Characterization of self-regulatory mechanisms and internal dynamics of ETS transcription factor PU.1

The ETS family of transcription factors bind to site-specific DNA via DNA-binding domains called the ETS domains. The ETS domains are structurally homologous but divergent in primary sequences. PU.1 is an essential transcription factor and its biological activity is primarily controlled by up- and down-regulation of its expression. Aside from down-regulated expression, only a few inhibitory mechanisms are known for PU.1. The most understood one involves PU.1 forming a heterodimer with other protein partners, such as GATA-1. However, unlike auto-inhibited ETS-family members whose activity is regulated by autoinhibitory elements that reduce the net affinity of binding to specific DNA, PU.1 has no such regulatory mechanism at the protein-DNA level. We report here that PU.1, unlike its auto-inhibited paralog Ets-1, forms a 2:1 complex with site-specific DNA (\u3e10 bp) in a negatively cooperative manner. We also detected potential interface (193DKDK196) of the PU.1 dimer by using heteronuclear single quantum correlation (HSQC) NMR. Self-titration of PU.1 is a negative feedback mechanism at the protein-DNA level. Following these findings, our group found the presence of the IDRs flanking the ETS domain does not change the DNA binding modes of the PU.1 ETS domain, yet the PEST domain modifies DNA recognition by the ETS domain through changing DNA binding affinities. We successfully assigned ~90% or more backbone amide resonances in the 1H-15N HSQC spectra of hPU.1 constructs with and without IDRs, in the absence and presence (1:1 complex) of DNA. Using the fully assigned HSQC spectra, we studied fast (ps to ns) time scale internal dynamics of PU.1 protein. Spin relaxation rates and heteronuclear 1H{15N}-NOE were acquired for the hPU.1 proteins with and without DNA by NMR. We demonstrated that the PEST domain remains disordered but becomes more dynamic upon specific DNA binding. In terms of DNA recognition, the presence of the PEST domain increases the affinity of 1:1 complex of the ETS domain with cognate DNA, without perturbing the structure or changing the fast time scale backbone motions of the ETS domain

Crystallographic and Spectroscopic Snapshots Reveal a Dehydrogenase in Action

Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NADþ-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation

Effects of Single Nucleotide Polymorphisms in the Human \u3ci\u3eHolocarboxylase Synthetase\u3c/i\u3e Gene on Catalytic Activity

Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to carboxylases and histones in eukaryotic cells. Biotinylated carboxylases play essential roles in the metabolism of fatty acids, amino acids, and glucose; biotinylated histones play essential roles in gene regulation and genome stability. HCS null individuals are not viable whereas HCS deficiency is linked to developmental delays and phenotypes such as short life span and low stress resistance. Greater than 2,500 single nucleotide polymorphisms (SNPs) have been reported for HCS, but the biological importance of these polymorphisms is unknown. We hypothesized that some of these SNPs impair catalytic activity and that this effect can be overcome by dietary intervention with biotin. Here, we analyzed the enzyme kinetics of five recombinant HCS variants using a propionyl-CoA carboxylase surrogate (“p67”) as substrate for biotinylation. Vmax of variants L216R, V96F and G510R were 6%, 78% and 73%, respectively, of the Vmax in wild-type HCS. The Km values of the variants V96F and G510R were not significantly different from wild-type HCS. The activity of L216R was too low to allow for meaningful analysis of Km. In contrast, the affinity of variant Q699R for biotin was significantly lower than that of wild type HCS (Km: 1.57 times that of wild type) and its Vmax could be restored to that of wild-type HCS by biotin supplementation. This is the first biochemical characterization of catalytic activities of HCS variants. Also, this is the first report to show that HCS activity can be restored to normal by biotin supplementation. Advisor: Janos Zemplen

Efficacy of Low-Pressure Inflation of Oversized Drug-Coated Balloon for Coronary Artery Disease

Objectives. This study sought to assess the efficacy of oversized drug-coated balloon (DCB) inflation at low pressure for the prevention of acute dissections and late restenosis. Background. The major limitation of DCB coronary angioplasty is the occurrence of severe dissection after inflation of DCB. Methods. Between 2014 and 2018, 273 consecutive patients were retrospectively studied. 191 lesions (154 patients) treated by oversized DCB inflation at low pressure (<4 atm, 2.4 ± 1.2 atm, DCB/artery ratio 1.14 ± 0.22; LP group) were compared with 135 lesions (119 patients) treated by the standard DCB technique (7.1 ± 2.2 atm, DCB/artery ratio 1.03 ± 0.16; SP group). Results. Although the lesions in the LP group were more complex than those in the SP group (smaller reference diameter (2.38 mm vs. 2.57 mm, P=0.011), longer lesions (11.7 mm vs. 10.5 mm, P=0.10), and more frequent use of rotational atherectomy (45.0% vs. 28.1%, P=0.003), there was no significant difference in the NHLBI type of dissections between the two groups (11.5%, 12.0%, 5.2% vs. 12.6%, 12.6%, 2.2% in type A, B, and C, P=0.61), and no bailout stenting was required. In 125 well-matched lesion pairs after propensity score analysis, the cumulative incidence of target lesion revascularization at 3 years was 4.5% vs. 7.0%, respectively (P=0.60). Late lumen loss (−0.00 mm vs. −0.01 mm, P=0.94) and restenosis rates (7.4% vs. 7.1%, P=1.0) were similar in both of the groups. Conclusion. The application of oversized DCB at low pressure is effective and feasible for preventing late restenosis comparative to the standard technique of DCB

ETS transcription factors induce a unique UV damage signature that drives recurrent mutagenesis in melanoma

Recurrent mutations are frequently associated with transcription factor (TF) binding sites (TFBS) in melanoma, but the mechanism driving mutagenesis at TFBS is unclear. Here, we use a method called CPD-seq to map the distribution of UV-induced cyclobutane pyrimidine dimers (CPDs) across the human genome at single nucleotide resolution. Our results indicate that CPD lesions are elevated at active TFBS, an effect that is primarily due to E26 transformation-specific (ETS) TFs. We show that ETS TFs induce a unique signature of CPD hotspots that are highly correlated with recurrent mutations in melanomas, despite high repair activity at these sites. ETS1 protein renders its DNA binding targets extremely susceptible to UV damage in vitro, due to binding-induced perturbations in the DNA structure that favor CPD formation. These findings define a mechanism responsible for recurrent mutations in melanoma and reveal that DNA binding by ETS TFs is inherently mutagenic in UV-exposed cells

Positive Cooperativity of the p97 AAA ATPase Is Critical for Essential Functions*

p97 is composed of two conserved AAA (ATPases associated with diverse cellular activities) domains, which form a tandem hexameric ring. We characterized the ATP hydrolysis mechanism of CDC-48.1, a p97 homolog of Caenorhabditis elegans. The ATPase activity of the N-terminal AAA domain was very low at physiological temperature, whereas the C-terminal AAA domain showed high ATPase activity in a coordinated fashion with positive cooperativity. The cooperativity and coordination are generated by different mechanisms because a noncooperative mutant still showed the coordination. Interestingly, the growth speed of yeast cells strongly related to the positive cooperativity rather than the ATPase activity itself, suggesting that the positive cooperativity is critical for the essential functions of p97