Mycobacterium tuberculosis (Mtb), kills more people worldwide than any other bacterial infectious disease. There has been a recent resurgence of TB drug discovery activities, resulting in the identification of a number of novel enzyme inhibitors. Many of these inhibitors target the electron transport chain complexes and the F1FO-ATP synthase; these enzymes represent new target spaces for drug discovery, since the generation of ATP is essential for the bacterial pathogen's physiology, persistence, and pathogenicity. The anti-TB drug bedaquiline (BDQ) targets the Mtb F-ATP synthase and is used as salvage therapy against this disease. Medicinal chemistry efforts to improve the physio-chemical properties of BDQ resulted in the discovery of 3,5-dialkoxypyridine (DARQ) analogs to which TBAJ-876 belongs. TBAJ-876, a clinical development candidate, shows attractive in vitro and in vivo antitubercular activity. Both BDQ and TBAJ-876 inhibit the mycobacterial F1FO-ATP synthase by stopping rotation of the c-ring turbine within the FO domain, thereby preventing proton translocation and ATP synthesis to occur. While structural data for the BDQ bound state are available, no structural information about TBAJ-876 binding have been described. In this study, we show how TBAJ-876 binds to the FO domain of the M. smegmatis F1FO-ATP synthase. We further calculate the binding free energy of both drugs bound to their target and predict an increased affinity of TBAJ-876 for the FO domain. This approach will be useful in future efforts to design new and highly potent DARQ analogs targeting F-ATP synthases of Mtb, nontuberculosis mycobacteria (NTM) as well as the M. leprosis complex.Tuberculosis (TB), the deadly disease caused by   •BDQ inhibits mycobacterial F-ATP synthase.•TBAJ-876 is a BDQ analogue with improved affinity for the enzyme.•O domain binding sites of TBAJ-876.Simulations help to structurally clarify the F•Insights will help guide development of multidrug-resistant tuberculosis drugs. Due to the lack of an effective fermentative process, the oxidative phosphorylation (OXPHOS) pathway is crucial to maintain redox homeostasis and the synthesis of sufficient quantities of ATP , which is essential for growth and viability of the pathogen  structure of the eriments . These eeriments . In addig sites’ ). The leytoplasm . Therefogradient .Fig. 2BDM. smegmatis FO domain.The successful therapeutic advance of BDQ has been overshadowed by the development of clinical resistance  along wi22.1O domain of the M. smegmatis F-ATP synthase (PDB-ID: 7JGC)  . This prD: 7JGC)  MembraneD: 7JGC) , as showeviously  and confeviously . A physiermostat  and the ermostat . We simutructure , and TBAtructure  dividingtructure  was set tructure , CHARMM3tructure  and the tructure  for the tructure  was usedtructure , using atructure  algorith2.2trj_cavity  approach, when drug and key-glutamate were in the neutral state; this approach was chosen as a deprotonated key-glutamate induces a potentially artefactual water funnel connecting the solvent and the key-glutamate . Althougj_cavity  and subtj_cavity . We repe33.1O domain in two states. First, the drug and key-glutamate (E65) were simulated in their charged states and second in their neutral states. Irrespective of the protonation state of E65/drugs, we observed a comparable number of hydrogen bonds and hydrophobic contacts between drug and protein (cut-off 4 Å) when comparing the same binding site  to derive the free energy estimate, as the charged state induces a potentially artefactual water funnel connecting the solvent and the key-glutamate in the absence of ligands  = 0.1 kcal/mol) and within the standard deviation. This is in agreement with experiments  the affinity measured for the c-ring is lower than for the whole enzyme  presence . Our calpresence . Being bubunit a .Fig. 5In4O domain from M. smegmatis, resolved the energetic basis for drug binding, and finally discussed this in the context of how the drugs likely inhibit the enzyme function. This represents a framework which may potentially be used to predict novel lead compounds that bind to other pathogenic bacterial F-ATP synthases essential for their survival (Acinetobacter baumannii, whose structure was recently solved (M. abscessus, the causative agent of pulmonary disease. The drug displays attractive in vitro and in vivo activities against the Mab complex strains, although with lower potency and reduced bactericidal activity, like BDQ (We have elucidated the binding sites of the drugs BDQ and TBAJ-876 bound to the Fsurvival , such asy solved . The datlike BDQ .Alexander Krah: Conceptualization, Formal analysis, Investigation, Visualization, Writing – original draft, Writing – review & editing. Gerhard Grüber: Writing – original draft, Writing – review & editing. Peter J. Bond: Formal analysis, Supervision, Funding acquisition, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Lymphangiomas are lymphatic malformations most commonly found in the head, neck and axilla. Their incidence in gastrointestinal tract, especially duodenum is very rare. They are mostly congenital and are usually seen in children. Owing to their asymptomatic nature, these tumors are often diagnosed incidentally. Surgical resection is the definite treatment.We represent a case of a 30-year-old male with duodenal lymphangioma with presentation of intermittent dyspepsia for 1 year, partially relieved by a proton pump inhibitor.Helicobacter pylori antigen test, the patient underwent an esophagogastroduodenoscopy which revealed H pylori gastritis and a duodenal lymphangioma.Upon physical examination, there was mild tenderness observed in the epigastrium. The rest of the physical examination was unremarkable. His complete blood count report was unremarkable. Upon a negative stool for Patient was put on triple therapy  for 14 days and his symptoms improved. The lymphangioma was not resected owing to small size.Patient was followed till 1 year and his symptoms had improved.H pylori gastritis and a duodenal lymphangioma. There is likely to be an association between the two and therefore, further studies are required to find out any relationship that may exist between the 2 conditions.The case describes a correlation between  The gastrointestinal tract is affected in less than 5% of the presentations.,2 Lymphangiomas of the duodenum are even more infrequent. In addition to duodenum, these lymphangiomas have also been reported in other parts of the gastrointestinal tract such as esophagus, stomach, jejunum and colon. Although lymphangiomas can present at any age, 50% are observed at birth and 90% by 2 years of age. Therefore, their diagnosis in adults is rare. These tumors are mostly asymptomatic or present with nonspecific symptoms and are often diagnosed incidentally. Duodenal lymphangiomas are sessile and although, can be picked up on radiological examination where they appear as sharply marginated round-to-oval filling defect with smooth surface, their diagnosis is confirmed on histological basis from either biopsy, surgery or autopsy. The endoscopic view depicts them as soft polypoid masses with smooth and yellowish surfaces. They have also been described as submucosal tumors with white colored spots. Because they are submucosal tumors and the overlying mucosa appears normal, these tumors are often difficult to diagnose until excised and examined under microscope. Previous reports show, these lymphangiomas have only been diagnosed incidentally on an autopsy or diagnosed histologically following a surgery. Endoscopic biopsy can allow a preoperative diagnosis without any complications. As lymphangiomas are known to be benign with no malignant potential, these tumors can be subjected to minimal operation, and although the course of treatment depends on the surgeon, an endoscopic polypectomy may be enough to remove the tumor. If, however, polypectomy fails to remove the complete tumor, additional surgery should be carried out as incomplete excision can lead to recurrence. Lymphangiomas of lower intestinal tract should be considered for resection owing to their symptomatic nature, however, lymphangiomas of upper intestinal tract can be left unattended as they are only mildly discomforting, unless they cause a mechanical gut obstruction. We report a lymphangioma of the duodenum incidentally diagnosed in an adult male.Lymphangiomas are rare, benign lymphatic malformations that arise due to the failure of the individual lymphatic tissues to connect with the general lymphatic system.A 30-year-old male previously healthy presented in the outpatient department with intermittent dyspepsia and heartburn for 1 year. The pain was more marked in the epigastrium and increased with food intake. There was no relationship of the pain to the use of any specific food type. The patient used proton pump inhibitor omeprazole 20 mg twice daily for it, however, the pain was only partially relieved. He is nonsmoker, not alcoholic, with no self-medication user history and no alarming signs or symptoms such as anemia, weight loss, nights sweat, dysphagia or odynophagia.The patient was vitally stable at the time of presentation. His pulse was 75/min, blood pressure was 120/80 mm Hg, respiratory rate was 15/min, and body temperature was 98°F. Physical examination revealed mild tenderness in epigastrium. The rest of the examination was unremarkable. His complete blood count was unremarkable . The gastrointestinal system is rarely involved. Although commonly believed to arise as obstruction of lymphatic tissue that fails to unite with the systemic lymphatic system, lymphangiomas can also arise secondary to abdominal trauma, underlying inflammation, surgery, radiation exposure, or a congenital defect in the lymphatic system. Pathologically, they are identified as dilated lymphatics in the submucosal layer. Lymphangiomas are categorized into 3 types. Cystic is the most common form and comprises cyst-like cavities filled with protein-rich fluid. Cavernous lymphangiomas are dilated lymphatic channels of varying sizes with an intact connection with normal lymphatic vessels. Capillary lymphangiomas consist of small thin-walled lymphatic vessels beneath the skin.Lymphangiomas are rare, benign fluid-filled cyst-like lesions arising from lymphatic vessels. so diagnosing them can be challenging. Our patient presented with dyspepsia possibly secondary to H pylori infection and responded to triple therapy. However, there may be an association between lymphangiomas and dyspepsia.Most of these tumors are asymptomatic or have vague, nonspecific symptoms like abdominal discomfort or a palpable mass, nausea, vomiting, and dyspepsia, Gangl et al reported one lymphangioma in 2100 esophagogastroduodenoscopies, 900 colonoscopies, and 5000 rectoscopies. The second part of the duodenum is a common site of occurrence and was noticed in 63.6% of duodenal involvement.Out of 567 patients who had an endoscopic biopsy performed, only 2 were reported to have benign duodenal lesions (0.35%). Duodenal lymphangiomas are submucosal tumors, often appearing as white-colored spots on the surface. It is believed that the accumulated chyle in the dilated lymphatics depicts itself as white spots macroscopically.Although lymphangiomas of the duodenum can present with symptoms of weight loss, anorexia, anemia, bleeding, epigastric pain, they are primarily an incidental finding in asymptomatic cases because of their relatively smaller size (<1 cm). Since our case had no such complication, no intervention was done for the lymphangioma.Because of its benign nature, lymphangioma seldom requires treatment unless complicated with infection, hemorrhage, or fistula formation.H pylori and lymphangioma of the duodenum should be further investigated. Endoscopy plays a significant role in diagnosing such tumors.Patients with dyspepsia can have an underlying lymphangioma. The association between Conceptualization: Naresh Kumar Seetlani, Vineeta Kumari, Summaiya Waheed.Data curation: Naresh Kumar Seetlani, Vineeta Kumari.Formal analysis: Naresh Kumar Seetlani.Investigations: Naresh Kumar Seetlani, Vineeta Kumari.Methodology: Summaiya Waheed, Zohaib Yousaf.Validation: Abdul Baqi, Summaiya Waheed, Fatima Ashfaq, Muhammad Junaid Tahir, Khabab Abbasher Hussien Mohamed Ahmed, Zohaib Yousaf.Writing – original draft: Summaiya Waheed, Muhammad Junaid Tahir, Zohaib Yousaf.Writing – review and editing: Zohaib Yousaf, Fatima Ashfaq, Khabab Abbasher Hussien Mohamed Ahmed, Muhammad Junaid Tahir.


