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Kidney ischemia and reperfusion injury (IRI) is a significant contributor to acute kidney injury (AKI), characterized by tubular injury and kidney dysfunction. Salvador family WW domain containing protein 1 (SAV1) is a key component of the Hippo pathway and plays a crucial role in the regulation of organ size and tissue regeneration. However, whether SAV1 plays a role in kidney IRI is not investigated. In this study, we investigated the role of SAV1 in kidney injury and regeneration following IRI. A proximal tubule-specific knockout of SAV1 in kidneys (SAV1ptKO) was generated, and wild-type and SAV1ptKO mice underwent kidney IRI or sham operation. Plasma creatinine and blood urea nitrogen were measured to assess kidney function. Histological studies, including periodic acid-Schiff staining and immunohistochemistry, were conducted to assess tubular injury, SAV1 expression, and cell proliferation. Western blot analysis was employed to assess the Hippo pathway-related and proliferation-related proteins. SAV1 exhibited faint expression in the proximal tubules and was predominantly expressed in the connecting tubule to the collecting duct. At 48 h after IRI, SAV1ptKO mice continued to exhibit severe kidney dysfunction, compared to attenuated kidney dysfunction in wild-type mice. Consistent with the functional data, severe tubular damage induced by kidney IRI in the cortex was significantly decreased in wild-type mice at 48 h after IRI but not in SAV1ptKO mice. Furthermore, 48 h after IRI, the number of Ki67-positive cells in the cortex was significantly higher in wild-type mice than SAV1ptKO mice. After IRI, activation and expression of Hippo pathway-related proteins were enhanced, with no significant differences observed between wild-type and SAV1ptKO mice. Notably, at 48 h after IRI, protein kinase B activation (AKT) was significantly enhanced in SAV1ptKO mice compared to wild-type mice. This study demonstrates that SAV1 deficiency in the kidney proximal tubule worsens the injury and delays kidney regeneration after IRI, potentially through the overactivation of AKT.

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Muscular dystrophy in the NH-413 chicken is caused by a missense mutation in the WWP1 gene. WWP1 is a HECT-type E3 ubiquitin ligase containing four tandem WW domains that interact with proline-rich peptide motifs of target proteins, and a short region connecting the second and third WW domains is crucial for the E3 ligase to maintain an autoinhibitory state. A mutation of the arginine in the WW2-WW3 linker to glutamine is thought to affect WWP1 function, but there is little information on this mutation to date. In this study, we generated a transgenic (Tg) mouse model expressing the WWP1 transgene with the R436Q mutation, which corresponds to the missense mutation found in the NH-413 chicken. Tg mice showed marked degradation of mutant WWP1 proteins in various tissues, particularly in striated muscle. Immunoprecipitation analysis using a WWP1-specific antibody demonstrated that the mutant WWP1 proteins lacked the C-terminal catalytic cysteine residue that is required for their binding to the E2-substrate complex during their degradation. In vitro analysis using the R436Q mutant of WWP1 lacking this catalytic cysteine residue showed no autodegradation, indicating that the loss-of-function degradation of this protein is caused by self-ubiquitination. Tg mice expressing R436Q WWP1 did not show stunted growth or premature death. Furthermore, histological analysis did not reveal any obvious changes. These observations suggested that the R436Q mutant WWP1 protein, which is released from autoinhibitory mode by its missense mutation, does not have abnormally activated enzyme function to substrates before its self-degradation and loss of enzyme function.

