Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5' leader regions.

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5' leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5' leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.

Excess body weight as a predictor of response to treatment with antidepressants in patients with depressive disorder.

BACKGROUND: Depression and obesity are debilitating conditions representing an enormous health and economic burden worldwide. Depression is common among patients with excess weight, but more importantly, these patients may be at risk for poor response when treated with antidepressant medications (AD). METHODS: We conducted a comprehensive scoping review to summarize the evidence regarding the difference in response to treatment of depression with AD among patients with excess weight as compared to normal weight patients and to identify knowledge gaps. RESULTS: The search of the Medline and PsycINFO (2004-2019) identified twelve relevant studies. Tabulation and frequency analysis of the charted data along with a narrative synthesis were performed. Nine studies (75%) reported clinically relevant negative association between patients' high BMI or obesity and treatment response to either nortriptyline, fluoxetine, or various AD; one study (8.3%) reported no difference in response to various AD combinations between BMI groups. One study showed benefits of bupropion and escitalopram combination in patients with morbid obesity (BMI > 35 kg/m2) as compared with escitalopram monotherapy. Another study reported benefits when using venlafaxine-XR in patients with morbid obesity. We also acknowledge the possible role of sex and genetic factors predicting AD treatment response. LIMITATIONS: The search was restricted to two most relevant sources, publications in four languages and adult population. CONCLUSION: The synthesized data may be useful to physicians in their decision regarding the choice of AD in patients with excess weight. Researchers need to address causality of association between obesity and treatment response to individual AD types.

A continuous assay for l-talarate/galactarate dehydratase using circular dichroism.

l-Talarate/galactarate dehydratase (TGD) is a member of the enolase superfamily of enzymes and catalyzes the dehydration of either meso-galactarate or l-talarate to form 5-keto-4-deoxy-d-glucarate (5-KDG). To facilitate study of this enzyme and other galactarate dehydratases, a continuous circular dichroism-based assay has been developed. Using recombinant enzyme from Salmonella typhimurium (StTGD), the rates of StTGD-catalyzed conversion of m-galactarate to 5-KDG were determined by following the change in ellipticity at 323 nm. The apparent molar ellipticity ([θ]323) for the 5-KDG formed was determined to be 202 ±â€¯2 deg cm2 dmol-1, which was used to convert observed rates (Δθ/Δt) into concentration-dependent rates (Δc/Δt). The kinetic parameters Km, kcat, and kcat/Km were 0.38 ±â€¯0.05 mM, 4.8 ±â€¯0.1 s-1, and 1.3 (±0.2) × 104 M-1s-1, respectively. These values are in excellent agreement with those published previously [Yew, W.S. et al. (2007) Biochemistry46, 9564-9577] using a coupled assay system. To demonstrate the utility of the assay, the inhibition constant (Ki = 10.7 ±â€¯0.4 mM) was determined for the competitive inhibitor tartronate. The continuous CD-based assay offers a practical and efficient alternative method to the coupled assay that requires access to 5-KDG aldolase, and to the labor-intensive, fixed-time assays.