Drosophila as a model organism. Finally, we show the relevance of this protein family to human myopathies and the development of muscle-related diseases.One of the most intriguing features of multicellular animals is their ability to move. On a cellular level, this is accomplished by the rearrangement and reorganization of the cytoskeleton, a dynamic network of filamentous proteins which provides stability and structure in a stationary context, but also facilitates directed movement by contracting. The ALP/Enigma family proteins are a diverse group of docking proteins found in numerous cellular milieus and facilitate these processes among others. In vertebrates, they are characterized by having a PDZ domain in combination with one or three LIM domains. The family is comprised of CLP-36 (PDLIM1), Mystique (PDLIM2), ALP (PDLIM3), RIL (PDLIM4), ENH (PDLIM5), ZASP (PDLIM6), and Enigma (PDLIM7). In this review, we will outline the evolution and function of their protein domains which confers their versatility. Additionally, we highlight their role in different cellular environments, focusing specifically on recent advances in muscle research using  Animal locomotion is one of the most fascinating mechanisms in nature. Evading an enemy, foraging for food, finding a mate; these fundamental processes of life all require locomotory abilities. On a cellular level, this is accomplished by the rearrangement and reorganization of the cytoskeleton, a dynamic network of filamentous proteins which provides stability and structure in a stationary context, but also facilitates directed movement by contracting. All animal cells require regulation of the cytoskeleton and their dynamics. This regulation is facilitated by numerous different classes of proteins, such as crosslinkers, kinases, docking proteins and more.Drosophila discs large, and zonula occludens-1 protein) domain, LIM  domains, a ZM (Zasp motif) domain, and an AM (ALP motif) domain  domain . While t) domain . ALP pro) domain   domain . Drosophentirely  , Mystique (PDLIM2), ALP (PDLIM3), RIL (PDLIM4), ENH (PDLIM5), ZASP (PDLIM6), and Enigma (PDLIM7) . The firDrosophila and vertebrates.Here we will provide an update of the current research on proteins of the ALP/Enigma family, focusing on their cytoskeletal functions in a-actinin, while also being able to interact with different cytoskeletal regulatory proteins  (where X denotes any amino acid) and are roughly 50–65 amino acids in length .  domains . The div domains .LIM domain proteins are critical to the ALP/Enigma proteins. They enable the proteins of the family to recognize stressed cytoskeletal areas, and interact with a slew of different proteins . In the Drosophila Zasp52, human ZASP, as well as the human ALP proteins PDLIM1 and PDLIM3, which indicates that this domain was likely present in the ancestor before the splitting of subfamilies  of adults  of Drosophila have strong homology to human sarcomeres  is a core component of the Z-disc. Ablation of ZASP (Cypher) in mouse heart muscles causes development of dilated cardiomyopathy with premature death . ZASP haDrosophila ZASP ortholog, is necessary for a myriad of processes in muscle cells. For starters, Zasp52 is essential for the maintenance and development of functional integrin adhesion sites, which is integral to cell spreading and the development of myotendinous junctions  experiments have demonstrated that integrins are more mobile in Zasp52 mutant embryos and talin mutants during later stages of Drosophila embryo development compared to their wild-type counterparts (Zasp52, the unctions . Moreoventegrins . More imterparts , which iterparts , and theterparts . Thus, Zterparts .Drosophila IFMs, Zasp52 localizes to Z-discs during early development and is necessary for the maintenance of the myofibril (Drosophila, Zasp66 and Zasp67, which are also necessary for the assembly of myofibrils (α-actinin null mutant phenotype, which is characterized by loss of Z-disc structure (Drosophila forms a multi-protein complex with α-actinin that is crucial for Z-disc formation during development. Recently, Zasp52 has also been shown to bind actin filaments directly (α-actinin binding domain. By being able to bind to actin directly, Zasp52 would still be able to anchor Z-discs to the cytoskeleton and retain the structure of the contractile apparatus in some cases of muscle diseases. The region required for actin binding in Zasp52 includes the extended PDZ domain, and the ZM region that immediately follows, although it is still unknown if amino acids of the 26 amino acid ZM domain itself contribute to actin binding (In addition to being involved in adhesion sites, Zasp52 is also a core regulator of Z-disc structure, which delineates the sarcomere and anchors the actin thin filaments in the contractile apparatus. In yofibril . In addiofibrils . Double tructure . This redirectly . This ma binding .Drosophila IFM as a model, the importance of this domain has been elucidated (α-actinin and associated proteins that grow in size and eventually dictate the final size of the sarcomere (Drosophila have a PDZ and ZM domain, while only Zasp52 contains additional LIM domains. Interestingly, temporal expression and localization of specific isoforms is what determines sarcomere growth. Zasp52, which contains LIM domains, has been shown to be a “growing” isoform of the Z-disc, while Zasp66 and Zasp67, lacking LIM domains, are “blocking isoforms.” The LIM domains and ZM domains interact, and it is this interaction that determines growth of the Z-disc. During the growth phase of the sarcomere, Zasp proteins with unbound LIM domains recruit additional Zasp proteins by the ZM domain. Each of the four LIM domains are able to interact with a ZM domain, which can increase the size of the sarcomere by recruiting additional growing isoforms. However, in the late phase, the ratio of growing to blocking isoforms changes, and more blocking isoforms lacking LIM domains begin binding to the already aggregated Zasp52 molecules. This terminates the growth of the sarcomere by impeding further growing isoform recruitment (Apart from the PDZ and LIM domains, Zasp52 also contains the muscle-specific ZM domain. Recently, using the ucidated . One comarcomere . As mentarcomere . All Zasruitment .Having shown the importance of Zasp in muscle cells and the sarcomere, we will now move on to what these discoveries mean for human myopathies, as well as other potential roles that ALP/Enigma proteins play in muscle-related diseases.The pathology of myofibrillar myopathies originates in the Z-disc. Often in myofibrillar myopathies, symptom onset is caused by disintegration of the Z-disc, then the myofibrils, followed by an abnormal ectopic accumulation of multiple proteins . In ZaspDrosophila muscle, vertebrate skeletal muscle has high regenerative properties due to the presence of satellite cells, a group of quiescent muscle progenitors that proliferate, differentiate and fuse into pre-existing myofibers upon muscle injury (reviewed in Drosophila sarcomeres in which the “blocking” isoform predominates, namely a smaller Z-disc diameter (Drosophila Z-disc diameter model which would function to end Z-disc growth at the specified diameter. Interestingly, in mouse models, when the larger form of ZASP containing all three LIM domains is knocked out, neonatal lethality and Z-disc perturbations are observed, while knock-out of the shorter ZASP isoform containing no LIM domains conversely has no phenotype (In contrast to diameter . In humadiameter . These phenotype . Moreovehenotype . In addihenotype  which leTTN gene (PDLIM5-knockout mouse model data (Recently, SNPs in PDLIM3 and PDLIM5 have been implicated in the development of idiopathic dilated cardiomyopathy . AdditioTTN gene . Additiodel data . TherefoDrosophila, showcasing the potential implications for muscle disorders. The mounting research in recent years on the ALP/Enigma family proteins is uncovering the families’ relevance for muscle-related diseases and possible therapeutic targets in a clinical setting. More research will need to be carried out in order to elucidate therapeutic avenues, as well as examining the possible genetic interactions between multiple members of the family in a myopathic context. Of particular interest should be future studies investigating if sarcomere diameter is controlled similarly in vertebrates as in insects and what this means for human myopathies.The ALP/Enigma proteins are a diverse group of adaptor proteins involved in numerous and vastly different cellular processes. In this review, we highlighted the evolution of their protein structure. It is from the complexity of their domains that their versatility is conferred, which enables them to bind a myriad of different proteins. We also covered recent advances in


The current study deciphers the processing of different proportions of white flour and whole wheat flour  into a pizza base using yeast-based fermentation. The bread making using the yeast system resulted in significant changes in the characteristics of bread, ranging from the porous structure development to the crumb cellular structure modifications. An increase in the proportions of whole wheat flour resulted in the formation of golden yellow pizza bases. The lightness of the crust was decreased, whereas the yellowness index was increased as the whole wheat flour contents were increased. The pore size of the pizza base was decreased while the pore density was increased as the whole wheat flour content was raised within the bread. The microscopic study also showed the formation of porous structures on the bulk of the pizza base. The texture analysis of the bread also suggested an increase in the formation of the rigid network structure when the amount of whole wheat flour was increased. The springiness, cohesiveness, and resilience were comparable for all the prepared samples. On the other hand, the values for hardness, gumminess, and chewiness showed an increasing trend with the increase in the whole wheat flour content. The impedance of the samples decreased when there was an increase in the whole wheat flour content. Overall, the pizza base that was developed with 50% whole wheat flour and 50% white flour ratio displayed acceptably firm yet sufficient viscoelastic properties for human consumption. The modern pizza, as we know it today, originated in