Tetratricopeptide Repeat Protein Research Articles

Abstract B018: KDM6A and MTAP expression loss enable identification of a large fraction of low grade non-invasive urothelial neoplasia of the urinary bladder

Abstract The identification of low-grade urothelial neoplasia in cytologic samples or in biopsies with dysplastic lesions poses a particular problem for pathologists in routine diagnostics. Improved diagnosis of low-grade urothelial neoplasia could help to reduce the number of cystoscopic follow-up examinations. KDM6A, also known as UTX (ubiquitously transcribed X chromosome tetratricopeptide repeat protein) is an epigenetic regulator which is frequently mutated in urothelial carcinoma. Because KDM6A is localized on chromosome Xp11.3, and because of the male predominance in bladder cancer, many KDM6A mutations should result in a complete loss of KDM6A protein expression and become detectable by immunohistochemistry (IHC). Similarly, S-methyl-5′-thioadenosine phosphorylase (MTAP), a critical enzyme for DNA and RNA synthesis, is co-deleted with the CDKN2A tumor suppressor located on chromosome 9p21 in about 25% of bladder cancers, resulting in reduced or lost protein expression. To study the prevalence and diagnostic potential of immunohistochemical loss of KDM6A and MTAP expression, 928 tumors were analyzed by IHC in a tissue microarray format. The cohort included 628 non-invasive papillary tumors (pTa) and 300 tumors from patients who underwent radical cystectomy for muscle-invasive disease (pT2-4). A complete KDM6A expression loss occurred in 35% of 342 pTa G2 low-grade, 23% of 150 pTa G2 high-grade, 17.8% of 90 pTa G3 tumors, and 20.1% of 243 interpretable muscle invasive (pT2-4) cancers (p<0.0001). Within pTa tumors, there was a strong correlation between KDM6A loss and low tumor grade (p=0.0003), whereas no significant difference was found between pT3 G3 and muscle invasive cancers (p=0.8828). Complete absence of MTAP expression occurred in 11% of 297 pTa G2 low-grade, 29.6% of 125 pTa G2 high-grade, 18.4% of 76 pTa G3 tumors, and 26.3% of 217 muscle invasive (pT2-4) cancers (p<0.0001). A combined analysis of 702 cancers with interpretable data for KDM6A and MTAP showed that 6.6% of tumors had expression loss of both KDM6A and MTAP, 20% had KDM6A loss only, and 13% had MTAP loss only. The rate of expression loss of KDM6A and/or MTAP was markedly higher in pTa G2 low (41.2%) and in pTa G2 high (40.3%) than in pTa G3 tumors (29%). In summary, our data demonstrate that about 40% of low-grade non-invasive urothelial neoplasias – which are usually not detected by urine cytology – had a completed expression loss of either KDM6A or MTAP. In patients with these alterations, follow-up could be potentially facilitated by immunohistochemistry. Citation Format: Florian Viehweger, Neele Heckmann, Natalia Gorbokon, Henning Plage, Sebastian Hofbauer, Sarah Weinberger, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Maximilian Lennartz, Tim Mandelkow, Elena Bady, Margit Fisch, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Tobias Klatte, Thorsten Schlomm, David Horst, Henrik Zecha, Martina Kluth, Sarah Minner. KDM6A and MTAP expression loss enable identification of a large fraction of low grade non-invasive urothelial neoplasia of the urinary bladder [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B018.

Multi-omic Analysis of Uterine Leiomyomas in Self-Described Black and White Women: Molecular Insights into Health Disparities

