RESUMO
Platelets contribute to a variety of physiological processes including inflammation, sepsis and cancer. However, due to their primary role in hemostasis, platelet transfusions are largely restricted to managing thrombocytopenia and bleeding. One way to expand the utility of platelet transfusions would be to genetically engineer donor platelets with new or enhanced functions. We have previously shown that lipid nanoparticles containing mRNA (mRNA-LNP) can be used to genetically modify authentic platelets in a non-clinical crystalloid solution. Currently, platelets collected for transfusion are stored in plasma or in plasma supplemented with platelet additive solution (PAS) at supraphysiological concentrations at room temperature, or at 4 ºC if intended for use in acute hemorrhage. Here we describe a new plasma-optimized mRNA-LNP for transfecting platelets directly in plasma and plasma supplemented with PAS that is scalable to physiological and supraphysiological platelet concentrations. Transfecting platelets in clinical solutions with mRNA-LNP does not affect aspects of in vitro physiology, and transfected platelets are storable. The compatibility of this transfection system with current clinical practices could enable future mRNA-LNP based platelet products and cell therapies.
RESUMO
Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by the loss of function of a set of imprinted genes on chromosome 15q11-15q13. One of these genes, NDN, encodes necdin, a protein that is important for neuronal differentiation and survival. Loss of Ndn in mice causes defects in the formation and function of the nervous system. Necdin is a member of the melanoma-associated antigen gene (MAGE) protein family. The functions of MAGE proteins depend highly on their interactions with other proteins, and in particular MAGE proteins interact with E3 ubiquitin ligases and deubiquitinases to form MAGE-RING E3 ligase-deubiquitinase complexes. Here, we used proximity-dependent biotin identification (BioID) and mass spectrometry (MS) to determine the network of protein-protein interactions (interactome) of the necdin protein. This process yielded novel as well as known necdin-proximate proteins that cluster into a protein network. Next, we used BioID-MS to define the interactomes of necdin proteins carrying coding variants. Variant necdin proteins had interactomes that were distinct from wildtype necdin. BioID-MS is not only a useful tool to identify protein-protein interactions, but also to analyze the effects of variants of unknown significance on the interactomes of proteins involved in genetic disease.
Assuntos
Substituição de Aminoácidos/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Mapas de Interação de Proteínas/genética , Ubiquitina-Proteína Ligases/genética , Animais , Biotinilação/genética , Diferenciação Celular/genética , Enzimas Desubiquitinantes/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Camundongos , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/ultraestrutura , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Neurônios/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/genética , Síndrome de Prader-Willi/genética , Conformação Proteica , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/químicaRESUMO
Lacking protein synthesis machinery and organelles necessary for autophagy or apoptosis, aged red blood cells (RBCs) are marked by circulating auto-antibodies for macrophage-mediated clearance. The antigen recognized by these auto-antibodies is the major protein of the RBC membrane, Band 3. To ensure regulation and specificity in clearance, the molecular "clock" must mark senescent cells in a way that differentiates them from younger cells, to prevent premature clearance. Predominant models of Band 3 senescence signaling are reviewed, and merits are discussed in light of the recently published crystal structure of the Band 3 membrane domain. © 2017 IUBMB Life, 70(1):32-40, 2018.