Naples, Italy, as an affordable, fast meal for working-class Neapolitans; however, the pizza in its most basic form as a seasoned flatbread has now been popularized worldwide, with different cultural versions of pizza based on local ingredients and flavors . A contiThe pizza base is made predominantly from refined wheat grain flour, which is also known as all-purpose flour or white flour (WF) . The wheThis phenomenon may be avoided by using WF in conjunction with whole wheat flour (WWF) . While tMany studies have attempted to analyze the changes in the structural and physical properties of food products due to WWF substitution. The alteration in the antioxidant  and carotenoid  concentrations due to the inclusion of the bran fraction of whole wheat into white flour has been studied . AnotherWhole grain wheat flour , all-purpose flour (generic brand), baking powder , salt , sunflower oil , and active dry yeast (generic brand) were sourced from the local market.The pizza base was prepared by mixing and kneading the wet and dry ingredients to form a dough. The dough was developed using different WF and WWF proportions. To the mixture, salt and baking powder were added. The ingredients were then mixed thoroughly for a minute with a whisk to ensure the formation of a homogenous mixture. Subsequently, water was warmed to bloom the yeast (37–45 °C). Accurately weighed active dry yeast and coarse grain sugar were added to the lukewarm water. The mix was then set to rest for 5 min. The bloomed yeast obtained was used for kneading dough. The process of kneading continued for 15 min. The finished dough was then kept in an oiled container, covered with cling wrap, and incubated for 3 h at room temperature (25 °C). After the incubation period, the dough was stretched out in a circular shape on a baking pan. The baking pan was then kept in the oven , which was pre-heated in the convection mode at 200 °C. Thereafter, baking was carried out at the said temperature for 12 min. At the end of the baking period, the finished pizza base was taken out and allowed to cool for 5 min. The composition of the prepared pizza bases is tabulated in The colorimetric study was performed on the pizza base crust formulations using a lab-developed colorimeter to acquire the CIELab  values. The detailed principle and working of the colorimeter are reported in the mentioned study . InitialThe surface topology of the crust and the bulk of the rectangular pizza base samples (length and width: 15 mm) were visualized using a Stereo Zoom Microscope . The microscope used an external eyepiece lens camera  to acquire images.The samples were subject to texture analysis to characterize the viscoelastic and textural properties.Stress relaxation of the samples was performed to characterize the viscoelastic properties. The experiment was carried out using a texture analyzer . The pizza bases were cut into individual rectangular cuboid pieces . The stress relaxation was performed with the flat probe (diameter: 63.5 mm). The probe pressed the samples down by a distance of 2 mm at a speed of 1 mm/s after a trigger force of 5 g. The deformation was then maintained for the 60 s, and the relaxation profiles were obtained by monitoring the force values over time. The probe was then redacted to its original height.Texture profile analysis was performed to study the various textural parameters such as hardness, springiness, cohesiveness, gumminess, chewiness, and resilience. The flat probe (diameter: 2.5 inch) was used to apply 50% strain to the sample for two compression cycles at a speed of 1 mm/sec. A time gap of 5 sec was maintained between the two cycles.Impedance analysis of the pizza base samples was performed using an impedance analyzer . The sample was kept on an insulator (cardboard), and the impedance probe  was inserted into the bulk of the samples. The impedance was measured at the frequency range of 1 Hz to 1 KHz.Data of different studies are represented as mean ± S.D of the triplicates.The differently formulated pizza bases were set to rest for 5 min after the baking process was complete. This was performed to ensure that the pizza bases reached a temperature that was safe to handle. The crust and bulk phase of the pizza bases were then imaged , with thColor analysis of a food product is an essential step that accounts for a crucial sensory characteristic. The CIELab color-space or L*a*b* color-space was developed by Commission Internationale de l’Eclairag, which defined and standardized a color measurement protocol that has been used for the color analysis of food products . The CIEThe average a* values of the samples were in the range of 2 and 25 b. This iThe above values  were used to calculate derived parameters such as whiteness index (WI) d and yelp > 0.05).Microscopic studies were carried out to visualize the surface microtopography of the crust and the bulk of the samples. The crust  showed tIn , the autp > 0.05). The unusual F0 value of WF50 can be explained by the formation of some associative interactions between gluten and dietary fiber. Further, the increased firmness of the WWF-containing pizza base can be explained by the increase in the bran fraction [SR is performed for many food products to characterize their viscoelastic properties. The highest force attained in the SR profile is used to forecast the firmness of the formulations (F0). The F0 values b showed fraction . The brafraction . As the The F0 and F60 were further used to calculate the %SR for all the formulations. The %SR values d of WF0,Texture profile analysis was performed on the pizza base crumb to extract six crucial parameters, i.e., hardness (g), springiness, cohesiveness, gumminess, chewiness, and resilience. Hardness refers to the force required to achieve a particular deformation . The harAnother parameter that was calculated from the TPA analysis was cohesiveness, the degree to which a material is fully compressed between teeth before rupturing. The cohesiveness values ranged from 0.80 to 0.86, as illustrated in c. It canThe last parameter of TPA was resilience, defined as the degree of resistance a material gives against deformation ,26. The Electrical impedance spectroscopy (EIS) is a technique that is used to measure the electrical characteristics of materials by applying a sinusoidal test voltage or current across the bulk. The impedance plots  show a dFurthermore, it should be noted that bakery products and the pizza bread manufacturing industry are automated, including high-speed production lines . The quaThe continuously changing human lifestyle has also changed the nature and globalization of food by demanding healthier qualities and nutritional values. This has also been portrayed in the worldwide tradition of flatbreads to the modern pizza as we know it today, due to its ease in preparation, portability, affordability, easy customization, and balanced composition. In most cases, the basic structure of pizza is based upon the bread prepared using WF; however, unbeknownst to many, the gluten content in WF brings both benefits and harm to the human body. For instance, the long-term and repetitive consumption of gluten is associated with allergic reactions, irritable bowel syndrome, gluten ataxia, etc. A gluten-free diet is the most widely recommended, and a large number of gluten-free products (GFP) are available on the market. These GFPs arguably mimic the macronutrient and dietary fiber composition of their gluten-containing counterparts (GCC) . A cross


To the editor,UBAP1 in autosomal dominant HSP pedigrees. These families are from Iran, USA, Germany, Canada, Bulgaria, Spain, and China, respectively, implying the diverse geographic origin for the UBAP1 variants. The phenotypes are predominantly pure early‐onset HSP in these families (MIM # 618418). In this study, we reported a novel UBAP1 (NM_016525.5) truncating variant c.371dupT (p.Leu124Phefs*15) in a Chinese autosomal dominant HSP pedigree  are a group of neurodegenerative diseases predominately presented with weakness and spasticity in lower extremities. HSPs have high clinical and genetic heterogeneity and over 80 genes or loci have been linked to HSP over the past two decades   Figure . We then) Figure . Accordi) Figure , this vaUBAP1, encodes the ubiquitin‐associated protein 1 (UBAP1), a subunit of ESCRT‐I complex. UBAP1 has two main domains, the UMA domain in the N‐terminal region (17–63 aa) and a SOUBA domain in the C‐terminal region. The former domain is associated with ESCRT‐I complex, while the latter domain maintains ubiquitin homeostasis of early endosome processing. We constructed plasmids containing wild‐type (WT) or mutant UBAP1 gene (NM_016525.5) and transfected the plasmids in HEK 293T cells. Western blot analysis revealed that this truncating variant actually led to the production of truncated mutant form of UBAP1, lacking the SOUBA domain (Figure n Figure . We perfn Figure  and promn Figure .UBAP1 variants including the one identified here have been described (Bian et al., UBAP1 (Figure UBAP1. In addition, all identified variants preserve the UMA domain but cause a loss of the SOUBA domain, implying that loss of ubiquitin binding would be detrimental. Further studies are required to elucidate the mechanism of SOUBA impairment causing HSP.To date, 18 1 Figure , implyinUBAP1 truncating variant in a Chinese autosomal dominant HSP pedigree. Our findings expanded variant spectrum of UBAP1 and further confirmed the pathogenicity of UBAP1 variants in HSP.In summary, we identified a novel The authors have declared no conflict of interest.Qiao Wei: data acquisition, analysis, and interpretation of data, statistical analysis, drafting the manuscript. Pei‐Shan Wang: data acquisition, analysis, and interpretation of data. Hai‐Lin Dong: data acquisition, interpretation of data. Wen‐Jiao Luo: data acquisition. Zhi‐Ying Wu and Hong‐Fu Li: funding, study design and conceptualization, data acquisition, analysis and interpretation of data, technical and material support, drafting, and critical revision of the manuscript.The study was approved by the Ethics Committees of Second Affiliated Hospital of Zhejiang University School of Medicine and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Specific national laws have been observed, too. Written informed consent was obtained for this study from all the patients prior to their inclusion in the study.


