Biology subjects

Nelson, S.

Publications and source records attributed to Nelson, S..

24 records · Page 2Linked to original sources

bioRxiv2022

A long-acting GDF15 analog causes robust, sustained weight loss and reduction of food intake in an obese non-human primate model

GDF15 is a circulating polypeptide associated with cellular stress, and recently linked to metabolic adaptation. GDF15 has a half-life of approximately 3 hours in and acts at the GFRAL receptor selectively expressed in the area postrema. To characterize the effects of sustained GFRAL agonism on food intake (FI) and body weight (BW), we developed a half-life extended analog of GDF15 (Compound H; CpdH) suitable for reduced dosing frequency and tested its effects in obese cynomolgus monkeys. Animals were treated once weekly for 12 weeks with 0.048, 0.16, or 1.6 mg/Kg of CpdH or with 0.02 mg/Kg of the long-acting GLP-1 analog dulaglutide as a positive control. FI was measured daily and BW was measured biweekly. Mechanism-based longitudinal exposure-response (E-R) modeling was performed to characterize the effects of CpdH and dulaglutide on FI and BW. The integrated novel model accounts for both acute, exposure-dependent effects of treatments to reduce FI and the compensatory changes in energy expenditure (EE) and FI that occur over time in response to weight loss. CpdH had approximately linear, dose-proportional pharmacokinetics with a half-life of {approx}8 days and treatment with CpdH led to dose- and exposure-dependent reductions in FI and BW. The 1.6 mg/Kg CpdH dose reduced mean FI by 57.5% at 1 week and provided sustained FI reductions of 31.5% from weeks 9-12, leading to a peak reduction in BW of 16{+/-}5%. Dulaglutide had more modest effects on FI (reductions between 15-40%) and peak BW loss was 3.8{+/-}4.0%. Longitudinal modeling of both the FI and BW profiles suggested reductions in BW observed with both CpdH and dulaglutide were fully explained by the exposure-dependent reductions in FI without any increase in EE. Upon verification of pharmacokinetic/pharmacodynamic relationship established in monkeys and human for dulaglutide, we predicted that CpdH could reach double digit BW loss in human. In summary, treatment with a long-acting GDF15 analog led to sustained dose- and exposure-dependent reductions in food intake in a monkey model of obesity and holds potential for effective clinical obesity pharmacotherapy. Significance StatementGDF15 activation of GFRAL receptors in the hindbrain controls food intake and body weight. Here we describe the effect and durability of a circulating half-life extended analog of GDF15 (Compound H) on food intake and body weight loss in a spontaneously obese cynomolgus monkey model. Inclusion of a translational treatment arm with a weight loss agent, dulaglutide, permitted pharmacokinetic/pharmacodynamic modeling and comparison of both GDF15 and GLP-1 based weight loss mechanisms, and development of an allometric scaling based mathematical model to estimate the efficacy of Compound H in human obese subjects.

pharmacology and toxicology↗

bioRxiv2022

Dynamic Phosphorylation Regulates Eukaryotic Translation Initiation Factor 4A Activity During the Cell Cycle

The eukaryotic translation initiation factor 4A (eIF4A) resolves mRNA structures to support protein synthesis, yet little is known about its regulation. Here we analyzed eIF4A phosphorylation during alternate stages of the cell cycle, and found three residues near the DEAD box motif (T73, T146, and S177) underwent substantial phosphorylation changes. Phosphomimetic mutations T73D and T146D led to G2/M phase arrest, and abolished eIF4A interaction with RNA, suggesting eIF4A activity is needed for completion of cell division. In addition to these repressive events, we found that S177, a site immediately adjacent to the DEAD-box, showed diametrically opposed phosphorylation, with only phosphorylated S177 present during G1/S arrest and dephosphorylated S177 peptides during G2/M arrest. Phosphomimetic S177D eIF4A increased polysome levels and enhanced normally reduced eIF4A-eIF4G-interaction during G2/M, while phosphodeficient S177A decreased polysome levels and reduced growth, suggesting phosphorylation of S177 enhances eIF4A-mediated translation during G1/S. Together these results suggest that dynamic phosphorylation of eIF4A S177 serves to stimulate translation during G1/S, while inhibitory phosphorylation of additional sites holds the potential to rapidly transition eIF4A to an inactive state and turn off translation. These results also suggest an important role for eIF4A in coupling translation to cell cycle stages.

