Search Results (400)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for Coronavirus Disease 2019 (COVID-19), utilizes its spike protein to infect host cells. In addition to angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1), AXL acts as a spike protein receptor and mediates infection, especially in respiratory cells with low ACE2 expression. Angiotensin II (1–8) can be cleaved into shorter peptides within the biological system. Antibody-based binding assays showed that angiotensin II causes a two-fold increase in the binding between the spike protein and AXL, but not ACE2 or NRP1. While a longer peptide, angiotensin I (1–10), did not affect the spike–AXL binding, shorter lengths of angiotensin peptides exhibited enhancing effects. The C-terminal deletions of angiotensin II to angiotensin (1–7) or angiotensin (1–6) resulted in peptides with enhanced activity toward spike–AXL binding with a similar capacity as angiotensin II. In contrast, the N-terminal deletions of angiotensin II to angiotensin III (2–8) or angiotensin IV (3–8) as well as the N-terminal deletions of angiotensin (1–7) to angiotensin (2–7) or angiotensin (5–7) produced peptides with a more potent ability to enhance spike–AXL binding (2.7-fold increase with angiotensin IV). When valine was substituted for tyrosine at position 4 in angiotensin II or when tyrosine at position 4 was phosphorylated, spike–AXL binding was increased, suggesting that modifications to tyrosine trigger enhancement. Angiotensin IV also enhances spike protein binding to ACE2 and NRP1. Thus, angiotensin peptides may contribute to COVID-19 pathogenesis by enhancing spike protein binding and thus serve as therapeutic targets. Full article

Casein Kinase II (CK2) is a ubiquitously present serine/threonine kinase essential for mammalian development. CK2 holoenzyme is a tetramer with two highly related catalytic subunits (α or α’) and two regulatory ß subunits. Global deletion of the α or β subunit in mice is embryonically lethal. We and others have shown that CK2 is overexpressed in leukemia cells and plays an important role in cell cycle, survival, and resistance to the apoptosis of leukemia stem cells (LSCs). To study the role of CK2α in adult mouse hematopoiesis, we generated hematopoietic cell-specific CK2α-conditional knockout mice (Vav-iCreCK2 ^f/f). Here we report the generation and validation of a novel mouse model that lacks CK2α in the hematopoietic compartment. Vav-iCreCK2α ^f/f mice were viable without dysmorphic features and showed a mild phenotype under baseline conditions. In Vav-iCreCK2α ^f/f mice, the blood count showed a significant decrease in total red blood cells and platelets. The spleen was enlarged in Vav-iCreCK2α ^f/f mice with evidence of extramedullary hematopoiesis. HSC and early progenitor cell compartments showed expansion in CK2α-null bone marrow, suggesting that the absence of CK2α impaired their proliferation and differentiation. Given the established roles of CK2 in cell cycle regulation and the findings reported here, further functional studies are warranted to investigate the role of CK2α in HSC self-renewal and differentiation. This mouse model serves as a valuable tool for understanding the role of CK2α in normal and malignant hematopoiesis. Full article

African swine fever virus (ASFV), the causative agent of African swine fever (ASF), poses a catastrophic threat to global swine industries through its capacity for immune subversion and rapid evolution. Multigene family genes (MGFs)-encoded proteins serve as molecular hubs governing viral evolution, immune evasion, cell tropism, and disease pathogenesis. This review synthesizes structural and functional evidence demonstrating that MGFs-encoded proteins suppress both interferon signaling and inflammasome activation, while their genomic plasticity in variable terminal regions drives strain diversification and adaptation. Translationally, targeted deletion of immunomodulatory MGFs enables the rational design of live attenuated vaccines that improve protective efficacy while minimizing residual virulence. Moreover, hypervariable MGFs provide strain-specific signatures for PCR-based diagnostics and phylogeographic tracking, directly addressing outbreak surveillance challenges. By unifying virology with translational innovation, this review establishes MGFs as priority targets for next-generation ASF countermeasures. Full article

