Biology subjects

Greenberg, L.

Publications and source records attributed to Greenberg, L..

16 recordsLinked to original sources

bioRxiv2026

Molecular and Cellular Determinants of Human Iron Overload Cardiomyopathy

Iron overload cardiomyopathy (IOC) is a serious heart condition that is caused by elevated levels of systemic iron. IOC is characterized by both systolic and diastolic dysfunction as well as arrhythmias. It has been challenging to isolate the cardiac-specific cellular and molecular mechanisms driving IOC because the disease affects multiple interconnected organ systems. Here, we leverage stem cell technologies, cardiac tissue engineering, and protein reconstitution assays to model key aspects of human IOC in vitro and to probe the cellular and molecular mechanisms driving cardiac dysfunction. We demonstrate that human engineered heart tissues consisting of both cardiomyocytes and cardiac fibroblasts faithfully recapitulate key aspects of the human disease, including reduced systolic function, impaired diastolic function, and increased prevalence of arrhythmogenic events. We demonstrate that while both cardiomyocytes and cardiac fibroblasts show increased intracellular iron levels, leading to reduced viability, cardiomyocytes show higher levels of iron accumulation and higher levels of reactive oxygen species production. Moreover, we show that in a tissue, iron overload has little effect on the action potential kinetics; however, it directly impacts the amplitude and kinetics of the calcium transient, potentially driving arrhythmogenesis. Finally, we demonstrate that iron overload decreases force production, in part, through oxidative damage of sarcomeric proteins and direct iron-based inhibition of myosin. In summary, our results reveal new insights into the cellular and molecular mechanisms of human IOC pathogenesis, and they establish new in vitro models that can be harnessed to faithfully recapitulate key aspects of the human disease phenotype. HighlightsO_LIContractile aspects of iron overload cardiomyopathy have been difficult to study in vitro. C_LIO_LIWe developed engineered heart tissues to model key aspects of the human disease. C_LIO_LIIn vitro iron overload reduces contractility and induces arrhythmogenesis. C_LIO_LIIron differentially affects cardiomyocytes and cardiac fibroblasts. C_LIO_LIIron overload directly impacts the actomyosin contractile apparatus. C_LI

biophysics↗

bioRxiv2025

Butterfly egg plasticity in physiologically and reproductive relevant traits: adaptation to climate?

Embryos of terrestrial animals developing inside immobile eggs face variable environmental conditions. Insect egg pores, critical for gas exchange (aeropyles) and fertilisation (micropyles), are ecologically important yet understudied. While aeropyles likely adapt to climatic respiratory demands, and micropyles may respond to environmental pressures, their inter- and intra-specific variation remains poorly studied. Here, we compared egg pore morphology of three closely related Pieris butterflies sampled in the Netherlands. In addition, we compared egg traits of different P. napi populations sampled along a 4,000-km latitudinal gradient from northern to southern Europe linked these traits with available climatic data. After several generations under identical greenhouse conditions, egg pore traits remained highly variable within and between species and groups. Remarkably, P. napi populations from divergent climates differed more in pore number and size than did co-occurring Pieris species. Aeropyle number and width were strongly associated with climatic variables such as maximum temperature, independent of egg size. Micropyle width varied with seasonality. Additionally, we observed the micropylar pit of older eggs covered by a substance more often than fresh eggs. Our findings indicate that insect egg pores are highly dynamic traits, shaped by ecological, behavioural and climatic factors and an under-recognised layer of egg adaptation.

ecology↗

bioRxiv2024

Loss of olfaction reduces caterpillar performance and increases susceptibility to a natural enemy

Insect herbivores such as caterpillars, are under strong selection pressure from natural enemies, especially parasitoid wasps. Although the role of olfaction in host-plant seeking has been investigated in great detail in parasitoids and adult lepidopterans, the caterpillar olfactory system and its significance in tri-trophic interactions remain poorly understood. In this study, we investigated the olfactory system of Pieris brassicae caterpillars and the importance of olfactory information in the interactions among this herbivore, its host plant Brassica oleracea and its primary natural enemy Cotesia glomerata. To examine the role of olfaction, we utilized CRISPR/Cas9 to knockout the odorant receptor co-receptor (Orco). This knockout (KO) impaired olfactory detection and primary processing in the brain. Orco KO caterpillars exhibited reduced weight and lost preference for their optimal food plants. Interestingly, the KO caterpillars also experienced reduced weight when challenged by the parasitoid C. glomerata whose ovipositor had been removed, and the mortality of the KO caterpillars under the attack of unmanipulated parasitoids increased. We then investigated the behavior of P. brassicae caterpillars in response to volatiles from plants attacked by conspecific caterpillars and volatiles from plants on which the caterpillars were themselves attacked by C. glomerata. After analyzing the volatile compounds involved in these interactions, we concluded that olfactory information enables caterpillars to locate suitable food sources more efficiently as well as to select enemy-free spaces. Our results reveal the crucial role of olfaction in caterpillar feeding and natural-enemy avoidance, highlighting the significance of chemoreceptor genes in shaping ecological interactions.