molecular biology↗

bioRxiv2021

Synthetic Natural Product-Inspired Peptides

Natural products (NPs) are a bountiful source of bioactive molecules. Unfortunately, discovery of novel bioactive NPs is challenging due to cryptic biosynthetic gene clusters (BGCs), low titers, and arduous purifications. Herein, we describe SNaPP (Synthetic Natural Product Inspired Peptides), a method for identifying NP-inspired bioactive molecules. SNaPP expedites bioactive molecule discovery by combining bioinformatics predictions of non-ribosomal peptide synthetases (NRPS) with chemical synthesis of the predicted NPs (pNPs). SNaPP utilizes a recently discovered cyclase, the penicillin binding protein (PBP)-like cyclase, as the lynchpin for the development of a library of cyclic peptide pNPs. Analysis of 500 BGCs allowed for identification of 131 novel pNPs. 51 diverse pNPs were synthesized using solid phase peptide synthesis and in-solution cyclization. Antibacterial testing revealed 14 pNPs with antibiotic activity, including activity against multidrug-resistant Gram-negative bacteria. Overall, SNaPP demonstrates the power of combining bioinformatics predictions with chemical synthesis to accelerate the discovery of bioactive molecules.

microbiology↗

bioRxiv2020

Precision Cas9 Genome Editing in vivo with All-in-one, Self-targeting AAV Vectors

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, [~]4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes.

molecular biology↗

bioRxiv2019

SKELETAL MyBP-C ISOFORMS TUNE THE MOLECULAR CONTRACTILITY OF DIVERGENT SKELETAL MUSCLE SYSTEMS

Skeletal muscle myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein; localized through its C terminus to distinct regions (C-zones) of the sarcomere. MyBP-C modulates muscle contractility, presumably through its N terminus extending from the thick filament and interacting with either the myosin head region and/or the actin thin filament. Two isoforms of MyBP-C (fast- and slow-type) are expressed in a muscle-type specific manner. Are the expression, localization, and Ca2+-dependent modulatory capacities of these isoforms different in fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles derived from Sprague-Dawley rats? By mass spectrometry, four MyBP-C isoforms (one fast-type MyBP-C and three N-terminally spliced slow-type MyBP-C) were expressed in EDL but only the three slow-type MyBP-C isoforms in SOL. Using EDL and SOL native thick filaments in which the MyBP-C stoichiometry and localization are preserved, native thin filament sliding over these thick filaments showed that only in the C-zone, MyBP-C Ca2+-sensitizes the thin filament and slows thin filament velocity. These modulatory properties depended on MyBP-Cs N-terminus, as N-terminal proteolysis attenuated MyBP-Cs functional capacities. To determine each MyBP-C isoforms contribution to thin filament Ca2+-sensitization and slowing in the C-zone, we used a combination of in vitro motility assays using expressed recombinant N-terminal fragments and in silico mechanistic modeling. Our results suggest that each skeletal MyBP-C isoforms N terminus is functionally distinct and has modulatory capacities that depend on the muscle-type in which they are expressed, providing the potential for molecular tuning of skeletal muscle performance through differential MyBP-C expression.\n\nSIGNIFICANCEMyosin-binding protein C (MyBP-C) is a critical component of the skeletal muscle sarcomere, muscles smallest contractile unit. MyBP-Cs importance is evident by genetic mutations leading to human myopathies, such as distal arthrogryposis (i.e. club foot). However, the molecular basis of MyBP-Cs functional impact on skeletal muscle contractility is far from certain. Complicating matters further is the expression of fast- and slow-type MyBP-C isoforms that depend on whether the muscle is fast- or slow-twitch. Using multi-scale proteomic, biophysical and mathematical modeling approaches, we define the expression, localization, and modulatory capacities of these distinct skeletal MyBP-C isoforms in rat skeletal muscles. Each MyBP-C isoform appears to modulate muscle contractility differentially; providing the capacity to fine-tune muscles mechanical performance as physiological demands arise.

physiology↗

bioRxiv2018

Reciprocal regulation among TRPV1 channels and phosphoinositide 3-kinase in response to nerve growth factor

Although it has been known for over a decade that the inflammatory mediator NGF sensitizes pain-receptor neurons through increased trafficking of TRPV1 channels to the plasma membrane, the mechanism by which this occurs remains mysterious. NGF activates phosphoinositide 3-kinase (PI3K), the enzyme that generates PIP3, and PI3K activity is required for sensitization. One tantalizing hint came from the finding that the N-terminal region of TRPV1 interacts directly with PI3K. Using 2-color total internal reflection fluorescence microscopy, we show that TRPV1 potentiates NGF-induced PI3K activity. A soluble TRPV1 fragment corresponding to the N-terminal Ankyrin repeats domain (ARD) was sufficient to produce this potentiation, indicating that allosteric regulation was involved. Further, other TRPV channels with conserved ARDs also potentiated NGF-induced PI3K activity whereas TRP channels lacking ARDs did not. Our data demonstrate a novel reciprocal regulation of PI3K signaling by the ARD of TRPV channels.

biochemistry↗