Black women are at an increased risk to develop uterine leiomyomas (ULMs) and to experience worse disease prognosis compared to White women. Epidemiological and molecular factors have been identified as underlying these disparities, but there remains a paucity of deep, multi-omic analysis investigating molecular differences in ULMs from Black and White patients. To identify molecular alterations within ULM tissues correlating with patient race by multi-omic analyses of ULMs collected from cohorts of Black and White women. We performed multi-omic analysis of ULMs from Black (42) and White (47) women undergoing hysterectomy for symptomatic uterine leiomyomata. Our analysis also included application of orthogonal methods to evaluate fibroid biomechanical properties, such as second harmonic generation microscopy, uniaxial compression testing, as well as sheer wave ultrasonography analyses. We found a greater proportion of mediator complex subunit 12 (MED12) mutant ULMs from Black women (>35% increase, Mann Whitney U p = 7E-4). MED12 mutant tumors exhibited elevated abundance of extracellular matrix proteins, including several collagen isoforms, involved in regulation of the core matrisome. Histological analysis of tissue fibrosis using trichrome staining and secondary harmonic generation microscopy confirmed that MED12 mutant tumors are more fibrotic than MED12 wildtype tumors. Using Sheer Wave ultrasonography in a prospectively collected cohort, Black patients had fibroids that were firmer when compared to White patients, even when similar in size. These analyses also uncovered expression quantitative trait loci (eQTL) associated with altered allele frequencies in African and European populations that correlated with differential abundance of several proteins in ULM that are independent of MED12 mutational status, including multiple eQTL mapping to tetratricopeptide repeat protein 38. Our study shows that Black women have a higher prevalence of ULMs harboring mutations in MED12 and that this mutational status correlates with increased tissue fibrosis in comparison with wildtype ULMs. Our study provides insights into molecular alterations underlying racial disparities in ULMs and improves our understanding of the molecular etiology underlying ULM development within these populations.

Diverse crystalline protein scaffolds through metal-dependent polymorphism.

As protein crystals are increasingly finding diverse applications as scaffolds, controlled crystal polymorphism presents a facile strategy to form crystalline assemblies with controllable porosity with minimal to no protein engineering. Polymorphs of consensus tetratricopeptide repeat proteins with varying porosity were obtained through co-crystallization with metal salts, exploiting the innate metal ion geometric requirements. A single structurally exposed negative amino acid cluster was responsible for metal coordination, despite the abundance of negatively charged residues. Density functional theory calculations showed that while most of the crystals were the most thermodynamically stable assemblies, some were kinetically trapped states. Thus, crystalline porosity diversity is achieved and controlled with metal coordination, opening a new scope in the application of proteins as biocompatible protein-metal-organic frameworks (POFs). In addition, metal-dependent polymorphic crystals allow direct comparison of metal coordination preferences.

Abstract 6449: KDM6A expression loss is a common feature in low grade non-invasive urothelial carcinomas of the urinary bladder

Abstract KDM6A, also known as UTX (ubiquitously transcribed X chromosome tetratricopeptide repeat protein) is an epigenetic regulator which is frequently mutated in urothelial carcinoma. Because KDM6A loss causes a dependency on EZH2, a potential therapeutic target, KDM6A analysis may have therapeutic importance. Most data on KDM6A mutations are derived from next generation sequencing (NGS). However, because of the combination of a high rate of truncating KDM6A mutations, the localization of KDM6A on chromosome Xp11.3, and the male predominance in bladder cancer, many KDM6A mutations should result in a complete loss of KDM6A protein expression and become detectable by immunohistochemistry (IHC). To study the prevalence and the potential diagnostic and clinical role of KDM6A expression loss, more than 2,100 tumors were analyzed by IHC in a tissue microarray format. The cohort included 1,128 patients who underwent radical cystectomy for muscle-invasive disease (pT2-4). A complete KDM6A expression loss occurred in 36% of 350 pTa G2 low-grade, 23% of 152 pTa G2 high-grade, and 18.5% of 92 pTa G3 tumors (p=0.0002). As compared to pTa G3 tumors, the frequency of KDM6A expression loss did not differ significantly in pT2 (17.2%), pT3 (21.9%), and pT4 (18.2%) cancers. Within pT2-4 carcinomas, KDM6A staining was unrelated to pT, pN, grade, L-status, V-status, overall survival, recurrence-free survival, and cancer-specific survival (p≥0.18 each). A KDM6A loss was more common in male (22.2%) than in female patients (15.4%; p<0,0001), and within male patients, a KDM6A loss was more frequent in tumors with Y-chromosome loss (36.1%) than in cancers without a Y-chromosome loss (16.3%; p<0.0001). KDM6A expression loss was significantly associated with increased p63 expression (p<0.0001) and high uroplakin 1b staining (p=0.0013) but unrelated to CK20, GATA3, UpK1b, and p53 staining. In summary, our data demonstrate that truncating KDM6A mutations occur in a significant subset of urothelial carcinomas which is linked to low-grade non-invasive cancer, male gender and loss of the Y chromosome. The predominance of KDM6A loss in low grade tumors makes KDM6A IHC a promising new tool for identification of low grade dysplasia in biopsies and early bladder cancer detection in cytology. Citation Format: Florian Viehweger, Natalia Gorbokon, Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberger, Bernhard Ralla, Antonia Franz, Annika Fendler, Michela de Martino, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Maximilian Lennartz, Tim Mandelkow, Elena Bady, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Joachim Weischenfeldt, Tobias Klatte, Thorsten Schlomm, David Horst, Henrik Zecha, Martina Kluth, Sarah Minner. KDM6A expression loss is a common feature in low grade non-invasive urothelial carcinomas of the urinary bladder [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6449.

