Titin role in muscle homeostasis: the kinase domain

The giant muscle protein titin is a central player in cardiovascular health and disease. Titin molecules spanning half of the sarcomere form a filament system in striated muscles. The titin filament is not only an essential structural component of sarcomere, but also plays a central role in myofibril signaling through its kinase domain (TK) and numerous protein ligands. Thus, not surprisingly, mutations in this molecule might have detrimental effects. In this work a structure-driven approach was taken to re-evaluate titin kinase catalytic activity and the pathogenicity of two cardiomyopathy-associated titin mutations. Comparison of recombinant preparations from E.coli and insect cells revealed intrinsic inactivity of TK. It was demonstrated that previously reported phosphotransfer activity towards Tcap (a small Z-disc protein) is due to contaminant kinase activity from insect cells but not TK itself. Next, it was established that the eukaryotic host produces structurally indistinguishable TK from that produced in insect cells, albeit inactive towards Tcap, classical kinase substrates or extracts from mature or differentiating muscles. Structural analysis identified evolutionary conserved residue substitutions converting vertebrate TKs to pseudokinases. The structural context of dilated cardiomyopathy associated mutation was revealed in the crystal structure of TK enclosing neighboring domains A170 and M1. The mutation site resides in a conserved helix located in the linker region between TK and the binding site of ubiquitin E3 ligase MuRF1. Aspartate to valine substitution causes disruption of a conserved hydrogen bond and detachment of the affected helix from TK. In the context of the titin filament, this causes dissociation of the binding site from TK and increases interdomain flexibility. Structural alterations translate into increased MuRF1-mediated degradation of mutant titin fragments through the ubiquitin-proteasome pathway. Speculatively, haploinsufficiency of mutant titin could be a possible pathomechanism leading to dilated cardiomyopathy associated with the mutation under study. Comprehensive analysis of arrhythmogenic left ventricular cardiomyopathy associated titin mutation generated a novel model of pathogenesis. In contrast to previous reports, we demonstrate that mutation does not cause affected domain I10 unfolding, and is structurally compatible. Observed destabilization of the domain was attributed to a disrupted hydrogen bond, causing increased flexibility. A crystal structure of the affected domain flanked by adjacent domains I9-I11 demonstrated that threonine to isoleucine substitution might have detrimental effects on interdomain arrangement, resulting in exposure of a hydrophobic patch. Functionally, differential localization of mutant protein was observed in transgenic muscles. Speculatively, mutation could result in impaired folding of mutant protein and lead to accumulation of degradation-resistant aggregates or cause an increased stickiness to thin filaments as a novel pathomechanism. Results presented in this work demonstrate that TK is a catalytically inactive pseudokinase acting as a molecular scaffold. It was demonstrated that TK and MuRF1 signaling modules are structurally interconnected and genetic perturbation of this link might lead to dilated cardiomyopathy. In similar fashion, genetic alteration of interaction between immunoglobulin domains might cause arrhythmogenic left ventricular cardiomyopathy

A Novel Murine Model of Parvovirus Associated Dilated Cardiomyopathy Induced by Immunization with VP1-Unique Region of Parvovirus B19

Background. Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results. We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions. Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

The effect of a unique region of parvovirus B19 capsid protein VP1 on endothelial cells

Parvovirus B19 (B19V) is a widespread human pathogen possessing a high tropism for erythroid precursor cells. However, the persistence or active replication of B19V in endothelial cells (EC) has been detected in diverse human pathologies. The VP1 unique region (VP1u) of the viral capsid has been reported to act as a major determinant of viral tropism for erythroid precursor cells. Nevertheless, the interaction of VP1u with EC has not been studied. We demonstrate that recombinant VP1u is efficiently internalized by rats’ pulmonary trunk blood vessel-derived EC in vitro compared to the human umbilical vein EC line. The exposure to VP1u was not acutely cytotoxic to either human- or rat-derived ECs, but led to the upregulation of cellular stress signaling-related pathways. Our data suggest that high levels of circulating B19V during acute infection can cause endothelial damage, even without active replication or direct internalization into the cells

Titin kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line

Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.publishe

The role of cardiac T-cadherin in the indicating heart failure severity of patients with non-ischemic dilated cardiomyopathy

Background and objectives: T-cadherin (T-cad) is one of the adiponectin receptors abundantly expressed in the heart and blood vessels. Experimental studies show that T-cad sequesters adiponectin in cardiovascular tissues and is critical for adiponectin-mediated cardio-protection. However, there are no data connecting cardiac T-cad levels with human chronic heart failure (HF). The aim of this study was to assess whether myocardial T-cad concentration is associated with chronic HF severity and whether the T-cad levels in human heart tissue might predict outcomes in patients with non-ischemic dilated cardiomyopathy (NI-DCM). Materials and Methods: 29 patients with chronic NI-DCM and advanced HF were enrolled. Patients underwent regular laboratory investigations, echocardiography, coronary angiography, and right heart catheterization. TNF- and IL6 in serum were detected by enzyme-linked immunosorbent assay (ELISA). Additionally, endomyocardial biopsies were obtained, and the levels of T-cad were assessed by ELISA and CD3, CD45Ro, CD68, and CD4- immunohistochemically. Mean pulmonary capillary wedge pressure (PCWP) was used as a marker of HF severity, subdividing patients into two groups: mean PCWP > 19 mmHg vs. mean PCWP 19 mmHg compared to those with mean PCWP 19 mmHg (p = 0.058). Cardiac T-cad levels correlate negatively with myocardial CD3 cell count (rho = 0.423, p = 0.028). Conclusions: Univariate Cox regression analysis did not prove T-cad to be an outcome predictor (HR = 1, p = 0.349). However, decreased T-cad levels in human myocardium can be an additional indicator of HF severity. T-cad in human myocardium has an anti-inflammatory role. More studies are needed to extend the role of T-cad in the outcome prediction of patients with NI-DCM

Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1

MuRF1 is an E3 ubiquitin ligase central to muscle catabolism. It belongs to the TRIM protein family characterized by a tripartite fold of RING, B-box and coiled-coil (CC) motifs, followed by variable C-terminal domains. The CC motif is hypothesized to be responsible for domain organization in the fold as well as for high-order assembly into functional entities. But data on CC from this family that can clarify the structural significance of this motif are scarce. We have characterized the helical region from MuRF1 and show that, contrary to expectations, its CC domain assembles unproductively, being the B2- and COS-boxes in the fold (respectively flanking the CC) that promote a native quaternary structure. In particular, the C-terminal COS-box seemingly forms an α-hairpin that packs against the CC, influencing its dimerization. This shows that a C-terminal variable domain can be tightly integrated within the conserved TRIM fold to modulate its structure and function. Furthermore, data from transfected muscle show that in MuRF1 the COS-box mediates the in vivo targeting of sarcoskeletal structures and points to the pharmacological relevance of the COS domain for treating MuRF1-mediated muscle atrophy.publishe