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Background and Objectives: Cardiac injury plays a critical role in contributing to the mortality associated with sepsis, a condition marked by various forms of programmed cell deaths. Previous studies hinted at the WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) involving in heart failure and endothelial injury. However, the precise implications of WWP2 in sepsis-induced cardiac injury, along with the underlying mechanisms, remain enigmatic. Methods: Sepsis induced cardiac injury were constructed by intraperitoneal injection of lipopolysaccharide. To discover the function of WWP2 during this process, we designed and performed loss/gain-of-function studies with cardiac-specific vectors and WWP2 knockout mice. Combination experiments were performed to investigate the relationship between WWP2 and downstream signaling in septic myocardium injury. Results: The protein level of WWP2 was downregulated in cardiomyocytes during sepsis. Cardiac-specific overexpression of WWP2 protected heart from sepsis induced mitochondrial oxidative stress, programmed cell death and cardiac injury, while knockdown or knockout of WWP2 exacerbated this process. The protective potency of WWP2 was predominantly linked to its ability to suppress cardiomyocyte ferroptosis rather than apoptosis. Mechanistically, our study revealed a direct interaction between WWP2 and acyl-CoA synthetase long-chain family member 4 (FACL4), through which WWP2 facilitated the ubiquitin-dependent degradation of FACL4. Notably, we observed a notable reduction in ferroptosis and cardiac injury within WWP2 knockout mice after FACL4 knockdown during sepsis. Conclusions: WWP2 assumes a critical role in safeguarding the heart against injury induced by sepsis via regulating FACL4 to inhibit LPS-induced cardiomyocytes ferroptosis.

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The multidomain protein BAG3 exerts pleiotropic oncogenic functions in many tumor entities including glioblastoma (GBM). Here, we compared BAG3 protein-protein interactions in either adherently cultured or stem-like cultured U251 GBM cells. In line with BAG3's putative role in regulating stem-like properties, identified interactors in sphere-cultured cells included different stem cell markers (SOX2, OLIG2, and NES), while interactomes of adherent BAG3-proficient cells indicated a shift toward involvement of BAG3 in regulation of cilium assembly (ACTR3 and ARL3). Applying a set of BAG3 deletion constructs we could demonstrate that none of the domains except the WW domain are required for suppression of cilia formation by full-length BAG3 in U251 and U343 cells. In line with the established regulation of the Hippo pathway by this domain, we could show that the WW mutant fails to rescue YAP1 nuclear translocation. BAG3 depletion reduced activation of a YAP1/AURKA signaling pathway and induction of PLK1. Collectively, our findings point to a complex interaction network of BAG3 with several pathways regulating cilia homeostasis, involving processes related to ciliogenesis and cilium degradation.

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Contact inhibition (CI) represents a crucial tumor-suppressive mechanism responsible for controlling the unbridled growth of cells, thus preventing the formation of cancerous tissues. CI can be further categorized into two distinct yet interrelated components: CI of locomotion (CIL) and CI of proliferation (CIP). These two components of CI have historically been viewed as separate processes, but emerging research suggests that they may be regulated by both distinct and shared pathways. Specifically, recent studies have indicated that both CIP and CIL utilize mechanotransduction pathways, a process that involves cells sensing and responding to mechanical forces. This review article describes the role of mechanotransduction in CI, shedding light on how mechanical forces regulate CIL and CIP. Emphasis is placed on filamin A (FLNA)-mediated mechanotransduction, elucidating how FLNA senses mechanical forces and translates them into crucial biochemical signals that regulate cell locomotion and proliferation. In addition to FLNA, trans-acting factors (TAFs), which are proteins or regulatory RNAs capable of directly or indirectly binding to specific DNA sequences in distant genes to regulate gene expression, emerge as sensitive players in both the mechanotransduction and signaling pathways of CI. This article presents methods for identifying these TAF proteins and profiling the associated changes in chromatin structure, offering valuable insights into CI and other biological functions mediated by mechanotransduction. Finally, it addresses unanswered research questions in these fields and delineates their possible future directions.

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The design of discrete ß-sheet peptides is far less advanced than e. g. the design of α-helical peptides. The reputation of ß-sheet peptides as being poorly soluble and aggregation-prone often hinders active design efforts. Here, we show that this reputation is unfounded. We demonstrate this by looking at the ß-hairpin and WW domain. Their structure and folding have been extensively studied and they have long served as model systems to investigate protein folding and folding kinetics. The resulting fundamental understanding has led to the development of hyperstable ß-sheet scaffolds that fold at temperatures of 100 °C or high concentrations of denaturants. These have been used to design functional miniproteins with protein or nucleic acid binding properties, in some cases with such success that medical applications are conceivable. The ß-sheet scaffolds are not always completely rigid, but can be specifically designed to respond to changes in pH, redox potential or presence of metal ions. Some engineered ß-sheet peptides also exhibit catalytic properties, although not comparable to those of natural proteins. Previous reviews have focused on the design of stably folded and non-aggregating ß-sheet sequences. In our review, we now also address design strategies to obtain functional miniproteins from ß-sheet folding motifs.