PDE11A is a little-studied phosphodiesterase sub-family that breaks down cAMP/cGMP, with the PDE11A4 isoform enriched in the memory-related hippocampal formation. Age-related increases in PDE11A expression occur in human and rodent hippocampus and cause age-related cognitive decline of social memories. Interestingly, age-related increases in PDE11A4 protein ectopically accumulate in spherical clusters that group together in the brain to form linear filamentous patterns termed “PDE11A4 ghost axons”. The biophysical/physiochemical mechanisms underlying this age-related clustering are not known. Here, we determine if age-related clustering of PDE11A4 reflects liquid–liquid phase separation (LLPS; biomolecular condensation), and if PDE11A inhibitors can reverse this LLPS. We show human and mouse PDE11A4 exhibit several LLPS-promoting sequence features, including intrinsically disordered regions, non-covalent pi–pi interactions, and prion-like domains that were particularly enriched in the N-terminal regulatory region. Further, multiple bioinformatic tools predict PDE11A4 undergoes LLPS. Consistent with these predictions, aging-like PDE11A4 clusters in HT22 hippocampal neuronal cells were membraneless spherical droplets that progressively fuse over time in a concentration-dependent manner. Deletion of the N-terminal intrinsically disordered region prevented PDE11A4 LLPS despite equal protein expression between WT and mutant constructs. 1,6-hexanediol, along with tadalafil and BC11-38 that inhibit PDE11A4, reversed PDE11A4 LLPS in HT22 hippocampal neuronal cells. Interestingly, PDE11A4 inhibitors reverse PDE11A4 LLPS independently of increasing cAMP/cGMP levels via catalytic inhibition. Importantly, orally dosed tadalafil reduced PDE11A4 ghost axons in old mouse ventral hippocampus by 50%. Thus, PDE11A4 exhibits the four defining criteria of LLPS, and PDE11A inhibitors reverse this age-related phenotype both in vitro and in vivo. Full article

The pandemic of Coronavirus Disease 2019 has triggered a worldwide public health emergency. Its pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed multiple strategies for effectively evading the host immune defenses, including inhibition of interferon (IFN) signaling. Several viral proteins of SARS-CoV-2 are believed to interfere with IFN signaling. In this study, we found that the SARS-CoV-2 accessory protein ORF7a considerably impaired IFN-activated Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling via suppression of the nuclear translocation of IFN-stimulated gene factor 3 (ISGF3) and the activation of STAT2. ORF7a dampened STAT2 activation without altering the expression and phosphorylation of Janus kinases (JAKs). A co-immunoprecipitation (co-IP) assay was performed to gather ORF7a protein, but it failed to precipitate STAT2. Interestingly, mass spectrometry and immunoblotting analyses of the ORF7a co-IP product revealed that ORF7a interacted with an RNA-binding protein, heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1), and HNRNPA2B1 was related to the inhibitory effect of ORF7a on STAT2 phosphorylation. Moreover, examination of ORF7a deletion constructs revealed that the C-terminal region of ORF7a (amino acids 96 to 122) is crucial for suppressing IFN-induced JAK/STAT signaling activation. In conclusion, we discovered that SARS-CoV-2 ORF7a antagonizes type I IFN-activated JAK/STAT signaling by interacting with HNRNPA2B1, and the C-terminal region of ORF7a is responsible for its inhibitory effect. Full article

The expansion of the polyglutamine tract in ATXN1 contributes to the pathogenesis of SCA1. ATXN1 functions as a transcriptional regulator that interacts with multiple transcription factors, and transcriptional dysregulation has been observed in SCA1. In addition, splicing dysregulation has been identified in cells derived from SCA1 patients and model mouse tissues. Although ATXN1 binds to RNA and splicing factors, its direct involvement in pre-mRNA splicing remains unclear. Here, we demonstrate that ATXN1 regulates the alternative splicing of several minigenes. Using an Mbnl1 minigene, we found that neither expansion nor deletion of the polyglutamine tract affected ATXN1-mediated splicing regulation. Deletion analysis revealed that its splicing regulatory activity involves a central region of ATXN1, the AXH domain, and a nuclear localization signal in the C-terminal region. The AXH domain alone failed to exhibit splicing regulatory activity, whereas the central region demonstrated weak but significant splicing regulation. Full regulatory function required at least one of these regions, suggesting their redundant role in splicing modulation. Importantly, we newly identified the central region as mediating RNA binding. These findings suggest a novel role for ATXN1 in alternative splicing, providing new insights into the mechanisms underlying SCA1 pathogenesis. Full article