ecology↗

bioRxiv2024

Multifunctional Roles of Sec13 Paralogues in the Euglenozoan Trypanosoma brucei

Secretory cargos are exported from the ER via COPII coated vesicles that have an inner matrix of Sec23/Sec24 heterotetramers and an outer cage of Sec13/Sec31 heterotetramers. In addition to COPII, Sec13 is part of the nuclear pore complex (NPC) and the regulatory SEA/GATOR complex in eukaryotes, which typically have one Sec13 orthologue. The kinetoplastid parasite Trypanosoma brucei has two paralogues: TbSec13.1, an accepted component of both COPII and the NPC, and TbSec13.2. Little is known about TbSec13.2, but others have proposed that it, and its orthologue in the distantly related diplonemid Paradiplonema papillatum, operate exclusively in the SEA/GATOR complex, and that this represents an evolutionary diversification of function unique to the euglenozoan protists (doi.org/10.1098/rsob.220364). Using RNAi silencing in trypanosomes we show both TbSec13s are essential. Knockdown of each dramatically and equally delays transport of GPI-anchored secretory cargo, indicating roles for both in COPII-mediated trafficking from the ER. Immunofluorescence and proximity labeling studies confirm that both TbSec13.1 and TbSec13.2 co-localize with TbSec24.1 to ER exit sites, and thus are functional components of the COPII machinery. Our findings indicate that TbSec13.2 function is not restricted to the SEA/GATOR complex in trypanosomes.

cell biology↗

bioRxiv2024

Danicamtiv reduces myosin's working stroke but enhances contraction by activating the thin filament

Heart failure is a leading cause of death worldwide, and even with current treatments, the 5-year transplant-free survival rate is only [~]50-70%. As such, there is a need to develop new treatments for patients that improve survival and quality of life. Recently, there have been efforts to develop small molecules for heart failure that directly target components of the sarcomere, including cardiac myosin. One such molecule, danicamtiv, recently entered phase II clinical trials; however, its mechanism of action and direct effects on myosins mechanics and kinetics are not well understood. Using optical trapping techniques, stopped flow transient kinetics, and in vitro reconstitution assays, we found that danicamtiv reduces the size of cardiac myosins working stroke, and in contrast to studies in muscle fibers, we found that it does not affect actomyosin detachment kinetics at the level of individual crossbridges. We demonstrate that danicamtiv accelerates actomyosin association kinetics, leading to increased recruitment of myosin crossbridges and subsequent thin filament activation at physiologically-relevant calcium concentrations. Finally, we computationally model how the observed changes in mechanics and kinetics at the level of single crossbridges contribute to increased cardiac contraction and improved diastolic function compared to the related myotrope, omecamtiv mecarbil. Taken together, our results have important implications for the design of new sarcomeric-targeting compounds for heart failure. SIGNIFICANCE STATEMENTHeart failure is a leading cause of death worldwide, and there is a need to develop new treatments that improve outcomes for patients. Recently, the myosin-binding small molecule danicamtiv entered clinical trials for heart failure; however, its mechanism at the level of single myosin crossbridges is not well understood. We determined the molecular mechanism of danicamtiv and showed how drug-induced molecular changes can mechanistically increase heart contraction. Moreover, we demonstrate fundamental differences between danicamtiv and the related myosin-binding small molecule omecamtiv mecarbil that explain the improved diastolic function seen with danicamtiv. Our results have important implications for the design of new therapeutics for heart failure.

biophysics↗

bioRxiv2024

An Unbiased Proteomic Platform for Activity-based Arginylation Profiling

Protein arginylation is an essential posttranslational modification (PTM) catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass in a protein sequence. Here we present a general ATE1-based arginylation profiling platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates (ex vivo) rather than live cells, thus eliminating translational bias derived from the ribosomal activity and enabling bona fide arginylation identification using isotopic features. The method has been successfully applied to an array of peptide, protein, cell, patient, and animal tissue samples using 20 {micro}g sample input, with 235 unique arginylation sites revealed from human proteomes. Representative sites were validated and followed up for their biological functions. The developed platform is globally applicable to the aforementioned sample types and therefore paves the way for functional studies of this difficult-to-characterize protein modification.