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Formins are a group of actin-binding proteins that mediate nascent actin filament polymerization, filament elongation, and barbed end-capping function, thereby regulating different cellular and developmental processes. Developmental processes like vertebrate gastrulation, neural growth cone dynamics, and limb development require formins functioning in a regulated manner. Formin-binding proteins like Rho GTPase regulate the activation of auto-inhibited conformation of diaphanous formins. Unlike other diaphanous formins, Formin1 (FMN1) a non-diaphanous formin is not regulated by Rho GTPase. FMN1 acts as an antagonist of the Bone Morphogenetic Protein (BMP) signaling pathway during limb development. Several previous reports demonstrated that WW domain-containing proteins can interact with poly-proline-rich amino acid stretches of formins and play a crucial role in developmental processes. In contrast, WW domain-containing Formin-binding Protein 4 (FNBP4) protein plays an essential role in limb development. It has been hypothesized that the interaction between FNBP4 and FMN1 can further attribute to the role in limb development through the BMP signaling pathway. In this study, we have elucidated the binding kinetics of FNBP4 and FMN1 using surface plasmon resonance (SPR) and enzyme-linked immunosorbent assays (ELISA). Our findings confirm that the FNBP4 exhibits interaction with the poly-proline-rich formin homology 1 (FH1) domain of FMN1. Furthermore, only the first WW1 domains are involved in the interaction between the two domains. Thus, this study sheds light on the binding potentialities of WW domains of FNBP4 that might contribute to the regulation of FMN1 function.

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Objective To investigate the expression level and clinical significance of WW domain-containing E3 ubiquitin protein ligase 1(WWP1)and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)in patients with heart failure with preserved ejection fraction(HFpEF).Methods A total of 153 patients with HFpEF admitted to Fengfeng General Hospital of North China Medical and Health Group from January 2021 to September 2022 were collected as the observation group.According to the New York Heart Association(NYHA)cardiac function grading of patients,they were grouped into cardiac function grading Ⅰ～Ⅱ group(n=64)and cardiac function grading Ⅲ～Ⅳ group(n=89),while 148 healthy volunteers were collected as the control group.The correlation between serum WWP1 and NLRP3 levels and cardiac function indexes of patients was explored by Pearson analysis.The diagnostic value of serum WWP1 and NLRP3 levels on the severity of heart failure in HFpEF patients was analyzed by the receiver operating characteristic(ROC)curve.Results Compared with the control group,the expression levels of WWP1(1.68±0.35 vs 1.04±0.19)and NLRP3(6.72±1.26 ng/ml vs 4.57±0.84 ng/ml)in the observation group were significantly increased,and the differences were statistically significant(t=19.623,17.359,all P<0.05).Compared with grade Ⅰ to Ⅱ groups,WWP1(1.87±0.39 vs 1.42±0.32)and NLRP3(7.53±1.40 ng/ml vs 5.59±1.18 ng/ml)expression levels in grade Ⅲ to Ⅳ groups were significantly increased and the differences were statistically significant(t=7.744,9.017,all P<0.05).The differences of heart rate,left atrial diameter(LAD),left ventricular end-diastolic diameter(LVEDD),left ventricular end-diastolic diameter(LVEDD),left atrial diameter(LAD),left ventricular end-diastolic diameter(LVEDD),left ventricular end-diastolic posterior wall thickness(LVPWT),left ventricular ejection fraction(LVPWT),left ventricular ejection fraction(LVEF),peak mitral early diastolic velocity(E)/peak late diastolic velocity(A)and the incidence of atrial fibrillation between the cardiac function grade Ⅰ to Ⅱ groups and the grade Ⅲ to Ⅳ groups were significant(t/χ2=2.757～7.069,all P<0.05).Serum WWP1 level in HFpEF patients was positively correlated with LAD,LVEDD and LVPWT(r=0.547,0.471,0.536,all P<0.05),and negatively correlated with LVEF and E/A(r=-0.485,-0.417,all P<0.05).Serum NLRP3 level was positively correlated with LAD,LVEDD and LVPWT(r=0.534,0.494,0.520,all P<0.05),and negatively correlated with LVEF and E/A(r=-0.462,-0.523,all P<0.05).ROC results showed that the area under the curve(AUC)of serum WWP1 and NLRP3 levels alone for diagnosing the severity of heart failure in HFpEF patients was 0.825 and 0.855,respectively,and the AUC(0.924)diagnosed by the combination of the two was significantly greater than that diagnosed by the serum WWP1 alone and the AUC diagnosed by the NLRP3 alone(Z=3.600,P<0.001;Z=3.053,P=0.002).Conclusion The levels of serum WWP1 and NLRP3 were increased in patients with HFpEF,which were closely related to the cardiac function of patients.Serum WWP1 and NLRP3 have certain diagnostic value for the severity of heart failure in patients with HFpEF.