Mild starvation due to low concentrations of an inhibitor of glycolysis, 2-deoxy-D-glucose, activates AMP-activated protein kinase (AMPK) and lysine-specific demethylase 2A (KDM2A) to reduce rRNA transcription and cell proliferation in breast cancer cells. However, the mechanisms of how AMPK regulates KDM2A are unknown. Here, we found that PHD finger protein 8 (PHF8) interacted with KDM2A and contributed to the reduction in rRNA transcription and cell proliferation by 2-deoxy-D-glucose in a breast cancer cell line, MCF-7. We analyzed how KDM2A bound PHF8 in detail and found that PHF8 interacted with KDM2A via two regions of KDM2A. One of the regions contained an intrinsically disordered region (IDR). IDRs can show rapidly switchable protein–protein interactions. Deletion of the PHF8-binding region activated KDM2A to reduce rRNA transcription, and 2-deoxy-D-glucose reduced the interaction between PHF8 and the KDM2A fragment containing the PHF8-binding region. A 2-deoxy-D-glucose or AMPK activator dephosphorylated KDM2A at Ser731, which is located on the N-terminal side of the PHF8-binding region. Replacement of Ser731 by Ala decreased binding of PHF8 to the KDM2A fragment that contains the PHF8-binding region and Ser731 and reduced rRNA transcription and cell proliferation. These results suggest that the mode of interaction between KDM2A and PHF8 is regulated via dephosphorylation of KDM2A through AMPK to control rRNA transcription, and control of the phosphorylation state of Ser731 would be a novel target for breast cancer therapy. Full article

Kindler Epidermolysis bullosa (KEB; OMIM 173650) is a rare autosomal recessive genodermatosis characterized by bullous poikiloderma and photosensitivity. Additional presentations include blistering, poor wound healing, skin atrophy, and increased risk of skin cancer. Most cases of KEB result from aberrations in the FERMT1 (Fermitin family member 1) gene encoding kindlin-1 and include nonsense, frameshift, splicing, and missense variants. Large deletion variants have been reported in nine cases to date. Most variants are predicted to lead to premature termination of translation and to loss of kindlin-1 function. In this study, we report on a 33-year-old male patient who presented with typical clinical manifestations of KEB. As routine molecular testing failed to obtain a diagnosis, Next Generation Sequencing (NGS) of an Epidermolysis Bullosa (EB)-specific panel was carried out followed by the determination of the deletion breakpoints and verification at the mRNA and protein levels. This approach revealed a new large homozygous deletion of ~9.4 kb in the FERMT1 gene involving exons 7 to 9. Finally, we performed a literature review on large FERMT1 deletions. The deletion is predicted to skip exons 7 to 9 within the mRNA, which results in a frameshift. The patient’s phenotype is likely caused by the resulting truncated and non-functioning protein. Our report further enriches the spectrum of FERMT1 gene variants to improve genotype–phenotype correlations. Full article

This study aims to enhance membrane protein production in HEK293T cells through genetic modification. HEK293T cells are used for recombinant protein and viral vector production due to their human origin and post-translational modification capabilities. This study explores enhancing membrane protein production in these cells by deleting the C-terminal of the ATF6B gene using CRISPR-Cas9 technology. The objective of this research is to investigate the effect of C-terminal deletion of the ATF6B gene on membrane protein production in HEK293T cells using CRISPR-Cas9 technology. To identify effective gene targets, sgRNAs were initially designed against multiple UPR-related genes, including ATF6A, IRE1A, IRE1B, PERK, and ATF6B. Among them, ATF6B was selected as the primary target for further investigation due to its superior editing efficiency. The efficiency of sgRNAs was evaluated using the T7E1 assay, and sequencing was performed to verify gene editing patterns. Membrane proteins were extracted from both ATF6B C-terminally deleted (ATF6B-ΔC) and wild-type (WT) cell lines for comparison. Flow cytometry was employed to assess membrane protein production by analyzing GFP expression in Membrane-GFP-expressing cells. HEK293T cells with C-terminally deleted ATF6B (ATF6B-ΔC) significantly increased membrane protein production by approximately 40 ± 17.6% compared to WT cells (p < 0.05). Sequencing revealed 11, 14, 1, and 10 bp deletions in the ATF6B-ΔC edited cells, which disrupted exon sequences, induced exon skipping, and introduced premature stop codons, suppressing normal protein expression. Flow cytometry confirmed a 23.9 ± 4.2% increase in GFP intensity in ATF6B-ΔC cells, corroborating the enhanced membrane protein production. These findings suggest that CRISPR-Cas9-mediated C-terminal deletion of the ATF6B gene can effectively enhance membrane protein production in HEK293T cells by activating the unfolded protein response pathway and improving the cell’s capacity to manage misfolded proteins. This strategy presents significant potential for the biotechnology and pharmaceutical industries, where efficient membrane protein production is essential for drug development and various applications. Full article