Sarcoptes scabiei. The scabies mites burrow is highly diagnostic but illegible by the naked eye, because it is tiny and may completely be obscured by scratch and crust. The classic technique is opening the end of an intact mite burrow with a sharp instrument and inspecting its contents in the light microscope under loupe vision. Dermatoscope is a new method to diagnose scabies, with the advantages of non-invasive and more sensitive. This study verified the characteristic manifestations of scabies under dermoscopy. Under the closer examination of the curvilinear scaly burrow, the scabies mite itself may be seen as a dark equilateral triangular structure, which is often referred to as a “jet with contrail.” Besides, this study found that the positive detection rate of microscopic characteristic manifestations under the dermoscopy ordered by the external genitals, the finger seams and the trunk, which were statistically different . Of note, this is the first study to explore the regional distribution of the characteristic dermoscopic manifestations of scabies. We are the first to propose to focus on examining the external genitalia and finger seams with dermoscopy.Scabies is a common skin disorder, caused by the ectoparasite  Sarcoptes scabiei. Clinically, it is characterized by erythematous papules or vesicles. The typical distribution of skin lesions includes finger seams, the wrists, armpits, groins, buttocks, genitals and the breasts in women. In infants and young children, the palms, soles, and head are also commonly involved, which is rare in adults , from August 2020 to October 2022. All the included patients met the 2018 The International Alliance for the Control of Scabies (IACS) criteria for the diagnosis of scabies . The defThe CBS dermoscopy detection system was used to scan the clinically suspicious skin lesions of the patients, including the finger seams, armpits, trunk, buttocks and genitalia, etc. The 50× lens was used to move back and forth on all clinical prone sites one by one. All dermoscopic pictures were saved from the computer after examination.The most arresting feature of scabies under dermoscopy was the burrow. Dermoscopy began with looking for the scabies burrow, which was the movement path of the scabies mite in the stratum corneum of the patients. The burrow entrance was usually where the scales was visible (the start of the sarcoptic curve), and the end of the burrow was usually where the blister was located (the end of the curve).Under the closer examination of the curvilinear scaly burrow, the scabies mite itself was usually located at the end of the burrow a few millimeters in front of the small blister. The end of the burrow was wiped with an alcohol cotton swab. Through the cuticles, the mite itself was seen at the end of the burrow as a dark equilateral triangular structure, corresponding to the pigmented head and anterior legs of the scabies mite. The burrow and the dark triangular structure were often referred to as a “jet with contrail” . BecauseP-value < 0.05 was selected as the threshold of statistical significance.Continuous variables were displayed as means and standard deviation (SD). Categorical variables were expressed as frequency counts and percentages, and chi-square was used to evaluate the differences between groups. All statistics were analysed by using the SPSS 22.0 software, and We collected a total of 56 cases with clinical scabies and suspected scabies. All of them received dermoscopy examination. Among them, 34 patients were male, accounting for 60.7%, and 22 patients were female, accounting for 39.3%. The eldest patient was 16 years old and the youngest patient was an 8-month-old infant. Their mean (SD) age was 6.14 ± 4.07 years old.In total, 50 patients presented the typical “jet with contrail” under dermoscopy. Those 50 cases were confirmed scabies. The burrow and dark triangular structure were shown on P-value < 0.05). The regional distribution of the characteristic dermoscopic manifestations of confirmed scabies was shown in Due to the small amounts, in this study, the neck, chest wall, abdomen, back, armpits and buttocks were all divided into the trunk. Only 6 patients could be found that the burrow and the dark triangular structure were on the trunk, versus 41 on the finger seams and 45 on the external genitals, relatively. The detection rates of characteristic dermoscopic manifestations of the finger seams and the external genitals were statistically significant compared with those of the trunk . Its high detection rate may stem from the fact that the epidermis thickness of finger seams and external genitals was thinner and the transmission of light was better. Therefore, skin can easily expose the scabies mite. In addition, genital area was more private with less scratching, resulting in less damagement of the primary skin lesions, so the burrows were more unbroken.This study found that the positive detection rate of microscopic structures under the dermoscopy ordered by the external genitals, the finger seams and the trunk, which were statistically different (It suggests that when we use dermoscopy in clinical examination of patients suspected of scabies, we should focus on observing the skin lesions of external genitalia and finger seams, which increased the credibility of dermoscopy and avoid missed diagnosis. Of note, this is the first study to explore the regional distribution of the characteristic dermoscopic manifestations of scabies. We are the first to propose to focus on examining the external genitalia and finger seams with dermoscopy in the diagnosis of scabies, which has great guiding value for clinical practice.This study revealed the characteristic manifestations of scabies under dermoscopy. It clearly showed the parasitic state of scabies, and had a more intuitive understanding of scabies. Dermoscopy can effectively decrease the misdiagnosis rate of clinical physicians. Besides, this study showed the non-characteristic manifestations under dermoscopy, which was neglected in the past studies. The significance of non-specific structures was to indicate the complicated infection and allergic status of different patients, and to indicate the skin barrier damage of different degrees in scabies patients .This study has limitations due to its retrospective design. And the relatively small size of sample is another limitation of our study. We look forward to multi-center, large sample prospective studies to enhance the confidence of the results. Furthermore, the limitations of dermoscopy are that eggs or fecal material are not visible . It can The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Y-LN and HY: design, analysis, and interpretation of data, and drafting of the manuscript. X-YX: acquisition of data and statistical analysis. G-LF and Y-QZ: critical revision of the manuscript for important intellectual content, obtaining funding, and supervision. All authors read and approved the final manuscript.


Interdomain interactions of spectrin are critical for maintenance of the erythrocyte cytoskeleton. In particular, “head-to-head” dimerization occurs when the intrinsically disordered C-terminal tail of β-spectrin binds the N-terminal tail of α-spectrin, folding to form the “spectrin tetramer domain”. This non-covalent three-helix bundle domain is homologous in structure and sequence to previously studied spectrin domains. We find that this tetramer domain is surprisingly kinetically stable. Using a protein engineering Φ-value analysis to probe the mechanism of formation of this tetramer domain, we infer that the domain folds by the docking of the intrinsically disordered β-spectrin tail onto the more structured α-spectrin tail. Legend: Protein engineering methods may be used to investigate the mechanism of complex formation from disordered partners, discriminating binding-and-folding from folding-and-binding mechanisms. Simulated structure of human erythroid β-spectrin C-terminal domains previously published as Shammas, S. L., Rogers, J. M., Hill, S. A., & Clarke, J. (2012). Slow, reversible, coupled folding and binding of the spectrin tetramerization domain. Biophys J 103, 2203–14. N-terminal domains of human erythroid α-spectrin PDB ID: 1OWA; erythroid spectrin tetramer domain PDB ID: 3LBX. Structures prepared in PyMol (DeLano Scientific). Spectrin is an elongated structural protein common to all animal species and requisite for life ,2. Altho2 and function to assemble the spectrin network onto the erythrocyte membrane t and F0 is the fluorescence at time zero, F is the fluorescence at time t, and k+ is the association rate constant. [AB]0, the concentration of dimer in the reaction mixture at time zero, is calculated from the Kd obtained from ITC experiments and the initial concentration of α0α1 ([A]t) in the protein solution prior to mixing with denaturant.Although one might expect dissociation to be well represented by single-exponential kinetics, we have demonstrated previously that a second-order reversible model is needed to accurately represent the disassociation data despite the relatively weak association between α0α1 and β16β17  are shown for the core and surface mutations in The calculated Φ-values and dissociation rate constants  fits well with the range of ΔGD–N for the three chicken brain spectrins. Further, visual inspection of the chevron curves in In this work, we have characterized three new spectrin domains: α1, β16, and the tetramer domain. Of these, α1 behaves most similarly to the previously described R15, R16, and R17. As shown in GD–N , and hence, k+ cannot be compared with folding rate constants for other spectrin domains (where kf has units of s−1). However, the first-order disassociation/unfolding rate constant, k−, has units of s−1 and thus can be directly compared to the unfolding rate constants  of the other spectrin domains. The non-covalent spectrin domain unfolds remarkably slowly, like the slow unfolding R17. With a k− about three orders of magnitude lower than the unfolding rate of α1, the tetramerization domain is actually significantly more kinetically stable than its direct  neighbor. It is interesting to note that the dependence of ln(k−) on urea concentration  is very similar to the other spectrin domains . As expected, this has a weaker concentration dependence than for the other spectrin domains . Given this, we were able to determine Φ-values for 26 positions in the tetramer domain. A Φ-value of 1 indicates that the residue is in a region of the protein that is fully structured at the transition state; a Φ-value of 0 indicates that the region of the protein is as unstructured in the transition state as it is in the starting  state. As is standard practice, we classify Φ-values as high (largely structured), medium (partly structured), or low (largely unstructured), to give a qualitative picture of the structure of the transition state. Only three Φ-values  were non-classical, that is, significantly greater than 1 or less than 0.We have previously found the association of the spectrin tetramer to be consistent with a two-state mechanism for association/dissociation, without populated intermediates; rate constants estimated from kinetic data collected at various subunit concentrations were consistent with each other, and equilibrium and kinetic estimates of complex stability were