Abstract 1049: Prevalence of KDM6A expression loss in human cancer: A tissue microarray study on 6,560 cancers from 114 different tumor types

Abstract KDM6A, also known as UTX (ubiquitously transcribed X chromosome tetratricopeptide repeat protein) is an epigenetic regulator constituting a critical part of the COMPASS-like complex. KDM6A mutations occur commonly in cancer. These are of interest because KDM6A loss results in a dependency on EZH2, a potential therapeutic target. Most data on KDM6A have been obtained by next generation sequencing (NGS). However, since most KDM6A mutations are truncating and KDM6A is located on the X chromosome, many mutations result in a complete expression loss which can be detected by immunohistochemistry (IHC). To learn more on the prevalence of KDM6A expression loss and its role in cancer, a tissue microarray containing 6,560 samples from 114 different tumor entities and 608 samples of 76 different normal tissue types was analyzed. In normal tissues, KDM6A staining was rather ubiquitously seen in nuclei. In cancer, KDM6A staining was considered weak (1+) in 581 (11.2%), moderate in 1,927 (37%), and strong in 2,362 (45.4%) of 5,206 interpretable tumors. A complete loss of KDM6A staining occurred in 336 (6.5%) of tumors. At least one case with a complete KDM6A expression loss was observed in 51 of 114 tumor categories. KDM6A staining was most commonly lost in different categories of urothelial carcinomas (19.5-39.2%), squamous cell carcinomas of the esophagus (27.1%), hepatocellular carcinoma (18.4%), gastric carcinoma (3.2-12.5%), papillary renal cell carcinoma (9.5%), adenocarcinoma of the prostate (5.9-9.4%), chromophobe renal cell carcinoma (8.9%), Ewing sarcoma (8.3%), pheochromocytoma (8.3%), and metastatic malignant melanoma (8%). A KDM6A expression loss in less than 8% of cases was observed in 32 further tumor entities. KDM6A expression loss was more than twice as frequent in tumors from male (8.4% of 2,526) than in tumors from female patients (3.9% of 2,024; p<0.0001). It is concluded that KDM6A IHC is a suitable method for the detection of truncating KDM6A mutations, especially in males. These make up for about 2/3 of all KDM6A mutations and may exert specific biological effects than differ from the various KDM6A missense mutations known to also occur. Citation Format: Florian Viehweger, Natalia Gorbokon, Maximilian Lennartz, Viktor Reiswich, Florian Lutz, Till Krech, Andreas M. Luebke, Claudia Hube-Magg, Guido Sauter, Ronald Simon, Stefan Steurer, Andreas H. Marx, Christian Bernreuther, Martina Kluth, Sarah Minner. Prevalence of KDM6A expression loss in human cancer: A tissue microarray study on 6,560 cancers from 114 different tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1049.

A pan-cancer analysis of TTC9A expression level and its correlation with prognosis and immune microenvironment

To investigate the expression level of tetratricopeptide repeat protein 9A in tumors and its association with the patients' prognosis and immune infiltration. TTC9A expression in different tumor tissues and its association with prognosis, DNA methylation, tumor mutation burden (TMB), and microsatellite instability (MSI) were analyzed based on data from TCGA and GTEx. TIMER and xCell were used to analyze the relationship between TTC9A expression and immune infiltration. Western blotting and RT-qPCR were used to detect the expression of TTC9A in 4 types of cancer cell lines. TTC9A expressions were significantly increased in many tumors and down-regulated in a few cancer types (P < 0.05). Western blotting and RT-qPCR showed that TTC9A expressions were elevated in lung, colon and liver cancer cells but decreased in bladder cancer cells. In head and neck squamous cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, low-grade glioma, malignant mesothelioma, and endometrial carcinoma tumors, a high expression of TTC9A was strongly correlated with better overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) (P < 0.05), but was correlated with worse OS, DSS, and PFI in lung adenocarcinoma, pancreatic adenocarcinoma, adrenal carcinoma, and rectal adenocarcinoma (P < 0.05). TTC9A hypermethylation was associated with a more favorable prognosis of glioblastoma multiforme, low- grade glioma, uveal melanoma, and ovarian plasmacytoid cystadenocarcinoma (P < 0.05) but with poor prognosis of squamous cell carcinoma of the uterine cervix and intracervical adenocarcinoma, squamous cell carcinoma of head and neck, squamous cell carcinoma of the lungs, adrenal carcinoma, and endometrial carcinoma (P < 0.05). In most of the cancer types, TTC9A was significantly correlated with the level of immune cell infiltration (P < 0.05). TTC9A can be used as a prognostic marker for a variety of cancers and is strongly associated with TBM, MSI and immune cell infiltration.

