RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.
Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , SARS-CoV-2/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas do Nucleocapsídeo de Coronavírus/química , Glicosilação , Células HEK293 , Humanos , Fosforilação , SARS-CoV-2/químicaRESUMO
The COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Similar to other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. S, E, and M proteins are glycosylated, and the N protein is phosphorylated. The S protein is involved in the interaction with the host receptor human angiotensin-converting enzyme 2 (hACE2), which is also heavily glycosylated. Recent studies have revealed several other potential host receptors or factors that can increase or modulate the SARS-CoV-2 infection. Interestingly, most of these molecules bear carbohydrate residues. While glycans acquired by the viruses through the hijacking of the host machinery help the viruses in their infectivity, they also play roles in immune evasion or modulation. Glycans play complex roles in viral pathobiology, both on their own and in association with carrier biomolecules, such as proteins or glycosaminoglycans (GAGs). Understanding these roles in detail can help in developing suitable strategies for prevention and therapy of COVID-19. In this review, we sought to emphasize the interplay of SARS-CoV-2 glycosylated proteins and their host receptors in viral attachment, entry, replication, and infection. Moreover, the implications for future therapeutic interventions targeting these glycosylated biomolecules are also discussed in detail.
Assuntos
COVID-19/virologia , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Configuração de Carboidratos , Glicosilação , Humanos , Polissacarídeos/química , Polissacarídeos/metabolismo , Conformação Proteica , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
BACKGROUND: Silk fibroin is an emerging biomaterial with enhanced properties of cellular regeneration, growth and proliferation. The use of a silk fibroin wound dressing has the potential to decrease the incidence of wound healing complications and to improve patient outcomes compared to synthetic dressing alternatives. METHODS: A prospective, randomized, single-blinded clinical trial was conducted on 50 patients who were dressed with a silk fibroin dressing on one side of their body and on the contralateral side with 3M Steri-Strips® after undergoing abdominoplasty, reduction mammaplasty, or brachioplasty procedures. Data was collected over 5 postoperative visits using photographs and an investigator administered questionnaire to monitor erythema, skin irritation, skin discomfort, the need for pharmaceutical intervention, wound dehiscence and mechanical skin injury. A comprehensive 75 patient statistical analysis was conducted combining the results with a previously published study comparing Dermabond® Prineo® to the silk dressing. RESULTS: 20.8% (10/48) of patients were assessed by surgeons as having skin erythema (7-10) on the Steri-Strip® control side and 0% (0/48) on the silk dressing side (p=0.002). The frequency of breast triple point separation in 43 cases was 30.2% (13/43) on the Steri-Strip® side and 9.3% (4/43) on the silk side (p=0.012). 75% (36/48) of patients had partial or total detachment of Steri-Strips® while 0% (0/48) had total detachment of the silk dressing and 18.8% (9/48) had partial detachment of the silk dressing within the first two weeks (p<0.001). CONCLUSION: A silk fibroin wound dressing significantly reduces the incidence of wound healing complications throughout the postoperative period.Clinical Relevance Statement: The adoption of a silk fibroin wound dressing into clinical practice has the potential to improve patient outcomes, decrease medical adhesive related skin injuries and reduce the rate of wound healing complications.
RESUMO
Background: Medical adhesive-related skin injuries (MARSIs) affect about 1.5 million patients annually in the United States. Complications include allergic contact dermatitis, skin blistering, skin tears, and surgical-site infections (SSIs). The authors hypothesize that a natural hypoallergenic silk bioprotein wound dressing will decrease the incidence of MARSI in comparison to a synthetic alternative. Objectives: This study aimed to assess the efficacy and safety of a silk bioprotein wound dressing compared to the Dermabond Prineo (Ethicon, Inc., Somerville, NJ) skin closure system. Methods: This prospective, randomized, single-blinded trial studied 25 patients who were dressed with Dermabond Prineo on one side of their body and on the contralateral side with the silk bioprotein dressing after undergoing abdominoplasty or reduction mammaplasty procedures. Data were collected over 5 postoperative visits using photographs and an investigator administered questionnaire to track rash, itch, discomfort, erythema, edema, SSIs, need for pharmaceutical intervention, mechanical injury, removal time, and bathing routines. Results: Sixty-four percent (16/25) of patients characterized the severity of discomfort as a score of 4 out of 10 or greater on the Dermabond Prineo control side and only 4% (1/25) for the silk-dressing side (P < .001). Fifty-two percent (13/25) had a visible rash of 4 or higher on the Dermabond Prineo side of their incision and 0% (0/25) had a rash on the silk side (P < .001). Fifty-two percent (13/25) required steroids or antibiotics to treat MARSI to Dermabond Prineo and 0% (0/25) required pharmaceutical intervention on the silk side (P < .001). Conclusions: The use of a silk bioprotein wound dressing significantly reduces the incidence of MARSI throughout the postoperative period.