RESUMO
The human prototypical SR protein SRSF1 is an oncoprotein that contains two RRMs and plays a pivotal role in RNA metabolism. We determined the structure of the RRM1 bound to RNA and found that the domain binds preferentially to a CN motif (N is for any nucleotide). Based on this solution structure, we engineered a protein containing a single glutamate to asparagine mutation (E87N), which gains the ability to bind to uridines and thereby activates SMN exon7 inclusion, a strategy that is used to cure spinal muscular atrophy. Finally, we revealed that the flexible inter-RRM linker of SRSF1 allows RRM1 to bind RNA on both sides of RRM2 binding site. Besides revealing an unexpected bimodal mode of interaction of SRSF1 with RNA, which will be of interest to design new therapeutic strategies, this study brings a new perspective on the mode of action of SRSF1 in cells.
Assuntos
Motivo de Reconhecimento de RNA/genética , Sítios de Splice de RNA/genética , Splicing de RNA , Fatores de Processamento de Serina-Arginina/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Substituição de Aminoácidos , Asparagina/genética , Biologia Computacional , Éxons/genética , Ácido Glutâmico/genética , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Ressonância Magnética Nuclear Biomolecular , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/isolamento & purificação , Fatores de Processamento de Serina-Arginina/ultraestrutura , Uridina/metabolismoRESUMO
Although smallpox was eradicated in 1980, it is still considered a potential agent of biowarfare and bioterrorism. Smallpox has the potential for high mortality rates along with a major public health impact, eventually causing public panic and social disruption. Passive administration of neutralizing monoclonal antibodies (mAbs) is an effective intervention for various adverse reactions caused by vaccination and the unpredictable nature of emerging and bioterrorist-related infections. Currently, vaccinia immune globulin (VIG) is manufactured from vaccinia vaccine-boosted plasma; however, this production method is not ideal because of its limited availability, low specific activity, and risk of contamination with blood-borne infectious agents. To overcome the limitations of VIG production from human plasma, we isolated two human single chain variable fragments (scFvs) (SC34 and SC212) bound to vaccinia virus (VACV) from a scFv phage library constructed from the B cells of VACV vaccine-boosted volunteers. The scFvs were converted to human IgG1 (VC34 and VC212). These two anti-VACV mAbs were produced in Chinese Hamster Ovary (CHO) DG44 cells. The binding affinities of VC34 and VC212 were estimated by competition ELISA to IC50 values of 2 µg/mL (13.33 nM) and 22 µg/mL (146.67 nM), respectively. Only the VC212 mAb was proven to neutralize the VACV, as evidenced by the plaque reduction neutralization test (PRNT) result with a PRNT50 of ~0.16 mg/mL (~1.07 µM). This VC212 could serve as a valuable starting material for further development of VACV-neutralizing human immunoglobulin for a prophylactic measure against post-vaccination complications and for post-exposure treatment against smallpox.
