SARS-CoV-2 ORF8 as a modulator of cytokine induction : evidence and search for molecular mechanisms

Funding: This study was supported by Federal University of ABC (UFABC), São Bernardo do Campo, São Paulo, Brazil and by Wellcome Trust ISSF 204821/Z/16/Z to Michael Nevels (Universityof St Andrews, UK).Severe cases of SARS-CoV-2 infection are characterized by an immune response that leads to the overproduction of pro-inflammatory cytokines, resulting in lung damage, cardiovascular symptoms, hematologic symptoms, acute kidney injury and multiple organ failure that can lead to death. This remarkable increase in cytokines and other inflammatory molecules is primarily caused by viral proteins, and particular interest has been given to ORF8, a unique accessory protein specific to SARS-CoV-2. Despite plenty of research, the precise mechanisms by which ORF8 induces proinflammatory cytokines are not clear. Our investigations demonstrated that ORF8 augments production of IL-6 induced by Poly(I:C) in human embryonic kidney (HEK)-293 and monocyte-derived dendritic cells (mono-DCs). We discuss our findings and the multifaceted roles of ORF8 as a modulator of cytokine response, focusing on type I interferon and IL-6, a key component of the immune response to SARS-CoV-2. In addition, we explore the hypothesis that ORF8 may act through pattern recognition receptors of dsRNA such as TLRs.Publisher PDFPeer reviewe

Biochemcal studies of erine proteases inhibitors from Boophilus microplus tick: gene silencing anchieved by RNA interference of a Kunitz-type trypsin inhibitor

BV UNIFESP: Teses e dissertaçõe

Molecular studies of serine protease inhibitors from cattle tick Boophilus microplus(larvae)

A partir de extratos de larvas e ovos do carrapato Boophilus microplus foram purificados inibidores de serinoproteases (BmTIs) utilizando cromatografia de afinidade em tripsina-Sepharose. Os BmTIs apresentaram massas moleculares entre 6,2 kDa e 18,4 kDa e atividade inibitoria sobre tripsina, HuPK (calicreina plasmatica humana) e elastase de neutrofilos. Utilizando cromatografia de troca-ionica coluna Resource Q, os BmTls foram separados nas formas proteicas denominadas BmTI-A, BmTI-B, BmTI-C, BmTI-D, BmTI-E e BmTI-F. O material nao-retido na cromatografia em coluna Resource Q foi separado em coluna Mono S obtendo-se as formas BmTI-1, BmTI-2, BmTI-3, BmTI-4 BmTI-5, BmTI-6 e BmTI-7. Todas as formas de BmTIs apresentaram atividade inibitoria sobre tripsina com constante de dissociacao (Ki) na ordem de nM. As formas BmTI-A, BmTI-D, BmTI-2, BmTI-3 e BmTI-4, inibem elastase de neutrofilos e HuPK com Ki na ordem de nM. O BmTI-D mostrou ser o melhor inibidor de HuPK e o BmTI-4 mais eficiente para elastase de neutrofilos, com constantes de dissociacao (Ki) de 11,6 nM e 0,2 nM, respectivamente. As formas BmTI-A, BmTI-D, BmTI-2, BmTI-3, BmTI-4 e BmTI6 tiveram as porcoes N-terminais determinadas por sequenciamento automatico de aminoacidos, estas sequencias parciais permitiram a classificacao destes BmTIs dentro da familia dos inibidores de serinoproteases tipo Kunitz de tecido animal. Os BmTIs pos tripsina-Sepharose foram utilizados na imunizacao de bovinos, mostrando acao imunogenica (producao de anticorpos anti-BmTIs) e protetora contra a infestacao por esta especie de carrapato. Na tentativa de clonar o gene de um BmTI foram utilizadas duas estrategias de clonagem baseadas na sintese do cDNA a partir do RNA mensageiro de larvas do carrapato 8. microplus. 1) A construcao de uma biblioteca de...(au)BV UNIFESP: Teses e dissertaçõe

Neutrophil elastase inhibitor purification strategy from cowpea seeds.

Serine proteases and its inhibitors are involved in physiological process and its deregulation lead to various diseases like Chronic Obstructive Pulmonary Disease (COPD), pulmonary emphysema, skin diseases, atherosclerosis, coagulation diseases, cancer, inflammatory diseases, neuronal disorders and other diseases. Serine protease inhibitors have been described in many species, as well as in plants, including cowpea beans (Vigna unguiculata (L.) Walp). Here, we purified and characterized a protease inhibitor, named VuEI (Vigna unguiculata elastase inhibitor), from Vigna unguiculata, with inhibitory activity against HNE (human neutrophil elastase) and chymotrypsin but has no inhibitory activity against trypsin and thrombin. VuEI was obtained by alkaline protein extraction followed by three different chromatographic steps in sequence. First, an ion exchange chromatography using Hitrap Q column was employed, followed by two reversed-phase chromatography using Source15RPC and ACE18 columns. The molecular mass of VuEI was estimated in 10.99 kDa by MALDI-TOF mass spectrometry. The dissociation constant (Ki) to HNE was 9 pM. These data indicate that VuEI is a potent inhibitor of human neutrophil elastase, besides to inhibit chymotrypsin

SARS-CoV-2 ORF8 as a Modulator of Cytokine Induction: Evidence and Search for Molecular Mechanisms.

Peer reviewed: TruePublication status: PublishedSevere cases of SARS-CoV-2 infection are characterized by an immune response that leads to the overproduction of pro-inflammatory cytokines, resulting in lung damage, cardiovascular symptoms, hematologic symptoms, acute kidney injury and multiple organ failure that can lead to death. This remarkable increase in cytokines and other inflammatory molecules is primarily caused by viral proteins, and particular interest has been given to ORF8, a unique accessory protein specific to SARS-CoV-2. Despite plenty of research, the precise mechanisms by which ORF8 induces proinflammatory cytokines are not clear. Our investigations demonstrated that ORF8 augments production of IL-6 induced by Poly(I:C) in human embryonic kidney (HEK)-293 and monocyte-derived dendritic cells (mono-DCs). We discuss our findings and the multifaceted roles of ORF8 as a modulator of cytokine response, focusing on type I interferon and IL-6, a key component of the immune response to SARS-CoV-2. In addition, we explore the hypothesis that ORF8 may act through pattern recognition receptors of dsRNA such as TLRs