Biology subjects

Previs, S. B.

Publications and source records attributed to Previs, S. B..

5 recordsLinked to original sources

bioRxiv2026

Identification of UCB-9721 as a potent inhibitor of MyoA, the essential class XIV myosin motor of apicomplexan parasites

The virulence of Toxoplasma gondii and other apicomplexan parasites relies on a unique form of cellular motility driven by MyoA, an unconventional class XIV myosin motor protein. To identify new chemical probes for investigating the molecular mechanisms of parasite motility, we screened over 50,000 small molecules for inhibitors of T. gondii MyoA (TgMyoA). The top hit from the screen, UCB-9721, is almost 40-fold more potent as an inhibitor of TgMyoA actin-activated ATPase activity than the previously described TgMyoA inhibitor, KNX-002, and 45-fold more potent at inhibiting parasite motility, with no detectable toxicity towards mammalian cells. UCB-9721 also inhibited the motility and/or growth of the related apicomplexan parasites Plasmodium falciparum, Cryptosporidium parvum, and Babesia duncani, suggesting that this compound will be a useful new chemical probe for studying motility and MyoA function in apicomplexan parasites more broadly. While UCB-9721 and KNX-002 were identified independently, they share a similar chemical scaffold. To determine why UCB-9721 is so much more potent than KNX-002 and to inform future development of this inhibitor class, we undertook comparative molecular docking analyses, targeted TgMyoA mutagenesis, and a directed structure-activity relationship analysis. The results identified the sulfonamide group of UCB-9721 and its hydrogen bond interactions with R249, E275 and a stabilized water network within the TgMyoA binding pocket as key to the compounds increased potency. Further development of UCB-9721, informed by the results presented here, may transform this promising new chemical class into actionable drug development leads against this important group of human and animal pathogens.

microbiology↗

bioRxiv2025

Kinesin-1 Autoinhibition Tunes Cargo Transport by Motor Ensembles

Intracellular vesicular transport by kinesin-1 motors through numerous 3-dimensional (3D) microtubule (MT) intersections must be regulated to support proper vesicle delivery. Knowing kinesin-1 can be regulated via autoinhibition, does kinesin-1 exhibit autoinhibition on cargo, and could this regulate vesicular transport through 3D MT intersections in vitro? To answer this question, we compared liposome transport by [~]10 nearly full-length kinesin-1 motors with KLC bound (Kin{Delta}C) versus constitutively active control (K543). In 3D MT intersections, Kin{Delta}C-liposomes terminate (48%), go straight (43%), but rarely turn (9%), starkly contrasting K543-liposomes which go straight (57%), turn (31%), but rarely terminate (12%). On single MTs, Kin{Delta}C-liposomes have reduced run lengths and detachment forces versus K543-liposomes, suggesting autoinhibition reduces MT engagement, as supported by 3-fold lower Kin{Delta}C MT landing rates versus K543, and mechanistic in silico modeling. Furthermore, kinesore, a small molecule that overcomes kinesin-1 autoinhibition, restores Kin{Delta}Cs MT engagement. Thus, we propose that partial kinesin-1 autoinhibition while cargo-bound may fine-tune cargo delivery to support physiological demands. Statement of significanceKinesin-1 vesicular transport through the cells complex microtubule (MT) network must be spatially and temporally regulated to meet the cells physiological demands. With kinesin-1 being autoinhibited, does this form of regulation impact the directional outcomes of vesicles encountering 3-dimensional (3D) MT-MT intersections as they do in cells? To address this in vitro, liposomes transported by kinesin-1 motors (team of [~]10), which exhibit autoinhibition, are challenged with 3D MT intersections and interestingly, preferentially terminate or go straight and rarely turn, while liposomes transported by constitutively active motors preferentially go straight or turn but rarely terminate. Therefore, autoinhibition reduces the number of MT-engaged motors within the team and provides the cell a means of fine-tuning cargo transport to its destination.

biophysics↗

bioRxiv2024

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development, and promoting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility from the C-zone? Globular domains critical to MyBP-Cs modulatory functions are absent from MyBP-H, suggesting MyBP-H may be functionally silent. However, our results suggest an active role. Small angle x-ray diffraction of intact larval tails revealed MyBP-H contributes to the compression of the myofilament lattice accompanying stretch or contraction, while in vitro motility experiments indicate MyBP-H shares MyBP-Cs capacity as a molecular "brake". These results provide new insights and raise questions about the role of the C-zone during muscle development.

biophysics↗

bioRxiv2022

Dissecting β-Cardiac Myosin and Cardiac Myosin-Binding Protein C Interactions using a Nanosurf Assay

Cardiac myosin-binding protein C (cMyBP-C) regulates cardiac contractility by slowing shortening velocity and sensitizing the thin filament to calcium. cMyBP-C has been shown to interact with the proximal myosin S2 tail and the thin filament. However, the relative contribution of these interactions to the collective modulation of actomyosin ensemble function remains unclear. Hence, we developed a "nanosurf" assay as a model system to interrogate cMyBP-C interactions with actin and/or myosin. Synthetic thick filaments were generated using recombinant human {beta}-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14 or 28 nm spacing, corresponding to the 14.3 nm myosin spacing found in native thick filaments. In vitro motility assays with myosin bound to the surrounding surface, exhibit enhanced thin filament interactions with synthetic thick filaments. No significant differences were observed in mean thin filament velocities between 14 and 28 nm spacing, consistent with our previous results for myosin V, VI, and {beta}-cardiac myosin S1. Our nanosurf assay demonstrates the slowing of actomyosin motility by cMyBP-C. Alternating {beta}-cardiac myosin HMM and cMyBP-C N-terminal fragments, C0-C2 or C1-C2, every 14 nm on the nanotube, reduced the mean thin filament velocity 4-6 fold relative to myosin alone. Interestingly, similar inhibition was observed using a {beta}-cardiac myosin S1 construct, which lacks the S2 region proposed to interact with cMyBP-C, suggesting the actin-cMyBP-C interactions may dominate the inhibitory mechanism. No significant inhibition of thin filament velocity was observed with a C0-C1f fragment, lacking the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A phosphomimetic C0-C2 fragment showed a 3-fold higher velocity compared to its phosphonull counterpart, further highlighting phosphorylation-dependent regulation via the M-domain. Together, we have established the nanosurf assay as a tool to precisely manipulate spatially dependent cMyBP-C binding partner interactions, shedding light on the molecular regulation of {beta}-cardiac myosin contractility. STATEMENT OF SIGNIFICANCECardiac myosin-binding protein C (cMyBP-C) is the most frequently mutated protein associated with hypertrophic cardiomyopathy (HCM), a common cause of sudden cardiac death. Despite the importance of cMyBP-C in cardiac contractility, the mechanisms underlying this regulation are unclear due to experimental challenges in studying the complex, transient, weak interactions of cMyBP-C with the contractile proteins of the sarcomere. In this study, we created a nanosurf synthetic DNA thick filament assay to dissect the cMyBP-C interactions with actin and human {beta}-cardiac myosin. We demonstrate actomyosin inhibition by cMyBP-C fragments regardless of recombinant human {beta}-cardiac myosin subfragment (HMM or S1) and highlight the importance of the cMyBP-C M-domain using cMyBP-C fragments and phosphomimetics.

biophysics↗

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↗