Search

Drug resistance remains a challenge for targeted therapy of cancers driven by EML4-ALK and related fusion oncogenes. EML4-ALK forms cytoplasmic protein condensates, which result from networks of interactions between oncogene and adapter protein multimers. While these assemblies are associated with oncogenic signaling, their role in drug response is unclear. Here, we use optogenetics and live-cell imaging to find that EML4-ALK assemblies suppress transmembrane receptor tyrosine kinase (RTK) signaling by sequestering RTK adapter proteins including GRB2 and SOS1. Furthermore, ALK inhibition, while suppressing oncogenic signaling, simultaneously releases the sequestered adapters and thereby resensitizes RTK signaling. Resensitized RTKs promote rapid and pulsatile ERK reactivation that originates from paracrine ligands shed by dying cells. Reactivated ERK signaling promotes cell survival, which can be counteracted by combination therapies that block paracrine signaling. Our results identify a regulatory role for RTK fusion assemblies and uncover a mechanism of tolerance to targeted therapies., (© 2024. The Author(s).)

Recently, targeted degradation has emerged as a powerful therapeutic modality. Relying on "event-driven" pharmacology, proteolysis targeting chimeras (PROTACs) can degrade targets and are superior to conventional inhibitors against undruggable proteins. Unfortunately, PROTAC discovery is limited by warhead scarcity and laborious optimization campaigns. To address these shortcomings, analogous protein-based heterobifunctional degraders, known as bioPROTACs, have been developed. Compared to small-molecule PROTACs, bioPROTACs have higher success rates and are subject to fewer design constraints. However, the membrane impermeability of proteins severely restricts bioPROTAC deployment as a generalized therapeutic modality. Here, we present an engineered bioPROTAC template able to complex with cationic and ionizable lipids via electrostatic interactions for cytosolic delivery. When delivered by biocompatible lipid nanoparticles, these modified bioPROTACs can rapidly degrade intracellular proteins, exhibiting near-complete elimination (up to 95% clearance) of targets within hours of treatment. Our bioPROTAC format can degrade proteins localized to various subcellular compartments including the mitochondria, nucleus, cytosol, and membrane. Moreover, substrate specificity can be easily reprogrammed, allowing modular design and targeting of clinically-relevant proteins such as Ras, Jnk, and Erk. In summary, this work introduces an inexpensive, flexible, and scalable platform for efficient intracellular degradation of proteins that may elude chemical inhibition., (© 2024. The Author(s).)

Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often too small to appear as puncta in conventional fluorescence microscopy. Here, we describe a fluorescent reporter strategy that detects protein clusters with high sensitivity called CluMPS (clusters magnified by phase separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, quantifiable fluorescence condensates. We use computational modeling and optogenetic clustering to demonstrate that CluMPS can detect small oligomers and behaves rationally according to key system parameters. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. CluMPS also detected and tracked clusters of unmodified and tagged endogenous proteins, and orthogonal CluMPS probes could be multiplexed in cells. CluMPS provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Extrusion three-dimensional (3D) bioprinting is a promising technology with many applications in the biomedical and tissue engineering fields. One of the key limitations for the widespread use of this technology is the narrow window of printability that results from the need to have bioinks with rheological properties that allow the extrusion of continuous filaments while maintaining high cell viability within the materials during and after printing. In this work, we use Carbopol (CBP) as rheology modifier for extrusion printing of biomaterials that are typically nonextrudable or present low printability. We show that low concentrations of CBP can introduce the desired rheological properties for a wide range of formulations, allowing the use of polymers with different cross-linking mechanisms and the introduction of additives and cells. To explore the opportunities and limitations of CBP as a rheology modifier, we used ink formulations based on poly(ethylene glycol)diacrylate with extrusion 3D printing to produce soft, yet stable, hydrogels with tunable mechanical properties. Cell-laden constructs made with such inks presented high viability for cells seeded on top of cross-linked materials and cells incorporated within the bioink during printing, showing that the materials are noncytotoxic and the printed structures do not degrade for up to 14 days. To our knowledge, this is the first report of the use of CBP-containing bioinks to 3D-print complex cell-laden structures that are stable for days and present high cell viability. The use of CBP to obtain highly printable inks can accelerate the evolution of extrusion 3D bioprinting by guaranteeing the required rheological properties and expanding the number of materials that can be successfully printed. This will allow researchers to develop and optimize new bioinks focusing on the biochemical, cellular, and mechanical requirements of the targeted applications rather than the rheology needed to achieve good printability.

The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive., (© 2023. The Author(s).)

The ability to deliver small protein scaffolds intracellularly could enable the targeting and inhibition of many therapeutic targets that are not currently amenable to inhibition with small-molecule drugs. Here, we report the engineering of small protein scaffolds with anionic polypeptides (ApPs) to promote electrostatic interactions with positively charged nonviral lipid-based delivery systems. Proteins fused with ApPs are either complexed with off-the-shelf cationic lipids or encapsulated within ionizable lipid nanoparticles for highly efficient cytosolic delivery (up to 90%). The delivery of protein inhibitors is used to inhibit two common proto-oncogenes, Ras and Myc, in two cancer cell lines. This report demonstrates the feasibility of combining minimally engineered small protein scaffolds with tractable nanocarriers to inhibit intracellular proteins that are generally considered "undruggable" with current small molecule drugs and biologics.

Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca 2+ -activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca 2+ activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC-cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium., (© 2019 Johnston et al.)

Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca 2+ sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca 2+ -sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca 2+ sensitizer. Our findings suggest that both methods of increasing myofilament Ca 2+ sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (k TR ); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I 1,1 /I 1,0 ratio from X-ray diffraction. Computational modeling suggests that Ca 2+ sensitization through this cTnC mutation or bepridil accelerates k TR primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in k TR , we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca 2+ activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca 2+ sensitization may act through stabilization of cTnC's D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca 2+ activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mutations in TNNC1 -the gene encoding cardiac troponin C (cTnC)-that have been associated with hypertrophic cardiomyopathy (HCM) and cardiac dysfunction may also affect Ca 2+ -regulation and function of slow skeletal muscle since the same gene is expressed in both cardiac and slow skeletal muscle. Therefore, we reconstituted rabbit soleus fibers and bovine masseter myofibrils with mutant cTnCs (A8V, C84Y, E134D, and D145E) associated with HCM to investigate their effects on contractile force and ATPase rates, respectively. Previously, we showed that these HCM cTnC mutants, except for E134D, increased the Ca 2+ sensitivity of force development in cardiac preparations. In the current study, an increase in Ca 2+ sensitivity of isometric force was only observed for the C84Y mutant when reconstituted in soleus fibers. Incorporation of cTnC C84Y in bovine masseter myofibrils reduced the ATPase activity at saturating [Ca 2+ ], whereas, incorporation of cTnC D145E increased the ATPase activity at inhibiting and saturating [Ca 2+ ]. We also tested whether reconstitution of cardiac fibers with troponin complexes containing the cTnC mutants and slow skeletal troponin I (ssTnI) could emulate the slow skeletal functional phenotype. Reconstitution of cardiac fibers with troponin complexes containing ssTnI attenuated the Ca 2+ sensitization of isometric force when cTnC A8V and D145E were present; however, it was enhanced for C84Y. In summary, although the A8V and D145E mutants are present in both muscle types, their functional phenotype is more prominent in cardiac muscle than in slow skeletal muscle, which has implications for the protein-protein interactions within the troponin complex. The C84Y mutant warrants further investigation since it drastically alters the properties of both muscle types and may account for the earlier clinical onset in the proband.

Background: Mutations in thin-filament proteins have been linked to hypertrophic cardiomyopathy, but it has never been demonstrated that variants identified in the TNNC1 (gene encoding troponin C) can evoke cardiac remodeling in vivo. The goal of this study was to determine whether TNNC1 can be categorized as an hypertrophic cardiomyopathy susceptibility gene, such that a mouse model can recapitulate the clinical presentation of the proband., Methods and Results: The TNNC1-A8V proband diagnosed with severe obstructive hypertrophic cardiomyopathy at 34 years of age exhibited mild-to-moderate thickening in left and right ventricular walls, decreased left ventricular dimensions, left atrial enlargement, and hyperdynamic left ventricular systolic function. Genetically engineered knock-in (KI) mice containing the A8V mutation (heterozygote=KI-TnC-A8V(+/-); homozygote=KI-TnC-A8V(+/+)) were characterized by echocardiography and pressure-volume studies. Three-month-old KI-TnC-A8V(+/+) mice displayed decreased ventricular dimensions, mild diastolic dysfunction, and enhanced systolic function, whereas KI-TnC-A8V(+/-) mice displayed cardiac restriction at 14 months of age. KI hearts exhibited atrial enlargement, papillary muscle hypertrophy, and fibrosis. Liquid chromatography-mass spectroscopy was used to determine incorporation of mutant cardiac troponin C (≈ 21%) into the KI-TnC-A8V(+/-) cardiac myofilament. Reduced diastolic sarcomeric length, increased shortening, and prolonged Ca(2+) and contractile transients were recorded in intact KI-TnC-A8V(+/-) and KI-TnC-A8V(+/+) cardiomyocytes. Ca(2+) sensitivity of contraction in skinned fibers increased with mutant gene dose: KI-TnC-A8V(+/+)>KI-TnC-A8V(+/-)>wild-type, whereas KI-TnC-A8V(+/+) relaxed more slowly on flash photolysis of diazo-2., Conclusions: The TNNC1-A8V mutant increases the Ca(2+)-binding affinity of the thin filament and elicits changes in Ca(2+) homeostasis and cellular remodeling, which leads to diastolic dysfunction. These in vivo alterations further implicate the role of TNNC1 mutations in the development of cardiomyopathy., (© 2015 American Heart Association, Inc.)