The Intracranial Aneurysm Gene THSD1 Connects Endosome Dynamics to Nascent Focal Adhesion Assembly

Abstract

Background/Aims: We recently discovered that harmful variants in THSD1 (Thrombospondin type-1 domain-containing protein 1) likely cause intracranial aneurysm and subarachnoid hemorrhage in a subset of both familial and sporadic patients with supporting evidence from two vertebrate models. The current study seeks to elucidate how THSD1 and patientidentified variants function molecularly in focal adhesions. Methods: Co-immunostaining and co-immunoprecipitation were performed to define THSD1 subcellular localization and interacting partners. Transient expression of patient-identified THSD1 protein variants and siRNA-mediated loss-of-function THSD1 were used to interrogate gene function in focal adhesion and cell attachment to collagen I in comparison to controls. Results: THSD1 is a novel nascent adhesion protein that co-localizes with several known markers such as FAK, talin, and vinculin, but not with mature adhesion marker zyxin. Furthermore, THSD1 forms a multimeric protein complex with FAK/talin/vinculin, wherein THSD1 promotes talin binding to FAK but not to vinculin, a key step in nascent adhesion assembly. Accordingly, THSD1 promotes mature adhesion formation and cell attachment, while its rare variants identified in aneurysm patients show compromised ability. Interestingly, THSD1 also localizes at different stages of endosomes. Clathrin-mediated but not caveolae-mediated endocytosis pathway is involved in THSD1 intracellular trafficking, which positively regulates THSD1-induced focal adhesion assembly, in contrast to the traditional role of endosomes in termination of integrin signals. Conclusions: The data suggest that THSD1 functions at the interface between endosome dynamics and nascent focal adhesion assembly that is impaired by THSD1 rare variants identified from intracranial aneurysm patients.

A recessive truncating variant in thrombospondin‐1 domain containing protein 1 gene THSD1 is the underlying cause of nonimmune hydrops fetalis, congenital cardiac defects, and haemangiomas in four patients from a consanguineous family

“…This distinct variant locations might be the reason for the variable phenotype, given that all heterozygous family members including the elderly as we mentioned earlier are healthy with no record of any cerebrovascular incidents. Rui and colleagues have recently reported that THSD‐1 protein interacts with nascent focal adhesion proteins in extracellular matrix and enhances the formation of a multimeric molecule of the scaffold proteins talin, vinculin and focal adhesion kinase (Figure f) that enhance nascent adhesions formation (Rui et al, ). It appears that these interactions are affected by pathogenic missense variants in intracellular domains as shown in the co‐immunoprecipitation experiments by Rui and co‐workers.…”

“…It appears that these interactions are affected by pathogenic missense variants in intracellular domains as shown in the co‐immunoprecipitation experiments by Rui and co‐workers. However, due to the truncation effect of the variant p.Arg450Ter they were not able to coimmunoprecipitate the mutant protein with any of the scaffold proteins (Rui et al, ). We predict that the truncated protein in our patients will similarly fail to interact with talin and other scaffold proteins affecting the integrin‐extracellular matrix interactions in blood vessels.…”

RNA-Seq Analysis of Cisplatin and the Monofunctional Platinum(II) Complex, Phenanthriplatin, in A549 Non-Small Cell Lung Cancer and IMR90 Lung Fibroblast Cell Lines

“…THSD1 is downregulated in A549 cells and other cancer cell lines, and may function as a tumor suppressor, but its role in cancer and interaction with other genes is not well understood [42]. However, THSD1 has recently been shown to form a protein complex with focal adhesion kinase (FAK), talin, and vinculin, where its role is to promote talin binding to FAK and functions in normal cell adhesion formation and attachment [43]. Therefore, our data suggests that unlike cisplatin, phenanthriplatin treatment might act to prevent the action of THSD1 in some cancers eliminating its role as a tumor suppressor, but the reason for the distinct action of the monofunctional complex compared to cisplatin on THSD1 is not clear.…”

Podosome formation impairs endothelial barrier function by sequestering zonula occludens proteins

“…Full length myc‐tagged ZO‐1 and FLAG‐tagged ZO‐2 are available from Addgene (#30317 and # 27415, respectively). The PDZ2 domain of ZO‐1 (nucleotide 514–753) was subcloned into pCMV5 by standard PCR protocol and verified by Sanger sequencing as we previously performed (Rui et al, ).…”