- reduction of O2 to form the peroxide intermediate (PI), whereas T3α remains reduced. This is supported by the spectroscopic features of PI in T1DT3αQ, which are identical to T1D PI. This selective redox activity of one edge of the trinuclear cluster demonstrates its asymmetry in O2 reactivity. The structural origin of this asymmetry between the T3α and T3β is discussed, as is its contribution to reactivity. [ABSTRACT FROM AUTHOR]

Transcription factor IIF (TFIIF) is required for transcription al protein-encoding genes by eukaryotic RNA polymerase II. In contrast to numerous studies establishing a rule for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae. In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed In probe the functional domains of the S. cerevisiae TFIIF subunits Tfg1 and Tfg2. Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo. Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfgl-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in viva. Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II. Importantly, biochemical results demonstrated an altered interaction between mutant TFIIIF protein and RNA polymerase II. These results provide direct evidence for the involvement of S. cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase It interaction is a determinant in this process. [ABSTRACT FROM AUTHOR]