in agreement . ApplyinFor the A-helix, we had good coverage: we determined ten Φ-values for nine buried residues (which monitor tertiary structure formation) and four surface Ala-to-Gly Φ-values (reporting on helix formation). The secondary structure Φ-values are higher than the tertiary Φ-values, and the Φ-values are higher in the middle/C-terminal end of the helix than at the N-terminal end .GD–N < 0.7 kcal mol−1. Thus, we could calculate only three surface and three buried Φ-values in this helix. Nevertheless, we can say that there is an indication of more structure formation at the N-terminal half of the B-helix  than for the C-terminal half, where Φ-values are close to 0.In Helix B, four buried and two surface residue mutations in this helix abolished binding, while two others had mutations with ΔΔGD–N), had a slope that was inconsistent with the other mutants  or by docking of partially preformed helices of A and B (R16 and R17). Here, we see yet another folding mechanism. The Φ-value analysis of the spectrin tetramer domain suggests a templating mechanism wherein transient elements of secondary structure in β17 dock onto the more highly structured C-helix. Our application of biophysical techniques and Φ-value analysis to the spectrin tetramer domain clearly demonstrates that enhanced understanding of IDP folding mechanisms can be achieved through the use of such methods, provided that care and stringent controls are used.We have previously shown that spectrin domains can fold All experiments were carried out in PBS [50 mM sodium phosphate and 150 mM NaCl (pH 7.04)]. The temperature for each experiment was held constant at 25 °C.Wild-type α1, β16, α0α1, and β16β17 were expressed from synthetic genes (GenScript USA) in a modified pRSETA plasmid. All mutagenesis was carried out using a Stratagene QuikChange kit; identity of mutants was confirmed by DNA sequencing.Escherichia coli C41(DE3) cultured in 2× TY media at 37 °C in the presence of 0.1 mg ml−1 ampicillin. Cells were induced with 0.1 mg ml–1 IPTG, and the temperature was reduced to 25 °C once culture optical density at 600 nm reached 0.4–0.6 AU. After overnight incubation, cells were collected by centrifugation, then lysed by sonication and centrifuged again to separate the soluble protein from insoluble cell debris. The protein of interest was purified from the soluble fraction by affinity chromatography on Ni+2 agarose resin. Bound protein was released from the resin by thrombin cleavage and further purified with gel-filtration chromatography. Proteins were frozen in liquid N2 and stored for long periods at −80 °C. Once thawed for use, proteins were stored at 4 °C. Typical yield for α0α1 and β16β17 is 50 and 35 mg l−1 culture, respectively.Protein expression and purification was carried out analogously to other spectrin domains . The proProtein samples were prepared at 10 μM in PBS buffer. Spectra were collected in a 4-mm-pathlength or 1-mm-pathlength cuvette as follows: core residues, Chirascan CD Spectrometer (Applied Photophysics); Scan 250–208 nm, 1 nm bandwidth; surface residues and wild-type constructs, CD Spectropolarimeter (JASCO); Scan 260–200 nm, 0.5 nm bandwidth. Minor differences in spectra obtained from the two instruments were observed; however, no significant changes were observed between mutants.The stability of individual wild-type subunits was determined using urea- or GdmCl-induced denaturation. After excitation at 280 nm, intrinsic tryptophan fluorescence was collected from 300 to 450 nm on a PerkinElmer LS-55 fluorescence spectrophotometer. Protein concentration for these experiments was 5 μM; temperature was 25 °C.The folding and unfolding kinetics of the individual wild-type proteins were monitored by change in fluorescence, using an Applied Photophysics SX.20 stopped-flow spectrophotometer at 25 °C. Trp residues were excited with 280 nm light, and fluorescence data were collected above 320 nm. α0α1 and β16β17 were unfolded in urea and GdmCl, respectively, at a final protein concentration of 1 μM. Six traces were averaged for each concentration of denaturant and fitted to a single-exponential model, using KaleidaGraph (Synergy Software). In keeping with previous work using this instrument, we removed the first 2.5 ms prior to fitting to avoid mixing artifacts .ITC experiments to determine the stability of the tetramer interaction were carried out on either an ITC200-Auto (core residues) or a VP-ITC (surface residues), both from MicroCal (GE). β16β17 at 25–50 μM in the cell was titrated with 15–20 injections to a total of 2–2.5 eq of α0α1, at 300 or 600 s intervals for core and surface residues, respectively. Each injection profile was integrated, and the resulting data were fitted with a one-site binding model in Origin 7 for MicroCal (OriginLab).Folding and unfolding kinetics were monitored by intrinsic tryptophan fluorescence on either an Applied Photophysics stopped-flow spectrophotometer model SX.18 or SX.20 or a Biologic rapid mixing device. For both instruments, Trp residues were excited with 280 nm light and fluorescence was collected above 320 nm. A solution of pre-mixed α0α1 and β16β17, equilibrated 2 h, was mixed 1:1 with a urea solution. For all mutant unfolding data, the initial protein solution was 40 μM in each subunit, resulting in a final concentration in the cell of 20 μM in each subunit. Experiments were conducted at 25 °C, and traces were fitted as described in Supporting information


The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cell-expressed developmentally downregulated 4-1 and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly; however, a direct interaction between them has not been described, and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the neural precursor cell-expressed developmentally downregulated 4-1 HECT domain and elements of the Tsg101 UEV domain involved in noncovalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention. The general process of Ub substrate modification involves a coordinated sequential action between E1 Ub-activating, E2 Ub conjugating, and E3 Ub ligating enzymes, where Ub is activated via thioester bond formation before being transferred to an E2 enzyme via a transthiolation reaction. The mechanism of Ub transfer to the target protein then differs depending on the type of E3 ligase which fall generally into the really interesting new gene (RING) monomer or dimer, RING between RING, and homologous to E6AP carboxyl terminus (HECT) classes (Ubiquitin (Ub) is a small (8.6 kDa) regulatory protein found in eukaryotic cells that is employed for signaling an array of cellular processes, including protein targeting for degradation, stress responses, DNA repair, and cell cycle progression , 2. Signvia single molecule Förster resonance energy transfer that this complex also involves additional scaffolding from an accessory ubiquitin E2 variant (UEV) domain  domain , 5. Such) domain .Here, we report the formation of an encounter-like complex between the HECT domain of the E3 ligase neural precursor cell-expressed developmentally downregulated 4-1 (Nedd4-1) and the UEV domain of the Tsg101 protein. The latter is a component of the endosomal sorting complex required for transport-I (ESCRT-I), one of four  complexes that comprise the endosomal trafficking machinery, in which Nedd4-1 or one of its isoforms functions in protein ubiquitylation . Tsg101/in trans with the UEV domain of Vps23  tags, bound to several sites on the HECT domain, to determine intermolecular paramagnetic relaxation enhancement (PRE) and pseudocontact shifts (PCSs) on the V domain . SimilarV domain .gag gene lacking the intact Tsg101 binding site (through substitution of LIRL). The Nedd4 isomer Nedd4-2s possesses a truncated C2 domain derived from alternative splicing  cells. pNL4-3-ΔPTAP encodes a splicing  and was splicing , 19.Here, we provide evidence that the interaction of the Tsg101 UEV domain with the catalytic domain of Nedd4-1 plays a key role in production of infectious HIV-1 particles and identify critical determinants of the rescue within the UEV and HECT domains. The UEV regions in proximity to the HECT lie on the β-hairpin and in proximity to the vestigial active site. These regions bind Ub noncovalently , 25. As A, red spheres) in the vicinity of the E2 binding location on the HECT domain. Site 867 is the location of the catalytic cysteine in the C-lobe, while site 627 is in the noncovalent Ub binding exosite of the N-lobe  and site 720 is situated in a proximal β-hairpin. When visualized on the structure of Nedd4-1 (PDB ID: 4BBN) having a “T” shaped interlobe configuration akin to the E2-bound structure of Nedd4L (PDB ID: 3JVZ), these three locations triangulate the E2 interaction region  , and 15N labeled Tsg101 UEV domain . Data for each site are shown in Fig. 1D, with the largest magnitudes seen for site 627, followed by site 867 and finally site 720. The profiles of all three datasets are similar with significant enhancements seen on the UEV side in the V43-S48 region and in an area flanking the vestigial active site (T99-H115). The former is part of an unique extended β-hairpin element in the UEV  that links β-strands 1 and 2. The latter region lies within the central active site region where structural similarity to canonical E2 is greatest (aa53–138)  , 30. We a53–138) .Figure 1in silico labeling of the HECT domain with the lanthanide tag at the relevant PRE sites, and then placing a single Tsg101 UEV domain 5 nm away from the HECT domain. Random rotation and translation of the UEV, followed by rigid body docking of the UEV domain to a fixed HECT domain, allowed an interaction of n UEV domains with a single HECT domain to be emulated with an ensemble of multiple HECT-UEV copies. In addition to the PRE data and the standard Xplor bond, angle, improper, and repel energy terms, previous NMR driven modeling of encounter complexes based on PRE or PCS restraints utilized a radius of gyration term to drive the initial docking between domains . The site 867 restraints in the C-lobe were almost immediately satisfied, and only two UEV copies were needed to achieve a mean correlation for the top 10% of ensembles of > 0.9. For site 627, five UEV copies were sufficient, and for the restraints from site 720, ten UEVs were required. Figure 2A shows the results visually for the case where five UEV domains are docked to the HECT domain, with the centers of mass (spheres) shown for the top 10% of UEV domains. In the front view , it is clear that with the current PRE restraints, the UEV domains clustered both in the Ub exosite and in the interlobe region around the canonical E2 site. From the side, the interaction surface extended from the C-lobe, down to below the N-lobe, again with the most density around the interlobe region and E2 binding site.We first screened the results of docking an increasing number of UEV domains to a 4BBN conformation HECT domain A. The siont view A, middle4BBN , the structure of Nedd4-1 with the A889F mutation  results in a “tilted” lobe conformation as in the structures of the Nedd4 isoform WWP1, including PDB ID: 1ND7 , and a visual representation of the results for five UEVs  again features clustering of the UEV domains into the interlobe region near the E2 binding site reaching toward the Ub exosite.The C-lobe of the HECT domain is thought to