Comparative Analysis of TPR Gene Family in Cucurbitaceae and Expression Profiling under Abiotic Stress in Cucumis melo L.

Tetratricopeptide repeat (TPR) proteins play numerous roles in plant growth and development by mediating protein–protein interactions in biological systems by binding to peptide ligands. Although genome-wide analyses of the TPR gene family in other species have been performed, its evolution and function in Cucurbitaceae remain unclear. In this study, 144 TPR genes from 11 genomes of eight Cucurbitaceae species with a heterogeneous distribution on the chromosomes were characterized. Based on the homology between Cucurbitaceae and Arabidopsis, the TPR genes were divided into four groups, and the evolutionary relationships of the Benincaceae and Cucurbitaceae tribes were also represented in a phylogenetic tree. Using the ‘DHL92′ genome as a reference, an integrated chromosome map was obtained containing 34 loci, 4 of which were common to the Cucurbitaceae. Cis-regulatory element analysis showed that these elements are essential for melon development and responses to light, phytohormones, and various stresses. CmTPR tissue- and development-specific expression analysis revealed differential expression patterns under normal growth conditions. Furthermore, the CmTPR genes responded to various abiotic stressors. Overall, this study offers insights into the evolutionary history of the TPR gene family in Cucurbitaceae and provides valuable information for elucidating the potential role of CmTPR genes during development and under different stresses in melon.

Engineered Conductive Proteins for Supercapacitors

Proteins can be used as building blocks of functional materials for electrochemical applications due to their structural properties and the possibility to tailor their ionic and electronic conductive properties.[1] One of the most attractive applications is in the bioelectronics field, for example as energy storage devices such as Electrical Double Layer Capacitors (EDLCs), due to the nontoxicity and biocompatibility of the materials.[2] EDLCs are primarily used for applications where high power is required during a short period of time. They present several advantages, such as fast charge-discharge cycling, high power density, and outstanding long-term stability.[3]Traditionally, the incorporation of biomaterials in EDCLs is limited because they can be denaturalized in contact with organic solvents or salts. In addition, they easily interact with other cell components resulting in parasitic reactions and their complex purification processes hinder their commercialization.[4] These drawbacks can be overcome by introducing engineered proteins, since their amino acid sequences can be tuned resulting in 3D structures with varying degrees of complexity. Based on this approach, engineered proteins with improved conductivity will be introduced as interlayers with good stability and wettability, to ensure that the ions can shuttle freely between the two electrodes.In our case, consensus tetratricopeptide repeat (CTPR) proteins have been chosen [5] and engineered with the aim of increasing the ionic conductivity of the resulting variants. CTPR variants have been mixed with 5% PEG solutions and drop cast to obtain self-standing films. After being cross-linked, the films have been sandwiched in a two electrodes Swagelok-cell configuration between two electrodes made of activated carbon. KCl (3M) aqueous solution has been used as electrolyte for evaluating the electrochemical performance of the devices.The electrochemical characterization of the EDLC devices has been carried out using cyclic voltammetry (CV) and galvanostatic cycling (GC) to study the effect of the of the engineering strategy of the CTPR proteins on the EDLC performances. The CV curves of the devices using thin films of proteins with distinct mutations show quadratic capacitive responses for all the assembled EDLCs, demonstrating both its ability to isolate the two electrodes and to allow the diffusion of ions. The EDLCs capacitances were calculated and analyzed from the GC characteristics at different charge-discharge currents and compared with commercial glass fiber separators confirming their feasibility as safe and environmentally friendly separators. The ionic diffusive behavior of the devices has been also studied by electrochemical impedance spectroscopy (EIS). Finally, the effect of the thickness, porosity, polarity, wettability, and ionic conductivity of the different protein separators has been addressed to explore the role of the engineered proteins in EDLC devices. Acknowledgements This abstract is part of R&D&I project TED2021-131641B-C44, funded by MCIN/AEI/10.13039/501100011033 and by European Union NextGenerationEU/PRTR. This project has received funding from the European Union’s Horizon 2020 FET Open under the grant agreement No: 964593

The CD40/CD40 ligand dyad and its downstream effector molecule ISG54 in relating acute neuroinflammation with persistent, progressive demyelination.

