Role of talin-1 in transition of liver fibrosis to cirrhosis开题报告

 2023-07-12 09:53:09

1. 研究目的与意义（文献综述包含参考文献）

Talin-1 role in liver disease.I. Talin-1 structure overview.Focal adhesions (FAs) are intracellular protein assemblies that serve as tension-sensing anchoring points to link cells to the extracellular environment [1,2]. FAs not only tether cells to the extracellular matrix (ECM), but also facilitate intracellular reorganization, resulting in dynamic changes in cell functions and cell morphologies [1-3]Talin is a large, 270 kDa protein with 18 domains comprising a 50 kDa globular head, a long rod made of 62 helices forming 13 helical bundle (rod) domains (R1R13) [4,5], and a dimerization (DD) motif at the C terminus [6]. It both activates integrins and couples them to the actin cytoskeleton [7].A unique conformational change of talin facilitates its spring-like behavior, through which talin can unfold into a linearly elongated 60100 nm rod-like shape [8-10].This allows it to bind to at least 11 different FA components including vinculin and actin [11] The talin head contains a 4.1-ezrin-radixin- moesin (FERM) domain with four subdomains (F0F3), which is a common structural feature of several integrin tail-binding proteins [12-15].While the active form of talin is well-characterized, the critical state of the inhibited form of talin is scarcely understood. The importance of talin inhibition is highlighted by the fact that disrup- tion of proper talin inhibition leads to morphogenetic defects dur- ing fly development [16] and it has been implicated in the migration of metastatic cancer cells. [17-19].Talin-1 is expressed mainly in the kidney, liver, spleen, stomach, lung and vascular smooth muscle and its overexpression can promote prostate cancer cell adhesion, migration and invasion [20-22]. Talin-1 was studied in some malignancies like colon [23], prostate [24] and oral squamous cell carcinoma [25]. II. Talin role in disease.Talin is accused in the cancer progression and metastasis [26]. The function of talin in cancerous phenotypes is not limited just to its influence on adhesion and motility but, rather, also depends on its downstream signaling, as emphasized by its overexpression in nonadherent cells.In addition, recently talin-1 was studied as biomarker for process of the carcinogenesis, infiltration and metastasis of HCC and the clinical diagnosis of HCC [27-28].ECM degradation is mediated by the sodium/ hydrogen exchanger 1 (NHE-1), which acidifies the peri- invadipodial microenvinonment and promotes protease activity [2931]. NHE-1 has therefore been implicated in the metastasis of melanoma, breast cancer, and pancreatic ductal adenocarcinoma as a result of stromal remodeling and often in partnership with other membrane proteins [3236]. NHE-1 inhibition has also been shown to pro- mote chemotherapy-induced apoptosis in vitro [37-39] determined that talin is required for invadapodia maturation and the recruitment of NHE-1 with the shared binding partner, moesin, to focal adhesions.Nevertheless, the silencing of talin prevents intracellular alkalinization at invadopodia, which is required for actin polymerization, motility, and metastasis in mammary adenocarcinoma cells [39].Many hallmark works that established paradigms in mechanobiology have transcended its field and have had wide-ranging impact on other disciplines. For instance, a study that demonstrated that alterations in the physical environment of a cell, specifically the extracellular matrix (ECM) alone, could encourage a malignant phenotype in cancer cells [40]; a second work demonstrated that similar alterations in the physical microenvironment could induce stem cells to differentiate into a range of cell types, from neurons to osteoblasts [41].What remains to be seen is how the role of talin as a key component of cellular mechanotransduction might impact its role in cancer progression. The effect of substrate stiffness on cancer metastasis is well documented [40], as is the fact that fibrotic environments (e.g., cirrhosis) are frequently associated with increased cancer development and progression [42].These studies demonstrate that the presence of talin is insufficient for proper cell function; talin must be subjected to force, whether by cell contraction or by the application of shear stress [43].III. Talin mechanotransduction.In addition to its role as an integrin activator, talin also acts as a mechanosensor; it stretches like a spring and transmits tension between the ECM and the actomyosin machinery within the FA [44,45], a process which is essential for regulating FA maturation and stability. As such, the transition between active and inactive talin likely plays a key regulatory role in FA dynamics, similar to the activation and inactivation of integrins.Integrins are a large family of heterodimeric cell surface receptors, which act as mechanoreceptors by relaying the information from cell to cell, and from the extracellular matrix (ECM) to the cell interior and vice versa [46]. Since integrin receptors directly bind to components of the ECM and control remodeling thereof, they may play a crucial role in the evolu- tion and progression of liver fibrosis [47].Talin is a key component in FAs, responsible for activating integrins and mediating both in- side-out and outside-in signaling [48-50]. Talin activates integrin by associating with the cytosolic tail of integrin beta-subunits. Once engaged, talin can assume an elongated conformation up to 100 nm in length [8] , directly linking the beta-integrin subunit in the first layer of the FA to actin bundles in the fourth layer [51]. By spanning all four layers of the FA, talin is in a unique position to act as a structural scaffold, greatly contributing to the overall composi- tion and organization of FA complexes [5].Talins location