have significant conformational flexibility that may pertain to its E3 ligase function, and structures of HECT ligases have been determined in a variety of N- to C-lobe orientations. To partially encode this flexibility into the modeling, we added two additional starting structures. In addition to the “T” shaped conformation seen in PDB ID: 4BBN A, left, ID: 1ND7 , 33. In ID: 1ND7 B, left, 5C7J , the C-lobe is twisted such that the catalytic cysteine points away from the E2 and noncovalent Ub-binding sites and instead points toward the α1-helix element on the opposite surface of the N-lobe , with the primary change in the visual results with five UEV domains (C) being an increase in the apparent spread of the ensembles. For the lobe conformation seen in 5C7J, the interaction at site 867 would indicate either the presence of a secondary binding surface on the opposite face of the N-lobe or the extension of the current binding surface to that face.Alternatively, in the “L” shaped HECT structures such as PDB ID: 5C7J C, left, e N-lobe . This moe N-lobe B, middle domains C being aA) in the α1-helix. The resulting PRE data (B) again showed a HECT-UEV interaction, with the same preferences for the β-hairpin and the region proximal to the vestigial active site as in the other datasets. Modeling this interaction showed good correlation for both five and 10 membered ensembles Fig. S1B, right). A representation is shown for the five UEV copies case in Fig. 3C, although with only two restraints the interaction appears to cover most of the surface of the HECT domain. Still, this points to either a secondary interaction near the α1-helix or to a contiguous interaction surface connecting the exosite, E2 site, and α1-helix region.To distinguish the presence of a second binding surface on the opposite face of the N-lobe from an extension of the front to the back surface, we added a PRE restraint at site 528 A in the PRE data B again s, i.e., below the cutoff used in a previous characterization of the adrenodoxin: cytochrome c complex  and 8-fold for a Gag variant encoding a disrupted PTAP motif (not shown). For infectivity, previous studies reported 15- to 40-fold increases while we observed 50- to 100-fold stimulation. As in previous studies, Nedd4L/Nedd4-2s overexpression did not alter cellular Gag protein levels, indicating that the titer increases were not simply due to elevated Gag expression. Finally, previous studies also found that deletion of the Nedd4L C2 conferred the ability to promote CA maturation inside the cell, compared to the full-length protein or deletion of the WW domain 2  conversion to mature CA to reduction in budding delay . This spPrevious studies demonstrated that Nedd4-mediated rescue requires active enzyme and Tsg101 expression, although Tsg101 binding to Gag is not required , 18. Thepanel A). Notably, only K673A strongly reduced viral particle release efficiency (panelB) with most other mutants having WT level efficiency, indicating that although both budding (through effective ESCRT III recruitment) and infectivity are aspects of Nedd4-2s-mediated rescue, the HECT domain and HECT-UEV interaction primarily contribute to maturation and infectivity. Consistent with this, two mutants: K673A and E755A, showed both a strong reduction in infectivity and an inability to promote dose-dependent CA maturation (panel C). K673A lies in the interlobe hinge while E755A is part of the E2 binding site in the structure of Nedd4-2 in complex with UbcH5 (PDB ID: 3JVZ). Still, in other lobe conformations, including the alternate tilted conformation (as in PDB ID: 4BE8) used in the modeling of the HECT-UEV interaction by NMR, E755A may form its own interlobe contacts (C).Starting with infectivity, severe impairments were seen for ala substitution of K673 (K673A), T688 in combination with D689 (TD688/689AA), and E755 (E755A) in the interlobe hinge and E2 binding regions of the HECT, respectively , panel Acontacts . The fincontacts C.Figure D onto the PDB ID: 3JVZ structure of Nedd4L. Determinants of infectivity (red) are localized in and around the interlobe hinge (orange), with K673A and TD688/689A being located in the N-lobe portion of the hinge region, while E755A straddles the hinge and the E2 binding site (green). Two determinants of CA maturation expand this picture, with Y917A in the C-lobe being proximal to the TD688/689AA pair, while E785A is located in the β-hairpin portion of the E2 binding site near E755A. Interestingly, while the HECT-UEV interaction identified by NMR included the noncovalent Ub binding exosite in the N-lobe (yellow), mutation of YY679/680 in this region had no significant impacts. These residues are the Nedd4-2s equivalents of YY604/605 which have previously been characterized as determinants of noncovalent Ub association at the exosite, implying that viral rescue by Nedd4-2s and the HECT-UEV interaction are not dependent on Ub binding in this region . The proximal E755A mutation, between the interlobe site and E2 binding site, does not appear to contact the C-lobe in this conformation, but introducing even a minimal representation of conformational heterogeneity by way of a morph to the tilted conformation seen in the 4BE8 structure of Nedd4-1 reveals that this location also likely contacts the C-lobe  or no (TD688/689AA) release efficiency defect compared to K673A. Additionally, both K673A and E755A also displayed strongly impaired ability to promote CA maturation. This range of observed phenotypes may stem from the conformational flexibility of the HECT, which is thought to regulate HECT function . Consiste C-lobe . Within These results suggest that stabilization or maintenance of HECT conformation is critical to our observed CA maturation and infectivity defects. On this basis, we propose that the Tsg101 interaction is involved in alteration of relative interlobe conformation, affecting HECT domain enzymatic activity pertinent to the rescue functions. We thus propose that the HECT–UEV interaction may modulate the conformational state of the HECT domain either in a regulatory fashion to alter HECT processivity or in the context of Tsg101 as a HECT substrate.in vivo. The solution NMR results with Nedd4-1 HECT are consistent with a broad interaction involving the canonical E2 binding site together with the noncovalent Ub exosite and the α1-helix region. Mutations in the E2-binding site and exosite of the related Nedd4-2s isoform tracked with an HIV-1 reporter assay revealed a narrower region of impact, where defects in CA maturation and specific infectivity are limited to the interlobe region. Together, our results suggest that the HECT-UEV interaction may play a role in the conformational landscape of the HECT protein, where the HECT-UEV interaction may either perturb the conformation of the HECT or stabilize it in an advantageous conformation. The results also provide novel insight into the manner in which Tsg101, a UEV protein, participates with the Ub E3 ligase Nedd4 in rescue of virus production. These studies could contribute to identification of targets in the enzyme useful for anti-viral drug design.In summary, the results presented here provide support for a novel protein–protein interaction involving the UEV domain of Tsg101 and the HECT domain of Nedd4-1 and its closely related isomers (Nedd4-2s and Nedd4-2 (Nedd4L). The encounter-like complex described here may be a transient state where one or more of the interfaces modeled precede a final, low energy state of the HECT-UEV interaction, or there may be one or more additional partner proteins Nedd4-1 (hNedd4-1) sequence. An N-terminal His6-tagged hNedd4-1 HECT domain expression plasmid was engineered using the In-Fusion (Takara Bio) recombinational cloning approach. The hNedd4-1 HECT domain coding sequence (corresponding to amino acid sequence 520–900) from the pCI-neo.mCherry-Nedd4 vector (6-tagged N-lobe HECT domain (corresponding to amino acid sequence 520–780) was produced by introducing a point-nonsense mutation at residue G781 in the null cysteine N-terminal His6-tagged hNedd4-1 HECT domain. Two overlapping primers containing an Opal codon were designed with the QuikChange Primer Design Program (Agilent) and synthesized by Eurofins; the QuikChange II XL Site-Directed Mutagenesis Kit was used following the manufacturer recommendation. Similarly, two sets of primers were designed to re-engineer two single cysteine mutants of the N-lobe HECT domain, namely F528C and S720C. The integrity of all the coding sequences was verified (Psomagen).All HECT domain constructs were created based on the human 4 vector  was seam6-tagged hNedd4-1 HECT domain or one of the N-terminal His6-tagged N-lobe HECT domain single cysteine constructs, were grown overnight at 37 °C in 1 L of Luria-Bertani broth  containing 50 μg/ml kanamycin (Sigma-Aldrich) and 34 μg/ml chloramphenicol (Sigma-Aldrich). After dilution with 1 L of corresponding media containing the antibiotics, the cells were grown at 37 °C for 2 h before induction with 1 mM IPTG (EMD Millipore) for 20 h at 18 °C. The cell pellets, harvested at 6000 rpm for 20 min at 15 °C, were resuspended in 100 mM Tris·HCl pH 7.4 buffer, containing 500 mM NaCl, 0.5 mM TCEP, 10% (v/v) glycerol, and one cOmplete EDTA-free Protease Inhibitor Cocktail tablet (MilliporeSigma) and disrupted by two passages through an M-110P Microfluidizer . Cell debris removal was carried out at 185,500×g for 60 min at 4 °C. A 5 M imidazole (Sigma-Aldrich) solution adjusted to pH 8.0 was added to the supernatants to a final concentration of 40 mM. The supernatants were loaded onto a 5 ml HisTrap HP column (Cytiva) equilibrated with 25 mM Tris·HCl pH 7.4 buffer containing 500 mM NaCl, 1 mM TCEP, and 20 mM imidazole. The proteins were eluted, using a 100 ml linear gradient to 500 mM imidazole in the same buffer, as single peaks at a maximum imidazole concentration of ∼300 mM. The protein-containing fractions considered pure by SDS-PAGE were pooled, and their concentrations were estimated using their measured A280 and their respective calculated molar extinction coefficient (http://web.expasy.org/protparam/). TEV protease cleavages, using 1:100 enzyme over protein at A280, were carried out in 10K MWCO Slide-A-Lyzer Dialysis Cassettes (ThermoFisher Scientific), at RT overnight with a concomitant buffer exchange to 25 mM Tris·HCl pH 7.4 buffer containing 500 mM NaCl and 1 mM TCEP. The His-tagged proteins—TEV and uncleaved HECT domains—were removed by filtering the dialysates through a 5 ml HisTrap HP column (Cytiva) equilibrated in 25 mM Tris·HCl pH 7.4 buffer containing 500 mM NaCl, 20 mM imidazole, and 1 mM TCEP. After SDS-PAGE analysis, the protein-containing fractions were pooled and buffer exchanged into 50 mM Tris·HCl pH 7.4 buffer, 500 mM NaCl, and 5% (v/v) glycerol by ultrafiltration using an Amicon, 3K MWCO (Millipore). The concentration of the samples was estimated using their A280, and the identity of the proteins was confirmed by MS analysis (Agilent 6224 ESI-TOF LC-MS). Wild-type Tsg101 UEV domain (residues 2–145) was expressed and purified as a 15N-labeled protein, as described previously (Rosetta 2 (DE3) pLysS competent cells (MilliporeSigma), transformed with the N-terminal Hiseviously .15N-Tsg101 and concentrated in an Amicon ultracentrifugal filter (3K MWCO) to 250 μl for NMR measurement in a Shigemi