Although Multiple Sclerosis (MS) is primarily thought to be an autoimmune condition, its possible viral etiology must be taken into consideration. When mice are administered neurotropic viruses like mouse hepatitis virus MHV-A59, a murine coronavirus, or its isogenic recombinant strain RSA59, neuroinflammation along with demyelination are observed, which are some of the significant manifestations of MS. MHV-A59/RSA59 induced neuroinflammation is one of the best-studied experimental animal models to understand the viral-induced demyelination concurrent with axonal loss. In this experimental animal model, one of the major immune checkpoint regulators is the CD40-CD40L dyad, which helps in mediating both acute-innate, innate-adaptive, and chronic-adaptive immune responses. Hence, they are essential in reducing acute neuroinflammation and chronic progressive adaptive demyelination. While CD40 is expressed on antigen-presenting cells and endothelial cells, CD40L is expressed primarily on activated T cells and during severe inflammation on NK cells and mast cells. Experimental evidences revealed that genetic deficiency of both these proteins can lead to deleterious effects in an individual. On the other hand, interferon-stimulated genes (ISGs) possess potent antiviral properties and directly or indirectly alter acute neuroinflammation. In this review, we will discuss the role of an ISG, ISG54, and its tetratricopeptide repeat protein Ifit2; the genetic and experimental studies on the role of CD40 and CD40L in a virus-induced neuroinflammatory demyelination model.

A novel tetratricopeptide-repeat protein, TTP1, forms complexes with glutamyl-tRNA reductase and protochlorophyllide oxidoreductase during tetrapyrrole biosynthesis.

The biosynthesis of the tetrapyrrole end-products chlorophyll and heme depends on a multifaceted control mechanism that acts primarily at the post-translational level upon the rate-limiting step of 5-aminolevulinic acid synthesis and upon light-dependent protochlorophyllide oxidoreductase (POR). These regulatory processes require auxiliary factors that modulate the activity, stability, complex formation, and subplastidal localization of the relevant proteins. Together, they ensure optimal metabolic flow during the day and at night. As an Arabidopsis homolog of the POR-interacting tetratricopeptide-repeat protein (Pitt) first reported in Synechocystis, we characterize tetrapyrrole biosynthesis-regulating tetratricopeptide-repeat protein1 (TTP1). TTP1 is a plastid-localized, membrane-bound factor that interacts with POR, the Mg protoporphyrin monomethylester cyclase CHL27, glutamyl-tRNA reductase (GluTR), GluTR-binding protein, and FLUORESCENCE IN BLUE LIGHT. Lack of TTP1 leads to accumulation of GluTR, enhanced 5-aminolevulinic acid synthesis and lower levels of POR. Knockout mutants show enhanced sensitivity to reactive oxygen species and a slower greening of etiolated seedlings. Based on our studies, the interaction of TTP1 with GluTR and POR does not directly inhibit their enzymatic activity and contribute to the control of 5-aminolevulinic acid synthesis. Instead, we propose that TTP1 sequesters a fraction of these proteins on the thylakoid membrane, and contributes to their stability.

Phytohormones as Regulators of Mitochondrial Gene Expression in Arabidopsis thaliana

The coordination of activities between nuclei and organelles in plant cells involves information exchange, in which phytohormones may play essential roles. Therefore, the dissection of the mechanisms of hormone-related integration between phytohormones and mitochondria is an important and challenging task. Here, we found that inputs from multiple hormones may cause changes in the transcript accumulation of mitochondrial-encoded genes and nuclear genes encoding mitochondrial (mt) proteins. In particular, treatments with exogenous hormones induced changes in the GUS expression in the reporter line possessing a 5′-deletion fragment of the RPOTmp promoter. These changes corresponded in part to the up- or downregulation of RPOTmp in wild-type plants, which affects the transcription of mt-encoded genes, implying that the promoter fragment of the RPOTmp gene is functionally involved in the responses to IAA (indole-3-acetic acid), ACC (1-aminocyclopropane-1-carboxylic acid), and ABA (abscisic acid). Hormone-dependent modulations in the expression of mt-encoded genes can also be mediated through mitochondrial transcription termination factors 15, 17, and 18 of the mTERF family and genes for tetratricopeptide repeat proteins that are coexpressed with mTERF genes, in addition to SWIB5 encoding a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein. These genes specifically respond to hormone treatment, displaying both negative and positive regulation in a context-dependent manner. According to bioinformatic resources, their promoter region possesses putative cis-acting elements involved in responses to phytohormones. Alternatively, the hormone-related transcriptional activity of these genes may be modulated indirectly, which is especially relevant for brassinosteroids (BS). In general, the results of this study indicate that hormones are essential mediators that are able to cause alterations in the transcript accumulation of mt-related nuclear genes, which, in turn, trigger the expression of mt genes.