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Constitutive androstane receptor (CAR), a nuclear receptor predominantly expressed in the liver, is activated by diverse chemicals and induces hepatocyte proliferation and hepatocarcinogenesis in rodents. However, the underlying mechanism responsible for CAR-dependent hepatocyte proliferation remains unclear. Importantly, this phenomenon has not been observed in the human liver. This study aimed to investigate the molecular mechanism underlying CAR-induced hepatocyte proliferation and to explore the species differences in hepatocyte proliferation between humans and rodents. Treatment of mice with the CAR activator TCPOBOP induced hepatocyte proliferation and nuclear accumulation of yes-associated protein (YAP), a known liver cancer inducer. This induction was abolished in CAR-knockout mice. Exogenously expressed YAP in cultured cells was accumulated in the nucleus by the coexpression with mouse CAR but not human CAR. Pull-down analysis of recombinant proteins revealed that mouse CAR interacted with YAP, whereas human CAR did not. Further investigations using YAP deletion mutants identified the WW domain of YAP as essential for interacting with CAR and showed that the PY motif (PPAY) in mouse CAR was crucial for binding to the WW domain, whereas human CAR with its mutated motif (PPAH) failed to interact with YAP. A mouse model harboring the Y150H mutation (PPAY to PPAH) in CAR displayed drastically attenuated TCPOBOP-induced hepatocyte proliferation and nuclear accumulation of YAP. CAR induces the nuclear accumulation of YAP through the PY motif-WW domain interaction to promote hepatocyte proliferation. The absence of this interaction in human CAR contributes to the lack of CAR-dependent hepatocyte proliferation in human livers.

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The design of metallo-miniproteins advances our understanding of the structural and functional roles of metals in proteins. We recently designed a metal-binding WW domain, WW-CA-Nle, which displays three histidine residues on its surface for coordination of divalent metals Ni(II), Zn(II) and Cu(II). However, WW-CA-Nle is a molten globule in the apo state and thus showed only moderate binding affinities with Kd values in the µM regime. In this report, we hypothesize that improved thermal stability of the apo state of the metal binding WW-domain scaffold should lead to improved preorganization of the metal-binding site and consequently to higher metal-binding affinities. By redesigning WW-CA-Nle, we obtained WW-CA variants, WW-CA-min and WW-CA-ANG, which were fully folded in the apo states and displayed moderate to excellent thermostabilities in the apo and holo states. We were able to show that the improved thermal stabilities led to improved metal binding, which was reflected in Kd values that were at least one order of magnitude lower compared to WW-CA-Nle. EPR spectroscopy and ITC measurements revealed a better defined and predisposed metal binding site in WW-CA-ANG.