tube. Final concentration of the NMR samples was 125 μM of 15N-Tsg101 to 150 μM of HECT. All samples were in a buffer containing 50 mM Tris (pH 7.4), 500 mM NaCl, 5% glycerol, and 7% 2H2O.Natural abundance HECTs with single cysteine at residue 528, 627, 720, and 867 were tagged with diamagnetic Lu-DOTA-M8-SPy or paramagnetic Tm-DOTA-M8-Spy for pseudo contact shifts, or Gd-DOTA-M8-Spy for PRE measurements, as described previously . BrieflyN-Γ2) were calculated as the difference in the transverse relaxation rates (R2) between the paramagnetic and diamagnetic samples (HN-Γ2 = R2para – R2dia). Amide proton transverse relaxation rates (R2) for paramagnetic and diamagnetic samples were acquired using an interleaved 15N–HSQC-based pulse sequence with two time points (20-ms separation) and 128 scans as previously described  to ensure that no disulfide bind formation occur between the DOTA tag and Cys residues in UEV domain, for which we observed no PRE.All NMR spectra were acquired at 300 K on Bruker 600 MHz spectrometer equipped with cryoprobe. Spectra were processed using NMRPipe  and analescribed . The sam4BBN, 4BE8, and 5C7J. The native cysteine at site 627 was missing from the 4BBN and 4BE8 structures, and so residues 620 to 630 from 5C7J were first spliced into the 4BBN and 4BE8 starting structures. All structures were then in silico labeled with two copies of the M8-DOTA-SPy tag (CTSA in Xplor-NIH) at the 528, 627, 720, and 867 positions, as needed. The UEV domain structure was taken from PDB ID: 4YC1. Initial ensemble inputs were generated by positioning the UEV domain 4 to 5 nm away from either the 627 or 528 sites on the HECT N-lobe, for the two binding representations shown in All structure calculations used Xplor-NIH, version 3.1. The initial structures of the NEDD4-1 HECT domain were taken from PDB IDs: Docking used the standard Xplor BOND, ANGL, IMPR, repel, and torsionDB energy terms. PRE restraints used the “SBMF” option, and the “correlation” mode PCS restraints used the rdcPot term with setUseDistance set to True. For all runs, backbone atoms were held rigid and sidechain atoms were mobile in both the HECT and UEV domains, with the exception of the CTSA tag DOTA cage and metal center atoms, which were grouped. The location of the HECT domain was then held fixed during docking, while the UEV domain was allowed to move as a rigid body.For the main docking protocol, following randomization of sidechain torsions, an initial 100 step sidechain-only minimization was used to orient sidechains. Subsequently, the UEV domain was translated by a random vector within a 6 nm sphere about its starting position, and 200 steps of joint minimization with repel and radius of gyration terms were used to “dock” the UEV domain to the HECT. After, the UEV domains were rotated randomly about their center of mass 20 times, selecting for the configuration that gave the highest joint PRE correlation. This initial process of translation, docking, and rotational search was repeated 10 times, with the best configuration used in subsequent steps.Following the initial docking search, 1500 steps of torsion-angle minimization with all potential terms were followed by an additional 1500 steps without the radius of gyration term, to fix residual clashes. Finally, 500 steps of cartesian minimization with all energy terms except the radius of gyration was perfomed with rigid backbone atoms and free sidechains, before final structures were written. For all runs, we used the best ensemble or statistics on the best 10% of ensembles, as written. All runs were a total of 100 written ensembles. The addition of simulated annealing was not found to significantly improve results over the use of Powell minimization.Results were analyzed using the standard Xplor-NIH outputs and in-house python scripts. All visualizations were generated using pymol and plots were generated using the seaborn, matplotlib, or plotly python packages.HIV-1 pNL4-3ΔEnv was as previously described .The p6 Lg for 15 min. Supernatants were added to sample buffer and examined by Western blotting. For analysis of the virus-like particles, media from the cells were filtered (0.45 micron) and then pelleted through a 20% sucrose cushion by centrifuging at 20,000g, 90 min. After centrifugation and washing, pellets were resuspended in sample buffer and examined by Western blotting. Primary antibodies were Rb anti-CA  were transfected using Roche X-tremeGene transfection reagent (Sigma-Aldrich). At 24 h, cells were collected, washed, and lysed  and centrifuged at 1000 anti-CA ; mouse aor assay . GraphPaAll data are contained within the article and the supporting information, except for raw NMR spectra. They are available upon request to the corresponding author.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Angelman Syndrome (AS) is a severe neurodevelopmental disorder, caused by the neuronal absence of the ubiquitin protein ligase E3A (UBE3A). UBE3A promotes ubiquitin-mediated protein degradation and functions as a transcriptional coregulator of nuclear hormone receptors, including the glucocorticoid receptor (GR). Previous studies showed anxiety-like behavior and hippocampal-dependent memory disturbances in AS mouse models. Hippocampal GR is an important regulator of the stress response and memory formation, and we therefore investigated whether the absence of UBE3A in AS mice disrupted GR signaling in the hippocampus. We first established a strong cortisol-dependent interaction between the GR ligand binding domain and a UBE3A nuclear receptor box in a high-throughput interaction screen. In vivo, we found that UBE3A-deficient AS mice displayed significantly more variation in circulating corticosterone levels throughout the day compared to wildtypes (WT), with low to undetectable levels of corticosterone at the trough of the circadian cycle. Additionally, we observed an enhanced transcriptomic response in the AS hippocampus following acute corticosterone treatment. Surprisingly, chronic corticosterone treatment showed less contrast between AS and WT mice in the hippocampus and liver transcriptomic responses. This suggests that UBE3A limits the acute stimulation of GR signaling, likely as a member of the GR transcriptional complex. Altogether, these data indicate that AS mice are more sensitive to acute glucocorticoid exposure in the brain compared to WT mice. This suggests that stress responsiveness is altered in AS which could lead to anxiety symptoms. UBE3A gene is located on chromosome 15 in a region (15q11.2–13.1) that is subjected to neuron– and paternal–specific genomic imprinting, making the maternal allele of UBE3A the sole source of UBE3A protein in mature neurons. Chromosomal aberrations such as deletions, uniparental disomy, imprinting defects, or point mutations that affect the integrity of the maternal UBE3A gene lead to the complete loss of neuronal UBE3A, resulting in AS [Angelman Syndrome (AS) is a neurodevelopmental disorder characterized by severe intellectual disability, motor deficits, anxiety, speech impairments, ataxia, sleep disturbances, and epilepsy ,2,3. AS ng in AS ,7,8,9,10egion 15q.2–13.1 tng in AS ,13,14. Mng in AS ,17,18. Nng in AS ,20,21,22ng in AS , the exaThe glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a critical role in stress response and subsequent adaptation ,24,25,26p = 0.004) , active nuclear (UBE3AWT) and cytosolic UBE3A (UBE3AdAZUL) modestly—but non-significantly—reduced GR levels, as compared to the empty vector (EMPTY). These modest effects could be rescued by inhibiting the proteasome  binding motifs, also known as nuclear receptor boxes (NR-boxes), of 64 known nuclear receptor coregulators were incubated with the GR LBD that was tagged with Glutathione S-transferase (GST). The binding of GR LBD to a coregulator NR-box was detected with an anti-GST antibody coupled to a fluorophore. The modulation index (MI) represents the normalized immunofluorescent signal in the presence of cortisol A. Out of= 0.004) . We furtse cells C. Since oteasome D. Howeveoteasome E. Additioteasome F. Taken p < 0.001, We next evaluated the role of UBE3A in GR signaling in vivo. To assess whether UBE3A deficiency in AS mice could interfere with HPA axis activity and alter circadian glucocorticoid release, we measured baseline plasma corticosterone (CORT) levels in UBE3A-deficient AS mice and WT littermates. As expected, WT and AS mice showed significantly higher plasma CORT levels at the beginning of their active phase (PM) as compared to the resting phase (AM) (p < 0.001) and found that UBE3A levels were not influenced by acute CORT treatment , the expression of 283 genes was altered in the hippocampus of WT mice , transcription coactivators , chromatin remodelers , transcription factors , proteins involved in nuclear receptor import (Ipo13) or protein stability . The top 10 enriched pathways according to the REACTOME database were predominantly involved in mRNA splicing, chromatid cohesion  and neurotransmitter signaling   . This suMir5125 which was not captured by 10× scRNA-seq, and Adnp. The expression of genes appeared heterogenous throughout different hippocampal cell types, e.g., Rfx3 and Rasal2 were highly expressed in the glutamatergic neurons of the dentate gyrus (DG), while Ccdc43 and Prpf33a expression was higher in CA2 glutamatergic neurons, and Ezr expression was specific for astrocytes , and that continuous CORT treatment did not influence UBE3A protein expression in the frontal cortex  and continuous CORT exposure led to a significant decrease in GR levels only in AS mice  was attributed to the genotype (AS vs. WT) and treatment (CORT vs. vehicle) effects. Comparable to hippocampal data, WT and AS mice showed most overlap upon vehicle treatment and diverged after CORT treatment, which means that most of the effects were driven by CORT treatment C. PC2 coUBE3A deficiency in AS has been previously associated with disrupted GR signaling in the hippocampus and impairments in circadian rhythm ,35. In tGR transcriptional activity depends on its interactions with coregulator proteins, which bind to either its N-terminal or its C-terminal coregulator binding domain. C-terminal coregulators, associated with the LBD and AF2, possess leucine-rich motifs (LxxLL) which are required to interact with the GR and determine the transcriptional outcome ,40. UBE3The interaction of UBE3A with GR could precede its ubiquitination and subsequent proteasomal degradation. However, the overexpression of active UBE3A did not significantly decrease GR stability in cells, which indicates that GR is not a typical UBE3A substrate and suggests that the coregulator function of UBE3A may be ligase independent ,22. In lWe established that AS mice displayed almost undetectable levels of CORT during the circadian trough, indicating that their HPA axis activity was influenced by UBE3A deficiency. The circadian dynamics of CORT release in the circulation are centrally regulated in the hypothalamic paraventricular nucleus (PVN), which receives negative feedback via the GR . PreviouKO cells, and previous findings in HeLa and Neuro2a cells, where UBE3A acted as a GR transcriptional coactivator [Our RNA-seq analysis provided genome-wide evidence of an altered hippocampal transcriptome in AS mice following acute CORT exposure. This treatment affected 10 times as