The Staphylococcus aureus cell wall protein serine rich adhesin for platelets (SraP) belongs to a large surface glycoprotein family of adhesins. Here, we provide experimental evidence that SraP mediates macrophage functions in a human monocyte-derived macrophage model via its N-terminal L-lectin module (LLM) in the ligand binding region. Our flow cytometry data demonstrated that macrophages infected by the LLM deletion strain profoundly impacted apoptosis, reducing the percentage of apoptotic cells by approximately 50%, whereas LLM overexpression significantly increased the percentage of early-stage apoptotic cells (p < 0.001). LLM deletion significantly enhanced phagocytosis by macrophages by increasing the number of engulfed bacteria, resulting in a significant increase in bacterial killing and leading to a notable decrease in bacterial survival within macrophages (p < 0.001). Furthermore, LLM modulated the ability of S. aureus to elicit inflammatory responses. The LLM deletion strain dampened the expression of proinflammatory factors but increased the expression of anti-inflammatory cytokines, such as IL10. Our evidence suggests that SraP likely plays a dual role in S. aureus pathogenesis, by acting as a virulence factor involved in bacterial adhesion and invasion and by mediating macrophage functions. Our future work will focus on the identification of small molecule inhibitors of LLM using molecular docking-based in silico screening and in vivo validation. Developing LLM inhibitors, alone or in combination with conventional antibiotics, may represent a novel strategy for combating S. aureus infections. Full article

In a previous outbreak of the human respiratory syncytial virus (HRSV), we identified a variant strain of genotype BA9 with a seven-amino-acid extension (Q-R-L-Q-S-Y-A) at the C-terminus of the attachment protein (G). To assess the impact of this extension on the virulence of HRSV, two full-length infectious clones using the wild strain of genotype BA9 as a backbone, one containing the seven-amino-acid extension (rRSV BA9 WT), and the other deleting this extension (rRSV BA9 Δ7AA), were successfully rescued using a reverse genetics system. The biological properties and virulence of the two rescued viruses were then compared and analyzed in vitro and in vivo. Compared to the rRSV BA9 Δ7AA, the rRSV BA9 WT exhibited a larger plaque size and a more pronounced suppression of the host cell innate immune response in vitro (IFN-β levels: 154.33 pg/mL vs. 11.27 pg/mL). The rRSV BA9 WT demonstrated increased adaptability in mice, with a 10-fold higher lung viral load and a stronger inflammatory response following intranasal exposure. Our study primarily demonstrated that the C-terminal extension of the G protein of the HRSV can enhance viral virulence, underscoring the importance of virological surveillance in the prevention and treatment of severe HRSV-related disease. Full article

Glial fibrillary acidic protein (GFAP) is classified as a type III intermediate filament protein predominantly expressed in mature astrocytes. It has the ability to self-assemble into 10 nm filaments in vitro, making it particularly valuable for elucidating the sequences essential for filament assembly. In this study, we created a series of deletion mutants targeting sequences in the N-terminal, C-terminal, and central rod domains to explore the sequences critical for the assembly of GFAP into 10 nm filaments. The impact of these deletions on filament formation was evaluated through in vitro assembly studies and transduction assays conducted with primary astrocytes. Our data revealed that deletions at the carboxy end resulted in abnormalities in either filament diameter calibration or lateral association, whereas deletions at the amino-terminal end significantly disrupted the filament assembly process, particularly restricting filament elongation. Furthermore, we discovered that the filament-forming sequences within the rod domain varied in their contributions to filament assembly and network formation. These findings enhance our understanding of the GFAP assembly process in vitro and provide a detailed mapping of the essential regions required for GFAP assembly. These insights hold significant implications for Alexander disease arising from deletion mutations in GFAP. Full article

Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, can rapidly degrade crystalline cellulose through direct cell-to-substrate contact. Most of its cellulases are secreted by the Type IX secretion system (T9SS) and anchored to the cell surface. Our previous study proved that the C-terminal domain (CTD) of the T9SS substrate cellulase Cel9A is glycosylated in C. hutchinsonii. However, its glycosylation mechanism has remained elusive. In this study, we found that chu_3394, which encodes UDP-glucose 6-dehydrogenase (Ugd), was important for the glycosylation of large amounts of periplasmic and outer membrane proteins in C. hutchinsonii. The contents of mannose, glucose, galactose, and xylose were detected to be reduced in the glycoproteins of the ∆ugd mutant compared to that of wild-type. They might be essential monosaccharides that contribute to the structure and function of glycans attached to proteins in C. hutchinsonii. The depletion of mannose, glucose, galactose, and xylose indicates a decrease in glycosylation modifications in the ∆ugd mutant strain. Then, we found that the deletion of ugd resulted in weakened glycosylation modification of the recombinant green fluorescent protein-tagged CTD of Cel9A. Additionally, the outer-membrane localization of Cel9A was affected in the mutant. Besides this, Ugd was also important for the synthesis of O-antigen of lipopolysaccharide (LPS). Thus, Ugd was involved in the synthesis of glycans in both glycoproteins and LPS in C. hutchinsonii. Moreover, the deletion of ugd affected the cellulose degradation, cell motility, and stress resistance of C. hutchinsonii. Full article

Background/Objectives: A mouse antibody directed against truncated Annexin A1 showed high tumor retention in pre-clinical cancer models and was approved by the National Cancer Institute Experimental Therapeutics (NExT) program for humanization and large batch cGMP production for toxicology and clinical trials. In this process, a contractor for Leidos accidentally produced a mutated version of humanized AnnA1 (hAnnA1-mut) with a single nucleotide deletion in the terminal Fc coding region that increased the translated size by eight amino acids with random alterations in the final twenty-four amino acids. We investigated the tissue distribution of hAnnA1-mut, hAnnA1, mAnnA1, and isotope-matched human IgG1 under various injection and conjugation conditions with C57BL/6, FVB, and BALB/c nude mice strains. Methods: Biodistribution studies were performed 24 h after injection of Tc-99m-HYNIC radiolabeled antibodies (purity > 98%). Non-reducing gel electrophoresis studies were conducted with IR680 labeled antibodies incubated with various mouse sera. Results: Our results showed that Tc-99m-HYNIC-hAnnA1 had low spleen and liver retention not statistically different from Tc-99m-HYNIC-IgG1 and Tc-99m-HYNIC-mAnnA1, with corresponding higher blood levels; however, Tc-99m-HYNIC-hAnnA1-mut had high levels in the spleen and liver with differences identified among the mouse strains, radiolabeling conditions, and injection routes. Histopathology showed no morphological change in the liver or spleen from any conditions. Gel electrophoresis showed an upward shift of hAnnA1-mut, consistent with the binding of blood serum protein. Conclusions: The changes in the Fc region of hAnnA1-mut led to higher liver and spleen uptake, suggesting the antibody’s recognition by the innate immune system (likely complement protein binding) and subsequent clearance. Future clinical translation using hAnnA1 and other antibodies needs to limit protein modifications that could drastically reduce blood clearance. Full article

Mowat–Wilson syndrome (MWS) is a rare multi-system genetic disorder caused by variants in the Zinc Finger E-Box-Binding Homeobox 2 (ZEB2) gene. ZEB2 is an autosomal dominant gene containing ten exons within the canonical version transcript (Isoform: O60315-1). The ZEB2 gene encodes six functional domains and seven non-domain regions. This review provides a comprehensive summary of pathogenic variants and their associated MWS clinical characteristics, focusing on ZEB2 pathogenic variants, functional protein domains and non-domain regions with clinical features. A systematic literature search from 2001 to 2023 and of unpublished datasets found 191 individuals with reported clinical features and genotypic data. Genetic defects and clinical manifestations were examined that presumably impact on the structure and function of the ZEB2 gene, thereby causing multiple developmental defects with corresponding clinical presentation. This study found more nonsense ZEB2 variants observed within exon 8, which encodes four of the six protein domains: the CtBP-interacting domain (CID), homeodomain (HD), SMAD-binding domain (SMD or SBD) and part of the N-terminal zinc finger cluster (N-ZF), suggesting exon 8 plays a crucial role in this protein structure and function with multi-organ involvement. Exon 8 defects were found to be statistically more represented for gastrointestinal findings when compared to other exons, while frameshift defects were more often seen for the typical MWS face in non-domain protein regions. In contrast, nonsense or other types of variants in exons 3, 4 and 5 which encode only flanking non-domain regions were observed more often, compared with other exons excluding exon 8, to be specifically involved in the MWS facial gestalt, brain malformations, developmental delay and intellectual disability. Deleterious ZEB2 frameshift (45%) and nonsense (38%) gene variants were most often observed with deletions at 6% and missense at 5%. The genotype and clinical relationships in MWS can provide insights into prognosis, morbidity, clinical surveillance strategies and counseling of family members. Full article