RESUMEN
How severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remains largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected Huh7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a dose-dependent activation of Akt, mTOR, S6K1 and 4E-BP1 at 24 hours post infection (hpi). However, we found a significant inhibition of HIF-1α through 24hpi and 48hpi of the infection, suggesting a crosstalk between the SARS-CoV-2 and the Akt/mTOR/HIF-1 signaling pathways. Inhibition of the mTOR signaling pathway using Akt inhibitor MK-2206 showed a significant reduction in virus production. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe coronavirus disease 2019 (COVID-19) patients.
Asunto(s)
Betacoronavirus/patogenicidad , Infecciones por Coronavirus/virología , Perfilación de la Expresión Génica/métodos , Neumonía Viral/virología , Proteómica/métodos , Transducción de Señal , COVID-19 , Línea Celular , Cromatografía Liquida , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/metabolismo , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismo , Pandemias , Neumonía Viral/genética , Neumonía Viral/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , SARS-CoV-2 , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Espectrometría de Masas en TándemRESUMEN
OBJECTIVE: Cleavage of IGFBPs by proteases results in IGFBP fragments that have altered IGF-binding affinity, and IGF-independent roles. We have previously purified a specific IGFBP-1 protease activity from the urine of an individual with multiple myeloma and dermatitis. The aim of this study was to determine whether IGFBP-1 protease activity and/or IGFBP-1 fragments were present in the circulation of this patient. METHODS: The size of immunoreactive IGFBP-1 in serum samples was determined after Superose 12 chromatography. Intact IGFBP-1 and IGFBP-1 fragments were characterized in four RIAs and after SDS-PAGE. RESULTS: Specific proteolysis of IGFBP-1 generated an N-terminal fragment (IGFBP-1(1-130)) with a predicted molecular mass of 13kDa but an apparent mass of 21kDa on SDS-PAGE. A C-terminal fragment (IGFBP-1(131-234)) produced in vitro migrated at 11.4kDa, close to its predicted size. However a C-terminal fragment of cleaved IGFBP-1 (IGFBP-1(142-234)) migrated at 14kDa on SDS-PAGE. Serum from the patient inhibited IGFBP-1 protease activity. Immunoreactive IGFBP-1 in patient serum was present at molecular masses consistent with IGFBP-1 fragments, in addition to intact IGFBP-1. CONCLUSIONS: Specific cleavage of IGFBP-1 occurs at the tissue level and not in the circulation in a patient with multiple myeloma and dermatitis. The fragments that are generated may have endocrine roles.
Asunto(s)
Dermatitis/sangre , Dermatitis/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Mieloma Múltiple/sangre , Mieloma Múltiple/metabolismo , Anciano , Péptidos Catiónicos Antimicrobianos/metabolismo , Biotinilación , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/metabolismo , Cromatografía/métodos , Femenino , Humanos , Isoformas de Proteínas , Radioinmunoensayo/métodos , Proteínas Recombinantes/química , Sefarosa/químicaRESUMEN
A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.
Asunto(s)
Técnicas Biosensibles , Biotecnología/métodos , Proteínas/metabolismo , Resonancia por Plasmón de Superficie/métodos , Adsorción , Anticuerpos Monoclonales/metabolismo , Avidina/farmacocinética , Biotinilación , Electroforesis en Gel de Poliacrilamida , Humanos , Ligandos , Espectrometría de Masas , Peso Molecular , Proinsulina/análisis , Proinsulina/aislamiento & purificación , Proinsulina/metabolismo , Unión Proteica , Proteínas/análisis , Proteínas/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/farmacologíaRESUMEN
A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.