Dissecting Distinct Roles of NEDDylation E1 Ligase Heterodimer APPBP1 and UBA3 Reveals Potential Evolution Process for Activation of Ubiquitin-related Pathways.

Despite the similar enzyme cascade in the Ubiquitin and Ubiquitin-like peptide(Ubl) conjugation, the involvement of single or heterodimer E1 activating enzyme has been a mystery. Here, by using a quantitative Förster Resonance Energy Transfer (FRET) technology, aided with Analysis of Electrostatic Similarities Of Proteins (AESOP) computational framework, we elucidate in detail the functional properties of each subunit of the E1 heterodimer activating-enzyme for NEDD8, UBA3 and APPBP1. In contrast to SUMO activation, which requires both subunits of its E1 heterodimer AOS1-Uba2 for its activation, NEDD8 activation requires only one of two E1 subunits, UBA3. The other subunit, APPBP1, only contributes by accelerating the activation reaction rate. This discovery implies that APPBP1 functions mainly as a scaffold protein to enhance molecular interactions and facilitate catalytic reaction. These findings for the first time reveal critical new mechanisms and a potential evolutionary pathway for Ubl activations. Furthermore, this quantitative FRET approach can be used for other general biochemical pathway analysis in a dynamic mode.

A real-world evaluation of oral vancomycin for severe Clostridium difficile infection: implications for antibiotic stewardship programs.

STUDY OBJECTIVE: To assess antibiotic treatment patterns and clinical outcomes of patients with Clostridium difficile infection (CDI) based on underlying severity of CDI disease. DESIGN: Retrospective analysis of data from a prospective cohort study. SETTING: Large tertiary care university hospital. PATIENTS: One hundred forty-four patients (mean ± SD age 63 ± 17 yrs) with CDI who received metronidazole (intravenous or oral) or oral vancomycin as their initial therapy option between 2006 and 2008. MEASUREMENTS AND MAIN RESULTS: Patients were stratified by severity of illness and treatment, and outcomes assessed were clinical response, relapse of disease, all-cause inpatient mortality, and length of hospital stay. Mild-moderate CDI disease was present in 85 patients (59%) and severe disease in 59 patients (41%). Overall, oral vancomycin was given to 16 patients (11%); use of this drug did not differ according to severity of infection. Among patients with severe disease, clinical success occurred in 32 (63%) of 51 patients given metronidazole and in all 8 patients (100%) given vancomycin (p=0.04). Inpatient mortality and hospital length of stay were lower in patients with severe CDI who were given oral vancomycin, although these results were not statistically significant. CONCLUSION: Oral vancomycin was not commonly used for severe CDI. An improved clinical response rate was observed in patients with severe CDI given oral vancomycin; this outcome supported results from clinical trials. Antibiotic stewardship teams could play a major role in providing guidance on CDI treatment based on severity.