Genomic Studies in a Large Cohort of Hearing Impaired Italian Patients Revealed Several New Alleles, a Rare Case of Uniparental Disomy (UPD) and the Importance to Search for Copy Number Variations.

Hereditary hearing loss (HHL) is a common disorder characterized by a huge genetic heterogeneity. The definition of a correct molecular diagnosis is essential for proper genetic counseling, recurrence risk estimation, and therapeutic options. From 20 to 40% of patients carry mutations in GJB2 gene, thus, in more than half of cases it is necessary to look for causative variants in the other genes so far identified (~100). In this light, the use of next-generation sequencing technologies has proved to be the best solution for mutational screening, even though it is not always conclusive. Here we describe a combined approach, based on targeted re-sequencing (TRS) of 96 HHL genes followed by high-density SNP arrays, aimed at the identification of the molecular causes of non-syndromic HHL (NSHL). This strategy has been applied to study 103 Italian unrelated cases, negative for mutations in GJB2, and led to the characterization of 31% of them (i.e., 37% of familial and 26.3% of sporadic cases). In particular, TRS revealed TECTA and ACTG1 genes as major players in the Italian population. Furthermore, two de novo missense variants in ACTG1 have been identified and investigated through protein modeling and molecular dynamics simulations, confirming their likely pathogenic effect. Among the selected patients analyzed by SNP arrays (negative to TRS, or with a single variant in a recessive gene) a molecular diagnosis was reached in ~36% of cases, highlighting the importance to look for large insertions/deletions. Moreover, copy number variants analysis led to the identification of the first case of uniparental disomy involving LOXHD1 gene. Overall, taking into account the contribution of GJB2, plus the results from TRS and SNP arrays, it was possible to reach a molecular diagnosis in ~51% of NSHL cases. These data proved the usefulness of a combined approach for the analysis of NSHL and for the definition of the epidemiological picture of HHL in the Italian population.

A Strategy to Enhance Secretion of Extracellular Matrix Components by Stem Cells: Relevance to Tissue Engineering.

The ability of cells to secrete extracellular matrix proteins is an important property in the repair, replacement, and regeneration of living tissue. Cells that populate tissue-engineered constructs need to be able to emulate these functions. The motifs, KTTKS or palmitoyl-KTTKS (peptide amphiphile), have been shown to stimulate production of collagen and fibronectin in differentiated cells. Molecular modeling was used to design different forms of active peptide motifs to enhance the efficacy of peptides to increase collagen and fibronectin production using terminals KTTKS/SKTTK/SKTTKS connected by various hydrophobic linkers, V4A3/V4A2/A4G3. Molecular dynamic simulations showed SKTTKS-V4A3-SKTTKS (P3), with palindromic (SKTTKS) motifs and SKTTK-V4A2-KTTKS (P5), maintained structural integrity and favorable surface electrostatic distributions that are required for functionality. In vitro studies showed that peptides, P3 and P5, showed low toxicity to human adipose-derived stem cells (hADSCs) and significantly increased the production of collagen and fibronectin in a concentration-dependent manner compared with the original active peptide motif. The 4-day treatment showed that stem cell markers of hADSCs remained stable with P3. The molecular design of novel peptides is a promising strategy for the development of intelligent biomaterials to guide stem cell function for tissue engineering applications.

Impact of disease-causing mutations on inter-domain interactions in cMyBP-C: a steered molecular dynamics study.

The molecular interactions of the sarcomeric proteins are essential in the regulation of various cardiac functions. Mutations in the gene MYBPC3 coding for cardiac myosin-binding protein-C (cMyBP-C), a multi-domain protein, are the most common cause of hypertrophic cardiomyopathy (HCM). The N-terminal complex, C1-motif-C2 is a central region in cMyBP-C for the regulation of cardiac muscle contraction. However, the mechanism of binding/unbinding of this complex during health and disease is unknown. Here, we study possible mechanisms of unbinding using steered molecular dynamics simulations for the complex in the wild type, in single mutations (E258K in C1, E441K in C2), as well as in a double mutation (E258K in C1 + E441K in C2), which are associated with severe HCM. The observed molecular events and the calculation of force utilized for the unbinding suggest the following: (i) double mutation can encourage the formation of rigid complex that required large amount of force and long-time to unbind, (ii) C1 appears to start to unbind ahead of C2 regardless of the mutation, and (iii) unbinding of C2 requires larger amount of force than C1. This molecular insight suggests that key HCM-causing mutations might significantly modify the native affinity required for the assembly of the domains in cMyBP-C, which is essential for normal cardiac function.

An Investigation of the Molecular Mechanism of Double cMyBP-C Mutation in a Patient with End-Stage Hypertrophic Cardiomyopathy.

Mutations in the gene coding for cardiac myosin binding protein-C (cMyBP-C), a multi-domain (C0-C10) protein, are a major causative factor for inherited hypertrophic cardiomyopathy. Patients carrying mutations in this gene have an extremely heterogeneous clinical course, with some progressing to end-stage heart failure. The cause of this variability is unknown. We here describe molecular modeling of a double mutation in domains C1 (E258K) and C2 (E441K) in a patient with severe HCM phenotype. The three-dimensional structure for the C1-motif-C2 complex was constructed with double and single mutations being introduced. Molecular dynamic simulations were performed for 10 ns under physiological conditions. The results showed that both E258K and E441K in isolation can predominantly affect the native domain as well as the nearby motif via conformational changes and result in an additive effect when they coexist. These changes involve important regions of the motif such as phosphorylation and potential actin-binding sites. Moreover, the charge reversal mutations altered the surface electrostatic properties of the complex. In addition, we studied protein expression, which showed that the mutant proteins were expressed and we can suppose that the severe phenotype was not due to haploinsufficiency. However, additional studies on human gene expression will need to confirm this hypothesis. The double mutation affecting the regulatory N-terminal of cMyBP-C have the potential of synergistically interfering with the binding to neighbouring domains and other sarcomeric proteins. These effects may account for the severe phenotype and clinical course observed in the complex cMyBP-C genotypes.