Targeting Esophageal Squamous Cell Carcinoma by Combining Copper Ionophore Disulfiram and JMJD3/UTX Inhibitor GSK J4.

The alcohol-averse drug disulfiram has been reported to have anti-tumor effects and is well suited for drug combinations. In order to identify potential drug combinations in esophageal squamous cell carcinoma (ESCC), we screened a bioactive compound library with the disulfiram copper chelation product CuET. The Jumonji domain-containing protein 3 (JMJD3) and the ubiquitously transcribed tetratricopeptide repeat protein X-linked (UTX) inhibitor GSK J4 were identified. To further understand the molecular mechanism underlying the efficient drug combination, we applied quantitative mass spectrometry to analyze the signaling pathway perturbation after drug treatment. The data revealed that the synergistic effect of GSK J4 and CuET was due to the interaction among JMJD3 and UTX, which may play important roles in maintaining endoplasmic reticulum (ER) homeostasis in tumor cells. Interestingly, our clinical data analysis showed that high expression of JMJD3 and UTX was associated with T stage and worse prognosis of ESCC patients, further supporting the importance of the above findings. In conclusion, our findings suggest that the combination of CuET and targeting JMJD3/UTX may be a safe, effective, and available treatment for ESCC.

Cross-scale design of energy dissipative composites using self-repairing interfaces based on sacrificial bonds

New composites with high energy dissipation and self-healing properties are required for structural materials, textiles, and protective equipment. This paper proposes a cross-scale strategy to design sacrificial bond composites (SBCs) using non-linear adhesive materials, like self-assembled proteins or mechanical adhesives, placed between opposite-facing magnets. Upon external loads, SBCs effectively dissipate deformation energy across their sacrificial bond interfaces following a biomimetic toughening mechanism similar to nacre’s. When the external load breaks the sacrificial bonds of a SBC, the opposite-facing magnets brings together the separated interface, allowing the reforming of its sacrificial bonds and the self-repairing of the composite after sustaining large strains. After mechanical failure at 600% strain, the consensus tetratricopeptide repeat (CTPR) protein films allows protein-based SBCs to recover 70% of their original tensile strength after letting their sacrificial bonds to reassemble for 1 h, at room temperature, in the presence of moisture. Mechanical adhesive-based SBCs, after their mechanical failure at 325% strain, are able to self-repair faster, regaining 85% of their tensile strength in less than 1 s. As a proof of concept, we demonstrate the fabrication of a reusable and lightweight fall arrest system exploiting mechanical adhesive interfaces and a protein-polyester yarn for the creation of high-energy dissipating textiles.

TTC6-Mediated Stabilization of the Flagellum Annulus Ensures the Rapid and Directed Motion of Sperm.

Sperm motility and structural integrity are essential for successful fertilization in vivo, and any hindrance of the correct assembly of the axoneme and peri-axonemal structures in the sperm flagellum can lead to fertility problems. While there has been considerable advancement in studying diseases related to the flagellum, the underlying mechanisms that control sperm movement are not yet fully understood. In this study, we reveal that the tetratricopeptide repeat protein 6 (Ttc6) gene, expressed mainly in the testes, plays a crucial role in maintaining male fertility in mice. We further demonstrate that the knockout of Ttc6 in mice results in decreased sperm motility and induces an abnormal circular swimming pattern, consequently leading to male subfertility. Morphological analysis showed an atypical hairpin-like appearance of the spermatozoa, and ultrastructural studies showed unsheathed flagella at the juncture between the midpiece and principal piece. Collectively, these findings suggest that TTC6 plays an essential role in maintaining the stability of the annulus region of the sperm flagellum, thus ensuring the swift and directed motion of sperm.

DTtc1, a conserved tetratricopeptide repeat protein, is required for maturation of Drosophila egg chambers via its role in stabilizing electron transport chain components.

