Finely tuned conformational dynamics regulate the protective function of the lncRNA MALAT1 triple helix.

Nucleic acid triplexes may regulate many important biological processes. Persistent accumulation of the oncogenic 7-kb long noncoding RNA MALAT1 is dependent on an unusually long intramolecular triple helix. This triplex structure is positioned within a conserved ENE (element for nuclear expression) motif at the lncRNA 3' terminus and protects the entire transcript from degradation in a polyA-independent manner. A requisite 3' maturation step leads to triplex formation though the precise mechanism of triplex folding remains unclear. Furthermore, the contributions of several peripheral structural elements to triplex formation and protective function have not been determined. We evaluated the stability, conformational fluctuations, and function of this MALAT1 ENE triple helix (M1TH) protective element using in vitro mutational analyses coupled with biochemical and biophysical characterizations. Using fluorescence and UV melts, FRET, and an exonucleolytic decay assay we define a concerted mechanism for triplex formation and uncover a metastable, dynamic triplex population under near-physiological conditions. Structural elements surrounding the triplex regulate the dynamic M1TH conformational variability, but increased triplex dynamics lead to M1TH degradation. Taken together, we suggest that finely tuned dynamics may be a general mechanism regulating triplex-mediated functions.

The ghrelin O-acyltransferase structure reveals a catalytic channel for transmembrane hormone acylation.

Integral membrane proteins represent a large and diverse portion of the proteome and are often recalcitrant to purification, impeding studies essential for understanding protein structure and function. By combining co-evolutionary constraints and computational modeling with biochemical validation through site-directed mutagenesis and enzyme activity assays, we demonstrate here a synergistic approach to structurally model purification-resistant topologically complex integral membrane proteins. We report the first structural model of a eukaryotic membrane-bound O-acyltransferase (MBOAT), ghrelin O-acyltransferase (GOAT), which modifies the metabolism-regulating hormone ghrelin. Our structure, generated in the absence of any experimental structural data, revealed an unanticipated strategy for transmembrane protein acylation with catalysis occurring in an internal channel connecting the endoplasmic reticulum lumen and cytoplasm. This finding validated the power of our approach to generate predictive structural models for other experimentally challenging integral membrane proteins. Our results illuminate novel aspects of membrane protein function and represent key steps for advancing structure-guided inhibitor design to target therapeutically important but experimentally intractable membrane proteins.

Fluorescence-based investigations of RNA-small molecule interactions.

Interrogating non-coding RNA structures and functions with small molecules is an area of rapidly increasing interest among biochemists and chemical biologists. However, many biochemical approaches to monitoring RNA structures are time-consuming and low-throughput, and thereby are only of limited utility for RNA-small molecule studies. Fluorescence-based techniques are powerful tools for rapid investigation of RNA conformations, dynamics, and interactions with small molecules. Many fluorescence methods are amenable to high-throughput analysis, enabling library screening for small molecule binders. In this review, we summarize numerous fluorescence-based approaches for identifying and characterizing RNA-small molecule interactions. We describe in detail a high-information content dual-reporter FRET assay we developed to characterize small molecule-induced conformational and stability changes. Our assay is uniquely suited as a platform for both small molecule discovery and thorough characterization of RNA-small molecule binding mechanisms. Given the growing recognition of non-coding RNAs as attractive targets for therapeutic intervention, we anticipate our FRET assay and other fluorescence-based techniques will be indispensable for the development of potent and specific small molecule inhibitors targeting RNA.

Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation.