in focal adhesions and its binding to both integrins and actin filaments positions it for bidirectional transmission of tension across the cell membrane, i.e. transmitting forces generated internally or imposed externally on cells [52,53].Indeed, later work showed that direct application of mechanical tension on cells promotes the growth of focal adhesions [54] and the recruitment of proteins such as vinculin to these sites [55].Talin also contains tandem repeat sequences within its rod domain and one of its unexpected features is that it contains 11 vinculin-binding sites, with most being cryptic within the purified protein [56]. These VBSs are buried within helical bundles and it is currently unclear how many, in fact, ever bind to vinculin in vivo [57]. The talin rod also contains numerous other binding sites, including a second actin binding site [58], an autoinhibition site that interacts with the talin head [59], a second integrin binding site [60], and binding sites for the intermediate filament protein, synemin [61], RIAM [62], and the tumor suppressor protein, deleted in liver cancer 1 (DLC-1) [63].Subsequent studies using single molecule atomic force microscopy revealed that, indeed, these vinculin-binding sites are exposed when talin experiences physiologically relevant mechanical tension [64,65].Tension on talin also affects the Rho GTPase signaling pathway, which regulates contractility and focal adhesion assembly. Deleted in liver cancer 1 (DLC1) is a RhoGAP and negative regulator of RhoA signaling that binds the unstretched form of talin [66,67]. It is released in response to tension [68]. Recent studies show that when bound to talin, DLC1 is active and antago- nizes RhoA activity, decreasing myosin-driven contractility [68].Tension on small adhesions is often greater than on large adhe- sions [53,69]. The initial positive feedback increasing contractility and tension on talin will promote growth, but as adhesions grow the load will be carried by more components, thereby decreasing the level of tension experi- enced by any single talin molecule. This is predicted to favor refolding of the talin domain to which DLC1 binds, consequently activating DLC1 to inhibit RhoA and decrease contractility [68]IV. Rap1 Talin-1 integrin activation pathway.The nature of the connection between Rap1 and talin1 in integrin activation is an important remaining gap in our understanding of this process. We recently identified an additional Rap1 binding site in talin1 F1 domain that makes a greater contribution than F0 in model systems (70).Rap is a member of the Ras family of small GTPases, with five different isoforms: Rap1A, Rap1B, Rap2A, Rap2B and Rap2C expressed in mammalian cells [71]. Rap proteins play a relevant role in cell adhesion [72,73], junction formation [7476], migration, invasion [77], cell polarity [78,79], exocytosis [80], apoptosis [81] and proliferation [82]. As a consequence, they are important for carcinogenesis [83,84] and cardiovascular function [85]. Rap proteins regulate liver physiopathology.For example, Rap2B promotes hepatocarcinoma (HCC) growth, while Rap1 might play a dual role. The RapGEF, Epac1, activates Rap upon cAMP binding, regulating metabolism, survival, and liver regeneration. [86]Rap1 was discovered nearly three decades ago[87], and its two isoforms Rap1a and Rap1b (with ~95% identity) are regarded as the key membrane-associated small GTPases to target proteins to the plasma membrane to regulate diverse cellular responses[88-91]. We found that the activated Rap1b interacts with the F0 domain of talinthe key mediator of integrin activation. Moreover, disruption of the Rap1b/talin interface substantially impaired the integrin αIIbβ3 activation, cell adhesion, and cell spreading [92]. Thus, talin-F0 binds favorably to the active form of Rap1b but, as shown in, the binding affinity is quite low (Kd ~ 162 μM) as previously concluded [15].Rap1b binding to talin is crucial for integrin activation. Membrane-anchored Rap1b shows enhanced binding to talin. While the forgoing data demonstrated the unique specificity of Rap1b/talin interaction and its importance in regulating integrin- mediated functions, a key issue still remains: how can such interaction with ~0.16mM affinity be effective since protein concentration in cells is typically much lower than 0.16 mM? We attempted to address this issue from two angles. First, both Rap1 and talin are highly abundant in cells [92]. For example, recent proteomic studies showed that Rap1b and talin belong to the top most abundant proteins in platelets with copy numbers of ~200,000, which are nearly 1/4 of the most abundant platelet protein actin [93].More recently, evidence has emerged that Rap1 can bind Talin directly and regulate its recruitment to the membrane [94]. Overall, our data suggest a crucial pathway by which agonist-activated Rap1 directly promotes the membrane recruitment of talin, which further leads to the talin unmasking and integrin activation. Intriguingly, a construct containing only a fusion of the membrane targeting sequence of Rap1 and the Talin binding sequence of RIAM is fully sufficient to recruit Talin to the membrane and induce integrin activation [8].

2. 研究的基本内容、问题解决措施及方案

The liver diseases are an emerging problem nowadays. People are in need of new ways to prevent and detect them on the early development stages. This project is focused on Talin-1 which acts as a mechanosensor in the cells. We suppose that with the development of liver fibrosis, increased stiffness of the liver cells can affect the concentration of Talin-1, thus making it possible to judge the patient's condition according to this value. It can help to prevent the transition of liver fibrosis to cirrhosis and make the treatment more effective.

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