We recently identified the Drosophila ortholog of TTC1 (dTtc1) as an interacting partner of Egalitarian, an RNA adaptor of the Dynein motor. In order to better understand the function of this relatively uncharacterized protein, we depleted dTtc1 in the Drosophila female germline. Depletion of dTtc1 resulted in defective oogenesis and no mature eggs were produced. A closer examination revealed that mRNA cargoes normally transported by Dynein were relatively unaffected. However, mitochondria in dTtc1 depleted egg chambers displayed an extremely swollen phenotype. Ultrastructural analysis revealed a lack of cristae. These phenotypes were not observed upon disruption of Dynein. Thus, this function of dTtc1 is likely to be Dynein independent. Consistent with a role for dTtc1 in mitochondrial biology, a published proteomics screen revealed that dTtc1 interacts with numerous components of electron transport chain (ETC) complexes. Our results indicate that the expression level of several of these ETC components was significantly reduced upon depletion of dTtc1. Importantly, this phenotype was completely rescued upon expression of wild-type GFP-dTtc1 in the depleted background. Lastly, we demonstrate that the mitochondrial phenotype caused by a lack of dTtc1 is not restricted to the germline but is also observed in somatic tissues. Our model suggests that dTtc1, likely in combination with cytoplasmic chaperones, is required for stabilizing ETC components.

An Emerging Nanozyme Class for à la carte Enzymatic‐Like Activities based on Protein‐Metal Nanocluster Hybrids

AbstractIn this study, the goal is to fabricate robust and highly efficient peroxidase‐like nanozymes that can ultimately be assembled into films for their easy reuse in catalytic cycles. Nanozymes are designed by mimicking the strategy adopted by metalloproteins to accommodate metal cofactors within their protein structure. The engineered consensus tetratricopeptide repeat (CTPR) protein module is selected as the scaffold to guide the growth and the stabilization of a library of in situ synthesized metal nanoclusters. A deep investigation of the interplay between the composition and function of the nanozymes reveals the impact of the protein templates and nanocluster composition on the peroxidase‐like activity of the hybrids. Moreover, among a total of 24 hybrids, a top‐performing nanozyme results from the growth of Au/Pt bimetallic nanoclusters on a CTPR protein with engineered histidine coordination sites. These nanozymes exhibit improved thermostability and resistance to hydrogen peroxide compared to natural peroxidases like horseradish peroxidase. Finally, it shows the easy fabrication of nanozyme composite films guided throughout the intrinsic self‐assembling properties of the CTPR scaffold. These heterogeneous solid materials are reused in several reaction cycles without significant loss of the catalytic performance, proving these protein‐templated nanozymes as an advantageous alternative to natural enzymes.

Insight into the genetic basis of ammonia tolerance in razor clam Sinonovacula constricta by genome-wide association study

With the development of high-density integrated farming model, the razor clam Sinonovacula constricta faces severe pressures from environmental factors, especially ammonia nitrogen, which is the top factor threaten the growth and survival of aquatic animals. Hence, ammonia tolerance has been one of the most important economic traits for razor clam. To cultivate new strains with ammonia tolerance trait, it is necessary to dissect genetic basis comprehensively underlying ammonia tolerance of S. constricta. In this study, we performed a genome wide association study (GWAS) to identify the single nucleotide polymorphisms (SNPs) and genes associated with ammonia tolerance through whole genome resequencing of 231 individuals. As a result, 56 associated SNPs (-log10P = 5) which were embedded in or adjacent to 236 genes were identified among a total of 4,577,102 SNPs. Most of these genes were related to various processes of metabolism, homeostasis, response to stress and apoptosis, suggesting that the polygenic architecture in the trait of ammonia tolerance was complex in razor clam. Among these sites, 11 genome-wide significant associated SNPs (-log10P = 6.66) adjacent to five candidate genes, including beta-galactosidase (glb1), tetratricopeptide repeat protein 28 (ttc28), Na+/K+-ATPase alpha-1 (nka), tubulin beta (tubb) and elongation factor 1-alpha (eef1a). In particular, NKA has been demonstrated to be involved in NH4+ excretion to reduce ammonia accumulation in mollusca. Our results provide insight into genetic basis of ammonia tolerance, and the SNPs would be valuable markers for selective breeding of ammonia-tolerant strain in S. constricta.

Tetratricopeptide repeat protein SlREC2 positively regulates cold tolerance in tomato.