The peptide hormone ghrelin plays a key role in regulating hunger and energy balance within the body. Ghrelin signaling presents a promising and unexploited target for development of small molecule therapeutics for treatment of obesity, diabetes, and other health conditions. Inhibition of ghrelin O-acyltransferase (GOAT), which catalyzes an essential octanoylation step in ghrelin maturation, offers a potential avenue for controlling ghrelin signaling. Through screening a small molecule library, we have identified a class of synthetic triterpenoids that efficiently inhibit ghrelin acylation by the human isoform of GOAT (hGOAT). These compounds function as covalent reversible inhibitors of hGOAT, providing the first evidence of the involvement of a nucleophilic cysteine residue in substrate acylation by a MBOAT family acyltransferase. Surprisingly, the mouse form of GOAT does not exhibit susceptibility to cysteine-modifying electrophiles, revealing an important distinction in the activity and behavior between these closely related GOAT isoforms. This study establishes these compounds as potent small molecule inhibitors of ghrelin acylation and provides a foundation for the development of novel hGOAT inhibitors as therapeutics targeting diabetes and obesity.

The octanoylated energy regulating hormone ghrelin: An expanded view of ghrelin's biological interactions and avenues for controlling ghrelin signaling.

Ghrelin is a small peptide hormone that requires a unique post-translational modification, serine octanoylation, to bind and activate the GHS-R1a receptor. Initially demonstrated to stimulate hunger and appetite, ghrelin-dependent signaling is implicated in a variety of neurological and physiological processes influencing diseases such as diabetes, obesity, and Prader-Willi syndrome. In addition to its cognate receptor, recent studies have revealed ghrelin interacts with a range of binding partners within the bloodstream. Defining the scope of ghrelin's interactions within the body, understanding how these interactions work in concert to modulate ghrelin signaling, and developing molecular tools for controlling ghrelin signaling are essential for exploiting ghrelin for therapeutic effect. In this review, we discuss recent findings regarding the biological effects of ghrelin signaling, outline binding partners that control ghrelin trafficking and stability in circulation, and summarize the current landscape of inhibitors targeting ghrelin octanoylation.

Discovery of C-Linked Nucleoside Analogues with Antiviral Activity against SARS-CoV-2.

The recent COVID-19 pandemic underscored the limitations of currently available direct-acting antiviral treatments against acute respiratory RNA-viral infections and stimulated major research initiatives targeting anticoronavirus agents. Two novel nsp5 protease (MPro) inhibitors have been approved, nirmatrelvir and ensitrelvir, along with two existing nucleos(t)ide analogues repurposed as nsp12 polymerase inhibitors, remdesivir and molnupiravir, but a need still exists for therapies with improved potency and systemic exposure with oral dosing, better metabolic stability, and reduced resistance and toxicity risks. Herein, we summarize our research toward identifying nsp12 inhibitors that led to nucleoside analogues 10e and 10n, which showed favorable pan-coronavirus activity in cell-infection screens, were metabolized to active triphosphate nucleotides in cell-incubation studies, and demonstrated target (nsp12) engagement in biochemical assays.

Ghrelin Octanoylation Is Completely Stabilized in Biological Samples by Alkyl Fluorophosphonates.

Ghrelin is a peptide hormone involved in multiple physiological processes related to energy homeostasis. This hormone features a unique posttranslational serine octanoylation modification catalyzed by the enzyme ghrelin O-acyltransferase, with serine octanoylation essential for ghrelin to bind and activate its cognate receptor. Ghrelin deacylation rapidly occurs in circulation, with both ghrelin and desacyl ghrelin playing important roles in biological signaling. Understanding the regulation and physiological impact of ghrelin signaling requires the ability to rapidly protect ghrelin from deacylation in biological samples such as blood serum or cell lysates to preserve the relative concentrations of ghrelin and desacyl ghrelin. In in vitro ghrelin O-acyltransferase activity assays using insect microsomal protein fractions and mammalian cell lysate and blood serum, we demonstrate that alkyl fluorophosphonate treatment provides rapid, complete, and long-lasting protection of ghrelin acylation against serine ester hydrolysis without interference in enzyme assay or ELISA analysis. Our results support alkyl fluorophosphonate treatment as a general tool for stabilizing ghrelin and improving measurement of ghrelin and desacyl ghrelin concentrations in biochemical and clinical investigations and suggest current estimates for active ghrelin concentration and the ghrelin to desacyl ghrelin ratio in circulation may underestimate in vivo conditions.