biochemistry↗

bioRxiv2024

Structural dynamics of the intrinsically disordered linker region of cardiac troponin T

The cardiac troponin complex, composed of troponins I, T, and C, plays a central role in regulating the calcium-dependent interactions between myosin and the thin filament. Mutations in troponin can cause cardiomyopathies; however, it is still a major challenge to connect how changes in sequence affect troponins function. Recent high-resolution structures of the thin filament revealed critical insights into the structure-function relationship of troponin, but there remain large, unresolved segments of troponin, including the troponin-T linker region that is a hotspot for cardiomyopathy mutations. This linker region is predicted to be intrinsically disordered, with behaviors that are not well described by traditional structural approaches; however, this proposal has not been experimentally verified. Here, we used a combination of single-molecule Forster resonance energy transfer (FRET), molecular dynamics simulations, and functional reconstitution assays to investigate the troponin-T linker region. We show that in the context of both isolated troponin and the fully regulated troponin complex, the linker behaves as a dynamic, intrinsically disordered region. This region undergoes polyampholyte expansion in the presence of high salt and distinct conformational changes during the assembly of the troponin complex. We also examine the {Delta}E160 hypertrophic cardiomyopathy mutation in the linker and demonstrate that it does not affect the conformational dynamics of the linker, rather it allosterically affects interactions with other troponin complex subunits, leading to increased molecular contractility. Taken together, our data clearly demonstrate the importance of disorder within the troponin-T linker and provide new insights into the molecular mechanisms driving the pathogenesis of cardiomyopathies. SIGNIFICANCE STATEMENTTroponin plays a central role in regulating heart contraction, and mutations in troponin can cause human cardiomyopathies. There are several functionally-significant regions of troponin that have not been structurally resolved, including the troponin T linker region that contains multiple cardiomyopathy mutations. In these unresolved regions, it is not possible to understand how changes in sequence affect function. We used computational and experimental techniques to demonstrate that this linker is dynamic and intrinsically disordered both in isolation and in the fully regulated thin filament. Moreover, we show how a cardiomyopathy mutation in this region affects function via allosteric disruption of intermolecular interactions. Our results highlight the need to consider how key mutations affect troponin disorder rather than the structure-function relationship.

biophysics↗

bioRxiv2024

Harnessing molecular mechanism for precision medicine in dilated cardiomyopathy caused by a mutation in troponin T

Familial dilated cardiomyopathy (DCM) is frequently caused by autosomal dominant point mutations in genes involved in diverse cellular processes, including sarcomeric contraction. While patient studies have defined the genetic landscape of DCM, genetics are not currently used in patient care, and patients receive similar treatments regardless of the underlying mutation. It has been suggested that a precision medicine approach based on the molecular mechanism of the underlying mutation could improve outcomes; however, realizing this approach has been challenging due to difficulties linking genotype and phenotype and then leveraging this information to identify therapeutic approaches. Here, we used multiscale experimental and computational approaches to test whether knowledge of molecular mechanism could be harnessed to connect genotype, phenotype, and drug response for a DCM mutation in troponin T, deletion of K210. Previously, we showed that at the molecular scale, the mutation reduces thin filament activation. Here, we used computational modeling of this molecular defect to predict that the mutant will reduce cellular and tissue contractility, and we validated this prediction in human cardiomyocytes and engineered heart tissues. We then used our knowledge of molecular mechanism to computationally model the effects of a small molecule that can activate the thin filament. We demonstrate experimentally that the modeling correctly predicts that the small molecule can partially rescue systolic dysfunction at the expense of diastolic function. Taken together, our results demonstrate how molecular mechanism can be harnessed to connect genotype and phenotype and inspire strategies to optimize mechanism-based therapeutics for DCM. Significance statementDilated cardiomyopathy (DCM), a leading cause of heart failure, is characterized by the inability of the heart to perfuse the body at normal filling pressures. There are multiple causes of DCM, including point mutations in sarcomeric proteins, but most patients receive similar courses of treatment, regardless of the underlying cause of the DCM. Many patients remain unserved by current therapies, and there is a need for new approaches. Here, we use multiscale experimental and computational approaches to demonstrate how knowledge of molecular mechanism can be harnessed to accurately predict the effects of a patient-specific mutation and responses to presumptive therapeutics. Our approach lays the foundation for a precision medicine approach to DCM.

