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Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, the subcellular structure that links the intermediate filament network of one cell to its neighbor. A mutation “hot-spot” within the NH2-terminal of the DSP protein (residues 299–515) is associated with arrhythmogenic cardiomyopathy. In a subset of DSP variants, disease is linked to calpain hypersensitivity. Previous studies show that calpain hypersensitivity can be corrected in vitro through the addition of a bulky residue neighboring the cleavage site, suggesting that physically blocking calpain accessibility is a viable strategy to restore DSP levels. Here, we aim to find drug-like molecules that also block calpain-dependent degradation of DSP. To do this, we screened ~2500 small molecules to identify compounds that specifically rescue DSP protein levels in the presence of proteases. We find that several molecules, including sodium dodecyl sulfate, palmitoylethanolamide, GW0742, salirasib, eprosarten mesylate, and GSK1838705A prevent wildtype and disease-variant-carrying DSP protein degradation in the presence of both trypsin and calpain without altering protease function. Computational screenings did not predict which molecules would protect DSP, likely due to a lack of specific DSP–drug interactions. Molecular dynamic simulations of DSP–drug complexes suggest that some long hydrophobic molecules can bind in a shallow hydrophobic groove that runs alongside the protease cleavage site. Identification of these compounds lays the groundwork for pharmacological treatment for individuals harboring these hypersensitive DSP variants.

 

The intercalated disk is a cardiac specific structure composed of three main protein complexes—adherens junctions, desmosomes, and gap junctions—that work in concert to provide mechanical stability and electrical synchronization to the heart. Each substructure is regulated through a variety of mechanisms including proteolysis. Calpain proteases, a class of cysteine proteases dependent on calcium for activation, have recently emerged as important regulators of individual intercalated disk components. In this review, we will examine how calcium homeostasis regulates normal calpain function. We will also explore how calpains modulate gap junctions, desmosomes, and adherens junctions activity by targeting specific proteins, and describe the molecular mechanisms of how calpain dysregulation leads to structural and signaling defects within the heart. We will then examine how changes in calpain activity affects cardiomyocytes, and how such changes underlie various heart diseases. 

The shift of funding organizations to prioritize interdisciplinary work points to the need for workflow models that better accommodate interdisciplinary studies. Most scientists are trained in a specific field and are often unaware of the kind of insights that other disciplines could contribute to solving various problems. In this paper, we present a perspective on how we developed an experimental pipeline between a microscopy and image analysis/bioengineering lab. Specifically, we connected microscopy observations about a putative mechanosensing protein, obscurin, to image analysis techniques that quantify cell changes. While the individual methods used are well established (fluorescence microscopy; ImageJ WEKA and mTrack2 programs; MATLAB), there are no existing best practices for how to integrate these techniques into a cohesive, interdisciplinary narrative. Here, we describe a broadly applicable workflow of how microscopists can more easily quantify cell properties (e.g., perimeter, velocity) from microscopy videos of eukaryotic (MDCK) adherent cells. Additionally, we give examples of how these foundational measurements can create more complex, customizable cell mechanics tools and models.

 

Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, a subcellular complex that links the cytoskeleton of one cell to its neighbor. A mutation ‘hot-spot’ within the NH2-terminal third of the DSP protein (specifically, residues 299–515) is associated with both cardiomyopathies and skin defects. In select DSP variants, disease is linked specifically to the uncovering of a previously-occluded calpain target site (residues 447–451). Here, we partially stabilize these calpain-sensitive DSP clinical variants through the addition of a secondary single point mutation—tyrosine for leucine at amino acid position 518 (L518Y). Molecular dynamic (MD) simulations and enzymatic assays reveal that this stabilizing mutation partially blocks access to the calpain target site, resulting in restored DSP protein levels. This ‘molecular band-aid’ provides a novel way to maintain DSP protein levels, which may lead to new strategies for treating this subset of DSP-related disorders. 

There are recent reports of associations of variants in the HPDL gene with a hereditary neurological disease that presents with a wide spectrum of clinical severity, ranging from severe neonatal encephalopathy with no psychomotor development to adolescent-onset uncomplicated spastic paraplegia. Here, we report two probands from unrelated families presenting with severe and intermediate variations of the clinical course. A homozygous variant in the HPDL gene was detected in each proband; however, there was no known parental consanguinity. We also highlight reductions in citrate synthase and mitochondrial complex I activity detected in both probands in different tissues, reflecting the previously proposed mitochondrial nature of disease pathogenesis associated with HPDL mutations. Further, we speculate on the functional consequences of the detected variants, although the function and substrate of the HPDL enzyme are currently unknown. 

The N‐terminal half of the giant cytoskeletal protein obscurin is comprised of more than 50 Ig‐like domains, arranged in tandem. Domains 18–51 are connected to each other through short 5‐residue linkers, and this arrangement has been previously shown to form a semi‐flexible rod in solution. Domains 1–18 generally have slightly longer ~7 residue interdomain linkers, and the multidomain structure and motion conferred by this kind of linker is understudied. Here, we use NMR, SAXS, and MD to show that these longer linkers are associated with significantly more domain/domain flexibility, with the resulting multidomain structure being moderately compact. Further examination of the relationship between interdomain flexibility and linker length shows there is a 5 residue “sweet spot” linker length that results in dual‐domain systems being extended, and conversely that both longer or shorter linkers result in a less extended structure. This detailed knowledge of the obscurin N terminus structure and flexibility allowed for mathematical modeling of domains 1–18, which suggests that this region likely forms tangles if left alone in solution. Given how infrequently protein tangles occur in nature, and given the pathological outcomes that occur when tangles do arise, our data suggest that obscurin is likely either significantly scaffolded or else externally extended in the cell.

 

Obscurin, a giant modular cytoskeletal protein, is comprised mostly of tandem immunoglobulin‐like (Ig‐like) domains. This architecture allows obscurin to connect distal targets within the cell. The linkers connecting the Ig domains are usually short (3–4 residues). The physical effect arising from these short linkers is not known; such linkers may lead to a stiff elongated molecule or, conversely, may lead to a more compact and dynamic structure. In an effort to better understand how linkers affect obscurin flexibility, and to better understand the physical underpinnings of this flexibility, here we study the structure and dynamics of four representative sets of dual obscurin Ig domains using experimental and computational techniques. We find in all cases tested that tandem obscurin Ig domains interact at the poles of each domain and tend to stay relatively extended in solution. NMR, SAXS, and MD simulations reveal that while tandem domains are elongated, they also bend and flex significantly. By applying this behavior to a simplified model, it becomes apparent obscurin can link targets more than 200 nm away. However, as targets get further apart, obscurin begins acting as a spring and requires progressively more energy to further elongate.