many genes in the AS hippocampus as in WT, which suggests that under normal circumstances UBE3A limits GR signaling in the mouse hippocampus. This is in contradiction with our in vitro results where GR response to cortisol was decreased in UBE3Activator ,33. The Smarce1, which was recently associated with an AS-like phenotype in patients with no molecular diagnostic [The majority of the genes that contributed to the acute CORT differences in the AS mouse hippocampus were associated with transcriptional activity; including transcription factors, coactivators, and chromatin remodelers such as agnostic . Furtheragnostic , and theagnostic . There iagnostic ,51,52,53agnostic ,55,56,57Ube3a during maturation and thus still express UBE3A [When mapping the genes that contributed to the differential transcriptomic effects in the hippocampus of AS and WT mice to our scRNA-seq database, we found that their expression was not restricted to neurons. Several corticosterone-responsive genes were also expressed—sometimes exclusively—in astrocytes, oligodendrocytes, endothelial cells, or microglia. In contrast with AS neurons that are devoid of UBE3A protein, non-neuronal AS cells like astrocytes and oligodendrocytes do not undergo epigenetic silencing of the paternal allele of ss UBE3A . Our resIn sharp contrast with our findings after acute treatment, no significant genotype-treatment interaction was found in the hippocampus after continuous CORT treatment. This confirms the differences in GR response to acute and continuous CORT treatment in the brain, as expected based on previous findings with acute and chronic stress paradigms ,60. Our In conclusion, our current study provides novel insight into the molecular interaction between the UBE3A and GR. We demonstrate that AS mice display reduced circulating corticosterone levels during the circadian trough, and a vastly different transcriptomic response in the hippocampus following acute corticosterone treatment. We believe that our findings contribute to a better understanding of the molecular mechanisms of altered glucocorticoid responsiveness in AS. For future research, the corticosterone treatment in AS mice provides a suitable model to investigate the effectiveness of GR antagonists that could compensate for the altered glucocorticoid sensitivity in AS mice, and such treatment strategies could potentially benefit AS patients that suffer from stress and anxiety symptoms ,63,64,65® array. The GR LBD tagged with Glutathione S-transferase (GST) was overexpressed in HEK 293T cells. The coregulator-derived motifs were incubated with the HEK 293T cell lysates containing GR LBD-GST, the ligand (DMSO or 1 µM cortisol), and a GST-specific antibody coupled to a fluorophore. The interaction between GR LBD and the coregulator motifs was assessed by detection of the fluorophore immunofluorescent signal. For all coregulator motifs, each treatment condition had four replicates from which the mean and the standard error of the immunofluorescent signal were calculated. The modulation index (MI) of each coregulator motif was calculated as the log10-transformed ratio of the mean signal in the presence of cortisol to the mean signal in the presence of DMSO. The standardized MARCoNI assay numbered the LxxLL domain amino acids based on the sequence of the human isoform 2 of UBE3A, however this isoform is lowly expressed [The GR is a nuclear receptor with two activation function domains (AF1 and AF2), a DNA-binding domain (DBD) and a ligand binding domain (LBD). GR interactions with coregulator motifs via its LBD were assessed in the presence of 1 µM cortisol compared to vehicle , as previously described . A totalxpressed . Therefoxpressed , was furKO) HEK 293T cells [®HD Transfection Reagent  according to the manufacturer’s instructions. Per well, 10 ng human GR plasmid, 25 ng tyrosine aminotransferase (Tat) GR response element coupled to the firefly luciferase (TAT1—GR-dependent reporter gene) [® Reporter Assay . The luminescent signal was measured with SpectraMax®iD3 , using SoftMax Pro 7.0.2. Each treatment condition had four replicates and the experiment was performed twice. All data are expressed as the ratio between the reporter gene and the control reporter gene luminescent signal.Wildtype (WT) and UBE3A knock-out (UBE3A3T cells  were seeer gene) , 1 ng CMKO HEK 293T cells were seeded at a density of 150,000 cells/well in 12-well plates and cultured in DMEM (1X) + GlutaMAXTM  supplemented with 10% fetal calf serum (FCS), penicillin, and streptomycin. After 24 h, the cells were transfected with plasmids containing the human GR (750 ng) in combination with different human UBE3A isoform–1 variants  (750 ng) using polyethylenimine (PEI) in a ratio DNA:PEI of 1:3. After 48 h, the cells were treated for eight hours with either vehicle (DMSO) or 1 µM cortisol, alone or in combination with 10 µM MG132 (proteasome inhibitor) or 70 µg/mL cycloheximide (CHX—de novo protein synthesis inhibitor). Hereafter the cells were harvested and snap-frozen in liquid nitrogen until further analysis. Cell pellets were lysed in lysis buffer , supplemented with the c0mplete Protease Inhibitor Cocktail . Protein concentrations were determined using BCA Protein Assay Kit  and a total of 10 µg protein per sample was loaded and run on a 26-well 4–15% Tris-Glycine gel . The separated proteins were transferred to a 0.2 mm nitrocellulose membrane  and blocked in TBST , supplemented with 5% (w/v) skim-milk powder  for 30 min at room temperature. Membranes were briefly rinsed with TBST, and incubated with primary antibodies overnight at 4 °C. The day after, the membranes were briefly washed in TBST, before incubation with secondary antibodies for an hour at room temperature. Finally, the membranes were washed three times with TBST and three times with TBS and analyzed by measuring enhanced chemiluminescence or infrared fluorescence . The primary and secondary antibodies listed in UBE3AProtein band intensities were measured using ImageStudioLite (Version. 5.x 4.0 3.1.) and normalized against β-Actin or GAPDH protein expression. The experiment was performed four times with three to four replicates per condition. The average of all replicates was calculated in each assessment giving a total N = 4 for the entire experiment. GR protein levels were expressed as a normalized percentage from the vehicle-treated control group.Ube3ap−m+/ mice  [Ube3am−/p+ (AS) offspring. The Ube3atm1Alb strain resulted from the deletion of the exon 5 of Ube3a, leading to an out-of-frame mutation and loss of UBE3A protein expression in neurons. Adult 10–18-week-old male mice were used in the experiments. Mice were housed in conventional cages with a 12:12 h light-dark cycle (7:00 AM lights on and 7:00 PM lights off) and ad libitum access to food and water. All animal experiments were conducted at the Erasmus MC in Rotterdam, in accordance with the European Commission Council Directive 2010/63/EU .WT C57BL/6J male and female 129sv 2181811) , were crn = 12) and AS (n = 12) mice via tail vein incision, at 8:00 (AM) and 18:00 (PM), to measure baseline circulating corticosterone. Blood samples were kept on ice and centrifuged at 12,000 rpm for five minutes. The plasma was then collected and stored at −20 °C until further processing. On day 5, animals were injected subcutaneously with 3 mg/kg corticosterone  or vehicle (5% ethanol in PBS) between 9:00 and 11:00 to mimic a transient increase in corticosterone, as previously described [On day 0 of the experiment, blood was withdrawn from WT  and AS (n = 10) mice underwent surgery to subcutaneously implant a corticosterone slow-release pellet  or a vehicle pellet (100 mg cholesterol) to mimic a sub-chronic corticosterone exposure, as previously described [On day 0 of the experiment, WT  according to the manufacturer’s instructions. To calculate the delta PM-AM at baseline, for each animal, the AM corticosterone levels (ng/mL) were subtracted from the PM corticosterone levels (ng/mL).Frozen frontal cortex and liver tissue were lysed with glass beads in RIPA buffer  supplemented with protease and phosphatase inhibitors  using the homogenizer  at 4 °C, with 1500 rpm for 40 s. Protein lysates were transferred in a fresh Eppendorf and protein concentrations were determined using a BCA protein assay kit . All protein lysates were diluted in RIPA to the final concentration of 0.8 µg/µL. Protein expression was measured using the Simple Western™ WES system  with the anti-mouse  or anti-rabbit  detection modules according to the procedure provided by the manufacturer. The primary antibodies listed in ® RNA kit  and RNA quality was assessed using the RNA 6000 Nano kit on the Bioanalyzer . All samples had an appropriate RNA Integrity Number (RIN) over 8 with a 28/18s ratio over 1 and were considered suitable for sequencing. Aliquots of total RNA samples were sent for RNA-seq at BGI Genomics . Stranded mRNA libraries were constructed, and 100-bp paired end sequencing was performed on the DNBseq platform resulting in over 20 million reads per sample. The BioWDL pipeline was used for reads quality control, alignment, and quantification. Quality control was performed using FastQC and MultiQC. Reads were aligned to mm10 using STAR and gene-read quantification was performed using HTSeq-count . Output files were merged into a count matrix as input for differential gene expression analysis. DESeq2 (version 1.30.1) was used for normalization of the data (median of ratio’s method) and identification of differentially expressed genes in R v4.0.2. Differential exon usage analysis was performed in the mouse cohort exposed to continuous corticosterone to validate UBE3A deficiency at the mRNA level in the AS model . The contrasts between groups were analyzed for differential expression in a pair-wise comparison.https://string-db.org/, accessed on 22 December 2022) [p-values are corrected for multiple testing using the Benjamini-Hochberg approach. Enrichment significance is expressed as the —log10(FDR).The subset of genes contributing to the differential response to acute corticosterone in AS mice underwent gene ontology (GO)-term and pathway enrichment analyses using the STRING database v11.5 (er 2022) . We seleThe top 20 downregulated and top 20 upregulated genes associated with the differential response to acute corticosterone in AS mice were selected for single-cell expression analysis in the adult mouse hippocampus, as we previously described . Our pubKO HEK-293T cells, statistical significance was calculated using the two-way analysis of variance (ANOVA) in GraphPad Prism 9. The EC50 values for both cell lines were determined using GraphPad Prism 9 non-linear fitting and expressed as mean and standard deviation (±SD). The results of GR stability in UBE3AKO HEK-293T cells, mouse plasma corticosterone and tissue protein measurements were corrected for outliers using the Grubb’s method, all results were expressed as mean and standard error of the mean (±SEM) and statistical significance was calculated using the two-way ANOVA or the Mann–Whitney test in GraphPad Prism 9. In the RNA-seq analysis, an FDR-adjusted p-value of 0.05 was used as a cut-off to determine differentially expressed genes.In the MARCoNI assay, statistical significance was determined by a t-test in R v.4.0.2. For the assessment of GR activity in WT and UBE3A