biophysics↗

bioRxiv2023

Divergent Molecular Phenotypes in Point Mutations at the Same Residue in Beta-Myosin Heavy Chain Lead to Distinct Cardiomyopathies

In genetic cardiomyopathies, a frequently described phenomenon is how similar mutations in one protein can lead to discrete clinical phenotypes. One example is illustrated by two mutations in beta myosin heavy chain ({beta}-MHC) that are linked to hypertrophic cardiomyopathy (HCM) (Ile467Val, I467V) and left ventricular non-compaction (LVNC) (Ile467Thr, I467T). To investigate how these missense mutations lead to independent diseases, we studied the molecular effects of each mutation using recombinant human {beta}-MHC Subfragment 1 (S1) in vitro assays. Both HCM-I467V and LVNC-I467T S1 mutations exhibited similar mechanochemical functions, including unchanged ATPase and enhanced actin velocity but had distinct effects on the basal activity of myosin. HCM-I467V S1 showed no change in basal ATPase activity of myosin while LVNC-I467T reduced the basal ATPase activity by 50%. Molecular dynamics simulations reveal that I467T allosterically disrupts nucleotide binding of myosin, which may contribute to the uncoupled reduced basal activity and enhanced actin velocity observed in this mutation. These contrasting molecular effects may lead to contractile dysregulation that initiates LVNC-associated signaling pathways that progress the phenotype. Together, analysis of these mutations provides evidence that phenotypic complexity originates at the molecular level and is critical to understanding disease progression and developing therapies.

biophysics↗

bioRxiv2022

Drug specificity and affinity are encoded in the probability of cryptic pocket opening in myosin motor domains

The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least 6 of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 milliseconds of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatins binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R2=0.82). To test our ability to make blind predictions, we predicted blebbistatins binding affinity for an isoform (Myh7b) whose blebbistatin sensitivity was unknown. Encouragingly, we find good agreement between the predicted and measured IC50 (0.67 {micro}M vs. 0.36 {micro}M). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets. SignificanceDrug development requires the discovery of compounds which specifically target one member of a protein family without triggering side effects that arise from interactions with other related proteins. Myosins are a family of motor proteins that are drug targets for heart diseases, cancer, and parasitic infections. Here, we investigate why the compound blebbistatin specifically inhibits some myosins more potently than others, even though its binding site is closed in all known experimental structures. We find that the blebbistatin binding pocket opens in molecular dynamics simulations of certain myosin motors, and that the probability of opening predicts how potently blebbistatin inhibits a particular motor. Our work suggests that differences in cryptic pocket formation can be exploited to develop specific therapeutics.

biophysics↗

bioRxiv2022

Sediment substrate size influences fish diversity in tributary mouth areas in impounded boreal rivers in Sweden

Large boreal rivers in Sweden are generally impounded by hydropower dams and a large proportion of their main stem shallow flowing habitats have been lost. Tributaries often contain relatively undisturbed habitats and could be important for the conservation of species diversity. Tributary mouth areas could be biodiversity hot-spots, due to the vicinity to the main stem and favorable environmental conditions. In this study, we investigate whether tributary mouth areas in two impounded boreal rivers (Ume- and Lule River) could be regarded as biodiversity hot spots for fish. Based on electrofishing in 20 tributary mouths, we found that overall fish diversity is generally low. The highest species richness and diversity was found in mouth areas dominated by intermediate substrate sizes (gravel - cobble). Few, if any, species were found in association with fine sediment substrates (smaller than sand). The tributary mouth areas had similar species richness and diversity as areas in the tributaries located 1-km upstream of the mouth, but the fish community composition often differed between these sites. Management action favoring fish diversity in the tributary mouth areas could include protection or rehabilitation of areas dominated by medium sized substrate and reduction of erosion and transport of fine sediments in the tributaries. Overall, we find no support for tributary mouths being hot-spots for fish biodiversity and while some patterns in diversity gives hints on suitable management action, it is important to further understand impacts in tributaries and their mouths especially in relation to temporal dynamics of the fish community.

ecology↗

bioRxiv2022

Structure and function of a Notch-like proteolytic switch domain in the extracellular matrix receptor dystroglycan