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Proteins gain optimal fitness such as foldability and function through evolutionary selection. However, classical studies have found that evolutionarily designed protein sequences alone cannot guarantee foldability, or at least not without considering local contacts associated with the initial folding steps. We previously showed that foldability and function can be restored by removing frustration in the folding energy landscape of a model WW domain protein, CC16, which was designed based on Statistical Coupling Analysis (SCA). Substitutions ensuring the formation of five local contacts identified as "on-path" were selected using the closest homolog native folded sequence, N21. Surprisingly, the resulting sequence, CC16-N21, bound to Group I peptides, while N21 did not. Here, we identified single-point mutations that enable N21 to bind a Group I peptide ligand through structure and dynamic-based computational design. Comparison of the docked position of the CC16-N21/ligand complex with the N21 structure showed that residues at positions 9 and 19 are important for peptide binding, whereas the dynamic profiles identified position 10 as allosterically coupled to the binding site and exhibiting different dynamics between N21 and CC16-N21. We found that swapping these positions in N21 with matched residues from CC16-N21 recovers nature-like binding affinity to N21. This study validates the use of dynamic profiles as guiding principles for affecting the binding affinity of small proteins.

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Aim To study the reversal effect of albiflorin(AL)on multidrug resistance of human ovarian cancer and the potential mechanism. Methods The drug resistance reversal effect of AL on SKOV3/DDP cells was detected by CCK-8 kit,and the effect of AL on P-glycoprotein(P-gp)function was detected by flow cytometry. The effects of AL on MYC,WWP1 and ABCB1 in SKOV3/DDP cells were detected by RT-qPCR and Western blot. The MYC-knockdown SKOV3/DDP cell line was constructed by RNA interference technology,and its drug resistance,P-gp function and related gene and protein expression changes were investigated. Results AL had a drug resistance reversal effect on SKOV3/DDP cells and a concentration-dependent inhibitory effect on P-gp function. The inhibitory effects of AL 25,50 and 100 μmol·L-1 on ABCB1/P-gp,MYC and WWP1 were gradually enhanced. The inhibitory effect of MYCi975,a MYC inhibitor,on ABCB1/P-gp,MYC and WWP1 was stronger than or equivalent to that of AL 100 μmol·L-1 group. After knockdown of MYC in SKOV3/DDP cells,cell drug resistance,P-gp function,and related gene and protein expression were inhibited. Conclusions The drug resistance reversal effect of AL on SKOV3/DDP cells may be related to the inhibition of P-gp function and the expression of ABCB1/P-gp,MYC and WWP1,which provides an experiment base for the development of AL as a drug resistance reversal agent for the clinical treatment of ovarian cancer.

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The Hippo pathway is an evolutionally conserved signaling cascade that controls organ size and tissue regeneration under physiological conditions, and its aberrations have been well studied to promote tumor initiation and progression. Dysregulation of the Hippo tumor suppressor signaling frequently occurs in gastric cancer (GC) and other solid tumors and contributes to cancer development through modulating multiple aspects, including cell proliferation, survival, metastasis, and oncotherapy resistance. In the clinic, Hippo components also possess diagnostic and prognostic values for cancer patients. Considering its crucial role in driving tumorigenesis, targeting the Hippo pathway may greatly benefit developing novel cancer therapies. This review summarizes the current research progress regarding the core components and regulation of the Hippo pathway, as well as the mechanism and functional roles of their dysregulation in gastrointestinal malignancies, especially in GC, and discusses the therapeutic potential of targeting the Hippo pathway against cancers.

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WW domain-containing oxidoreductase (WWOX) spans the common fragile site FRA16D. There is evidence that translocations and deletions affecting WWOX accompanied by loss of expression are frequent in many cancers and often correlate with a worse prognosis. Additionally, WWOX germline mutations were also found to be the cause of pathologies of brain development. Because WWOX binds to some transcription factors, it is a modulator of many cellular processes, including metabolic processes. Recently, studies have linked WWOX to familial dyslipidemias, osteopenia, metabolic syndrome, and gestational diabetes, confirming its role as a regulator of steroid, cholesterol, glucose, and normal bone metabolism. The WW domain of WWOX is directly engaged in the control of the activity of transcription factors such as HIF1α and RUNX2; therefore, WWOX gene alterations are associated with some metabolic abnormalities. Presently, most interest is devoted to the associations between WWOX and glucose and basic energy metabolism disturbances. In particular, its involvement in the initiation of the Warburg effect in cancer or gestational diabetes and type II diabetes is of interest. This review is aimed at systematically and comprehensively presenting the current state of knowledge about the participation of WWOX in the metabolism of healthy and diseased organisms.