Cold stress is a key environmental constraint that dramatically affects the growth, productivity, and quality of tomato (Solanum lycopersicum); however, the underlying molecular mechanisms of cold tolerance remain poorly understood. In this study, we identified REDUCED CHLOROPLAST COVERAGE 2 (SlREC2) encoding a tetratricopeptide repeat protein that positively regulates tomato cold tolerance. Disruption of SlREC2 largely reduced abscisic acid (ABA) levels, photoprotection, and the expression of C-REPEAT BINDING FACTOR (CBF)-pathway genes in tomato plants under cold stress. ABA deficiency in the notabilis (not) mutant, which carries a mutation in 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (SlNCED1), strongly inhibited the cold tolerance of SlREC2-silenced plants and empty vector control plants and resulted in a similar phenotype. In addition, foliar application of ABA rescued the cold tolerance of SlREC2-silenced plants, which confirms that SlNCED1-mediated ABA accumulation is required for SlREC2-regulated cold tolerance. Strikingly, SlREC2 physically interacted with β-RING CAROTENE HYDROXYLASE 1b (SlBCH1b), a key regulatory enzyme in the xanthophyll cycle. Disruption of SlBCH1b severely impaired photoprotection, ABA accumulation, and CBF-pathway gene expression in tomato plants under cold stress. Taken together, this study reveals that SlREC2 interacts with SlBCH1b to enhance cold tolerance in tomato via integration of SlNCED1-mediated ABA accumulation, photoprotection, and the CBF-pathway, thus providing further genetic knowledge for breeding cold-resistant tomato varieties.

Genetic mapping of a single nuclear locus determines the white flesh color in watermelon (Citrullus lanatus L.).

Flesh color is an important trait in watermelon (Citrullus lanatus L.). Several flesh color genes have been identified in watermelon; however, the inheritance of and the molecular basis underlying the white flesh trait remain largely unknown. In this study, segregation populations were constructed by crossing the canary yellow flesh line HSH-F with the white flesh line Sanbai to fine-map the white flesh gene in watermelon. Genetic analysis indicated that the white flesh trait is controlled by a single recessive locus, termed Clwf2. Map-based cloning delimited the Clwf2 locus to a 132.3-kb region on chromosome 6. The candidate region contains 13 putative genes, and four of them-Cla97C06G121860, Cla97C06G121880, Cla97C06G121890, and Cla97C06G121900-were significantly downregulated in the white flesh compared to the canary yellow flesh watermelon fruits. The Cla97C06G121890 gene, which encodes a tetratricopeptide repeat protein, showed almost no expression in the white flesh fruit before maturity, whereas it had a very high expression in the canary yellow flesh fruit at 18 days after pollination. Transmission electron microscopy revealed rounded and regularly shaped chromoplasts in both the canary yellow and white flesh fruits. Further quantitative real-time PCR analysis showed that the expression levels of several key plastid division genes and almost the entire carotenoid biosynthesis pathway genes were downregulated in the white flesh compared to the canary yellow flesh fruits. This study suggests that the proliferation inhibition of chromoplasts and downregulation of the CBP genes block the accumulation of carotenoids in watermelon and lead to white flesh. These findings advance and extend the understanding of the molecular mechanisms underlying white flesh trait formation and carotenoid biosynthesis in watermelon.

Recruiting the autophagy-lysosome pathway to engineered condensate-forming proteins.

Biomolecular condensates (BCs) are membraneless, subcellular compartments segregated from the cytoplasm by liquid-liquid phase separation, a thermodynamic process driven by the chemical properties of the constituent biomolecules. BCs are involved in a range of cellular processes such as signalling and transcriptional regulation, as well as the autophagy-lysosome pathway, one of the cell's main degradation mechanisms. Selective autophagy can recognise BCs containing cargo proteins through direct interactions with LC3, a protein present on the inner membrane of phagophores. Consensus tetratricopeptide repeat proteins (CTPRs) are highly stable proteins composed of repeated helix-turn-helix subunits that are useful in protein engineering as a result of their modular architecture and amenability to rational design. In this project, the CTPR scaffold that has been engineered to bind LC3 through grafted LIR motifs is fused to low complexity domains (LCDs) to induce BC formation. Here we investigate the effects of different LIR motifs within the engineered BCs to understand how LC3 interactions can change the physico-chemical properties of the condensates. Using these BCs we hope to gain a better understanding of how and why the autophagy-lysosome pathway utilises BCs for client degradation, with the potential for adapting the engineered proteins as targeted protein degradation therapeutics.