The dystrophin glycoprotein complex (DGC), anchored by the transmembrane protein dystroglycan, functions to mechanically link the extracellular matrix to the actin cytoskeleton to drive critical aspects of development and adult homeostasis. Breaking this connection via mutation of the actin adaptor protein dystrophin or impaired glycosylation of dystroglycan are strongly associated with diseases such as Muscular Dystrophy, yet cleavage of the dystroglycan protein by matrix metalloproteinases (MMPs) remains an understudied mechanism to disrupt the DGC. We solved X-ray structures of the membrane-adjacent domain of dystroglycan to understand the molecular underpinnings of dystroglycan MMP cleavage regulation. Dystroglycan proteolysis occurs within the versatile SEAL domain, which supports proteolysis in diverse receptors to facilitate mechanotransduction, protection of cell membranes, and even viral entry. The structure reveals a c-terminal extension of the SEAL domain that buries the MMP cleavage site by packing into a hydrophobic pocket, a unique mechanism of MMP cleavage regulation. We further demonstrate that structure-guided and disease-associated mutations disrupt proteolytic regulation using a new cell-surface proteolysis assay. Finally, we find that disruption of proteolysis leads to altered cellular mechanics and migration using high-throughput DNA tension probe and wound healing assays. These findings highlight that disrupted proteolysis is a relevant mechanism for "breaking" the DGC link to contribute to disease pathogenesis and may offer new therapeutic avenues for dystroglycanopathies.

biochemistry↗

bioRxiv2020

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis

Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.

immunology↗

bioRxiv2020

Mechanical dysfunction induced by a hypertrophic cardiomyopathy mutation is the primary driver of cellular adaptation

Familial hypertrophic cardiomyopathy (HCM), a leading cause of sudden cardiac death, is primarily caused by mutations in sarcomeric proteins. The pathogenesis of HCM is complex, with functional changes that span scales from molecules to tissues. This makes it challenging to deconvolve the biophysical molecular defect that drives the disease pathogenesis from downstream changes in cellular function. Here, we examined a HCM mutation in troponin T, R92Q. We demonstrate that the primary molecular insult driving the disease pathogenesis is mutation-induced alterations in tropomyosin positioning, which causes increased molecular and cellular force generation during calcium-based activation. We demonstrate computationally that these increases in force are direct consequences of the initial molecular insult. This altered cellular contractility causes downstream alterations in gene expression, calcium handling, and electrophysiology. Taken together, our results demonstrate that molecularly driven changes in mechanical tension drive the early disease pathogenesis, leading to activation of adaptive mechanobiological signaling pathways.

biophysics↗

bioRxiv2019

Disrupted mechanobiology links the molecular and cellular phenotypes in familial dilated cardiomyopathy

Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a major indicator for heart transplant. The disease is frequently caused by mutations of sarcomeric proteins; however, it is not well understood how these molecular mutations lead to alterations in cellular organization and contractility. To address this critical gap in our knowledge, we studied the molecular and cellular consequences of a DCM mutation in troponin-T, {Delta}K210. We determined the molecular mechanism of {Delta}K210 and used computational modeling to predict that the mutation should reduce the force per sarcomere. In mutant cardiomyocytes, we found that {Delta}K210 not only reduces contractility, but also causes cellular hypertrophy and impairs cardiomyocytes ability to adapt to changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and disease). These results link the molecular and cellular phenotypes and implicate alterations in mechanosensing as an important factor in the development of DCM.

biophysics↗

bioRxiv2019

Computational tool to study perturbations in muscle regulation and its application to heart disease

Striated muscle contraction occurs when myosin thick filaments bind to thin filaments in the sarcomere and generate pulling forces. This process is regulated by calcium, and it can be perturbed by pathological conditions (e.g., myopathies), physiological adaptations (e.g., {beta}-adrenergic stimulation), and pharmacological interventions. Therefore, it is important to have a methodology to robustly determine the mechanism of these perturbations and statistically evaluate their effects. Here, we present an approach to measure the equilibrium constants that govern muscle activation, estimate uncertainty in these parameters, and statistically test the effects of perturbations. We provide a MATLAB-based computational tool for these analyses, along with easy-to-follow tutorials that make this approach accessible. The hypothesis testing and error estimation approaches described here are broadly applicable, and the provided tools work with other types of data, including cellular measurements. To demonstrate the utility of the approach, we apply it to determine the biophysical mechanism of a mutation that causes familial hypertrophic cardiomyopathy. This approach is generally useful for studying the mechanisms of muscle diseases and therapeutic interventions that target muscle contraction.

biophysics↗