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More than 20 years since its discovery, our understanding of Pin1 function in various diseases continues to improve. Pin1 plays a crucial role in pathogenesis and has been implicated in metabolic disorders, cardiovascular diseases, inflammatory diseases, viral infection, cancer and neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's disease. In particular, the role of Pin1 in neurodegenerative diseases and cancer has been extensively studied. Our understanding of Pin1 in cancer also led to the development of cancer therapeutic drugs targeting Pin1, with some currently in clinical trial phases. However, identifying a Pin1-specific drug with good cancer therapeutic effect remains elusive, thus leading to the continued efforts in Pin1 research. The importance of Pin1 is highlighted by the presence of Pin1 orthologs across various species: from vertebrates to invertebrates and Kingdom Animalia to Plantae. Among these Pin1 orthologs, their sequence and structural similarity demonstrate the presence of conservation. Moreover, their similar functionality between species further highlights the conservancy of Pin1. As researchers continue to unlock the mysteries of Pin1 in various diseases, using different Pin1 models might shed light on how to better target Pin1 for disease therapeutics. This review aims to highlight the various Pin1 orthologs in numerous species and their divergent functional roles. We will examine their sequence and structural similarities and discuss their functional similarities and uniqueness to demonstrate the interconnectivity of Pin1 orthologs in multiple diseases.

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BACKGROUND/AIM: Deletions in the q arm of chromosome 3 have been reported in uterine leiomyomas, also as sole anomalies. Because some neoplasia-associated deletions may give rise to tumorigenic fusion genes, we chose to investigate thoroughly one such tumor. MATERIALS AND METHODS: A uterine leiomyoma obtained from a 45-year-old woman had the karyotype 46,XX,del(3)(q?)[11]. The tumor was further studied using array comparative genomic hybridization, RNA sequencing, reverse transcription polymerase chain reaction, Sanger sequencing, and fluorescence in situ hybridization methodologies. RESULTS: The deletion was shown to be from 3q22.2 to 3q26.32. Unexpectedly, a cryptic balanced t(2;3)(p21;q25) translocation was also found affecting two otherwise normal chromosomes 2 and 3, i.e., the der(3)t(2;3) was not the deleted chromosome 3. The translocation generated two chimeras between the genes WW domain containing transcription regulator 1 (WWTR1) from 3q25.1 and protein kinase C epsilon (PRKCE) from 2p21. The WWTR1::PRKCE fusion would code for a chimeric serine/threonine kinase, whereas the reciprocal PRKCE::WWTR1 fusion would code for a chimeric transcriptional coactivator protein. CONCLUSION: Leiomyomas carrying a deletion on 3q may also have a balanced t(2;3)(p21;q25) leading to fusion of WWTR1 with PRKCE.

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WW domain-containing E3 Ubiquitin-protein ligase 2 (WWP2) has been found to positively regulate odontoblastic differentiation by monoubiquitinating the transcription factor Kruppel-like factor 5 (KLF5) in a cell culture system. However, the in vivo role of WWP2 in mouse teeth remains unknown. To explore this, here we generated Wwp2 knockout (Wwp2 KO) mice. We found that molars in Wwp2 KO mice exhibited thinner dentin, widened predentin, and reduced numbers of dentinal tubules. In addition, expression of the odontoblast differentiation markers Dspp and Dmp1 was decreased in the odontoblast layers of Wwp2 KO mice. These findings demonstrate that WWP2 may facilitate odontoblast differentiation and dentinogenesis. Furthermore, we show for the first time that phosphatase and tensin homolog (PTEN), a tumor suppressor, is expressed in dental papilla cells and odontoblasts of mouse molars and acts as a negative regulator of odontoblastic differentiation. Further investigation indicated that PTEN is targeted by WWP2 for degradation during odontoblastic differentiation. We demonstrate PTEN physically interacts with and inhibits the transcriptional activity of KLF5 on Dspp and Dmp1. Finally, we found WWP2 was able to suppress the interaction between PTEN and KLF5, which diminished the inhibition effect of PTEN on KLF5. Taken together, this study confirms the essential role of WWP2 and the WWP2-PTEN-KLF5 signaling axis in odontoblast differentiation and dentinogenesis in vivo.