Molecular modeling of disease causing mutations in domain C1 of cMyBP-C.

Cardiac myosin binding protein-C (cMyBP-C) is a multi-domain (C0-C10) protein that regulates heart muscle contraction through interaction with myosin, actin and other sarcomeric proteins. Several mutations of this protein cause familial hypertrophic cardiomyopathy (HCM). Domain C1 of cMyBP-C plays a central role in protein interactions with actin and myosin. Here, we studied structure-function relationship of three disease causing mutations, Arg177His, Ala216Thr and Glu258Lys of the domain C1 using computational biology techniques with its available X-ray crystal structure. The results suggest that each mutation could affect structural properties of the domain C1, and hence it's structural integrity through modifying intra-molecular arrangements in a distinct mode. The mutations also change surface charge distributions, which could impact the binding of C1 with other sarcomeric proteins thereby affecting contractile function. These structural consequences of the C1 mutants could be valuable to understand the molecular mechanisms for the disease.

Molecular modeling study on orphan human protein CYP4A22 for identification of potential ligand binding site.

A molecular structure is an essential source to identify ligand binding sites in orphan human cytochrome P450 4A22 (CYP4A22) that belongs to family 4, which is known to be involved in the regulation of blood pressure. Thus, a homology model has been constructed for CYP4A22 and refined by molecular dynamics simulation (MDS). Subsequently, molecular docking was performed with possible substrates, arachidonic acid (essential fatty acid, AA) and erythromycin (therapeutic drug, ERY). These complexes were also subjected to MDS, which helped in predicting the energetically favorable binding sites for these ligands. Putative substrate recognition sites (SRSs) of this protein provide highly hydrophobic binding pockets for the target ligands. A few key ligand binding residues identified in this study indicates that they could also play a major role in ligand-channeling (F122, L132 and C230). Furthermore, it appears that they might serve critical support for the catalytic reaction center (E321, F450, P449 and R455). Structural analysis of channels proposed that the conformational changes might have originated from the active site upon ligand binding and transferred to the rest of the protein via SRSs, which could thereby regulate the channels in CYP4A22. Most of our prediction results are supported by other research groups. In summary, the first molecular modeling study of CYP4A22 yields structural knowledge, which would be helpful to design structure-based-drugs and functional experiments for the target protein.

Probing possible egress channels for multiple ligands in human CYP3A4: a molecular modeling study.

Human cytochrome P450 (CYP) 3A4 extensively contributes to metabolize 50% of the marketed drugs. Recently, a CYP3A4 structure with two molecules of ketoconazole (2KT) was identified. However, channels for egresses of these inhibitors are unexplored. Thus, we applied molecular dynamics simulations followed by channel analyses. Two simulations of empty and 2KT-bound CYP3A4 results revealed the multiple ligand-induced conformational changes in channel forming regions, which appear to be important for the regulation of channels. In addition, we observed that the channel-3 entrance is closed due to the large structural deviation of the key residues from Phe-cluster. F215 and F220 are known as entrance blockers of channel-2 in metyrapone-bound CYP3A4. Currently, F220 blocks the channel-3 along with F213 and F241. Therefore, it suggested that channel-1 and 2 could potentially serve as egress routes for 2KT. It is also supported by the results from MOLAxis analyses, in which the frequency of channel occurrence and bottleneck radius during simulation favor channel-1 and 2. Several bottleneck residues of these channels may have critical roles in 2KT egresses, especially S119. Our modeling study for multiple ligand-channeling of CYP3A4 could be very helpful to gain new insights into channel selectivity of CYP3A4.

Molecular modeling study of CodX reveals importance of N-terminal and C-terminal domain in the CodWX complex structure of Bacillus subtilis.

In Bacillus subtilis, CodW peptidase and CodX ATPase function together as a distinctive ATP-dependent protease called CodWX, which participates in protein degradation and regulates cell division. The molecular structure of CodX and the assembly structure of CodW-CodX have not yet been resolved. Here we present the first three-dimensional structure of CodX N-terminal (N) and C-terminal (C) domain including possible structure of intermediate (I) domain based on the crystal structure of homologous Escherichia coli HslU ATPase. Moreover, the biologically relevant CodWX (W(6)W(6)X(6)) octadecamer complex structure was constructed using the recently identified CodW-HslU hybrid crystal structure. Molecular dynamics (MD) simulation shows a reasonably stable structure of modeled CodWX and explicit behavior of key segments in CodX N and C domain: nucleotide binding residues, GYVG pore motif and CodW-CodX interface. Predicted structure of the possible I domain is flexible in nature with highly coiled hydrophobic region (M153-M206) that could favor substrate binding and entry. Electrostatic surface potential observation unveiled charge complementarity based CodW-CodX interaction pattern could be a possible native interaction pattern in the interface of CodWX. CodX GYVG pore motif structural features, flexible nature of glycine (G92 and G95) residues and aromatic ring conformation preserved Y93 indicated that it may follow the similar mode during the proteolysis mechanism as in the HslU closed state. This molecular modeling study uncovers the significance of CodX N and C domain in CodWX complex and provides possible explanations which would be helpful to understand the CodWX-dependent proteolysis mechanism of B. subtilis.