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Human KIBRA, a member of the WWC family proteins, is an upstream regulator of the Salvador/Warts/Hippo (SWH) signaling pathway and predominately expressed in nervous system. The protein has two functionally regulatory domains WW1 and WW2 at N-terminal region, which recognize and bind to the PY-motif segments of their partner proteins to serve as a signaling scaffold role in the SWH pathway. The two domains are highly conserved, but their downstream ligands and biological functions may not be fully consistent. In this study, we attempted to systematically profile the PY-motif affinity to and selectivity between KIBRA WW1 and WW2 domains involved in partner recognition sites. Ontology mining was used to enrich the KIBRA-interacting proteins in literature libraries, from which a variety of PY-motif peptide segments were identified, and their binding behavior to each domain was then analyzed by integrating computational modeling and experimental assay. Most PY-motif peptides were found to interact potently with WW1 and WW2, but they generally only exhibit a moderate or modest selectivity between the two domains. Subsequently, several representative peptides were further examined in detail to elucidate the molecular mechanism underlying their affinity and selectivity. It is revealed that the middle motif region of PY-motif peptides is primarily responsible for the affinity and stability of peptide binding, but only contributes marginally to peptide selectivity. Instead, the N-terminal region and, particularly, C-terminal region of PY-motif peptides play a crucial role in the selectivity. Hydrophobic contacts and hydrogen bonds confer stability and specificity to the domain-peptide interaction, respectively.

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Genetic alterations in the WW domain-containing oxidoreductase (WWOX) gene cause autosomal recessive developmental and epileptic encephalopathy, characterized by the onset of refractory seizures in infants, along with severe axial hypotonia and profoundly impaired psychomotor development. It has also been expanded to include metabolism and endocrine systems. Despite its function as a tumor suppressor gene, genetic alterations in WWOX have been found in several metabolic disorders and neural diseases related to brain development. Whole-exome sequencing (WES) was performed on the patient sample. Genomic DNA was fragmented, and the exons of known genes in the human genome, as well as the corresponding exon-intron boundaries,were enriched using Roche KAPA capture technology (KAPA hyperExome Library, WES identifying the homozygous variant c.406A>G in WWOX (OMIM:605131). This variant of WWOX was also observed in the prenatal WES data, indicating that both parents were heterozygous carriers and the detected variant was homozygous. This study highlighted the importance of the human WWOX gene in brain development and the association between WWOX gene mutations and developmental delay. We recommend performing WES as a primary screening before the final diagnosis, particularly in populations with high rates of consanguinity and in clinically challenging cases.

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Radiation therapy is one of the major treatment modalities for patients with cancers. However, ionizing radiation (IR) damages not only cancer cells but also the surrounding vascular endothelial cells (ECs). Hippo pathway effector genes Yap1 and Taz are the two transcriptional coactivators that have crucial roles in tissue homeostasis and vascular integrity in various organs. However, their function in adult ECs at the steady state and after IR is poorly understood. Here, we report sex- and context-dependent roles of endothelial YAP1/TAZ in maintaining vascular integrity and organismal survival. EC-specific Yap1/Taz deletion compromised systemic vascular integrity, resulting in lethal circulation failure preferentially in male mice. Furthermore, EC-specific Yap1/Taz deletion induced acute lethality upon sublethal IR that was closely associated with exacerbated systemic vascular dysfunction and circulation failure. Consistent with these findings, RNA-seq analysis revealed downregulation of tight junction genes in Yap1/Taz-deleted ECs. Collectively, our findings highlight the importance of endothelial YAP1/TAZ for maintaining adult vascular function, which may provide clinical implications for preventing organ injury after radiation therapy.

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