RESUMO
Unconventional myosins are a superfamily of actin-based motors implicated in diverse cellular processes. In recent years, much progress has been made in describing their biophysical properties, and headway has been made into analyzing their cellular functions. Here, we focus on the principles that guide in vivo motor function and targeting to specific cellular locations. Rather than describe each motor comprehensively, we outline the major themes that emerge from research across the superfamily and use specific examples to illustrate each. In presenting the data in this format, we seek to identify open questions in each field as well as to point out commonalities between them. To advance our understanding of myosins' roles in vivo, clearly we must identify their cellular cargoes and the protein complexes that regulate motor attachment to fully appreciate their functions on the cellular and developmental levels.
Assuntos
Actinas/metabolismo , Proteínas Motores Moleculares/metabolismo , Miosinas/metabolismo , Isoformas de Proteínas/metabolismo , Actinas/ultraestrutura , Animais , Cálcio/metabolismo , Adesão Celular , Movimento Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , GTP Fosfo-Hidrolases/metabolismo , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Motores Moleculares/ultraestrutura , Miosinas/ultraestrutura , Isoformas de Proteínas/ultraestrutura , Processamento de Proteína Pós-TraducionalRESUMO
XTENs are unstructured, nonrepetitive protein polymers designed to prolong the in vivo half-life of pharmaceuticals by introducing a bulking effect similar to that of poly(ethylene glycol). While XTEN can be expressed as a recombinant fusion protein with bioactive proteins and peptides, therapeutic molecules of interest can also be chemically conjugated to XTEN. Such an approach permits precise control over the positioning, spacing, and valency of bioactive moieties along the length of XTEN. We have demonstrated the attachment of T-20, an anti-retroviral peptide indicated for the treatment of HIV-1 patients with multidrug resistance, to XTEN. By reacting maleimide-functionalized T-20 with cysteine-containing XTENs and varying the number and positioning of cysteines in the XTENs, a library of different peptide-polymer combinations were produced. The T-20-XTEN conjugates were tested using an in vitro antiviral assay and were found to be effective in inhibiting HIV-1 entry and preventing cell death, with the copy number and spacing of the T-20 peptides influencing antiviral activity. The peptide-XTEN conjugates were also discovered to have enhanced solubilities in comparison with the native T-20 peptide. The pharmacokinetic profile of the most active T-20-XTEN conjugate was measured in rats, and it was found to exhibit an elimination half-life of 55.7 ± 17.7 h, almost 20 times longer than the reported half-life for T-20 dosed in rats. As the conjugation of T-20 to XTEN greatly improved the in vivo half-life and solubility of the peptide, the XTEN platform has been demonstrated to be a versatile tool for improving the properties of drugs and enabling the development of a class of next-generation therapeutics.
Assuntos
Antivirais/química , HIV-1/efeitos dos fármacos , Fragmentos de Peptídeos/química , Polímeros/química , Proteínas Recombinantes de Fusão/química , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Feminino , Peptídeo 2 Semelhante ao Glucagon/química , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Meia-Vida , Humanos , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/farmacologia , Polietilenoglicóis/química , Polímeros/farmacocinética , Polímeros/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Solubilidade , Espectrometria de Massas por Ionização por Electrospray , Distribuição TecidualRESUMO
Myosin VI is a molecular motor implicated in many processes, and it likely associates with a variety of cargoes that specify its functions. Although it is critical to Drosophila development, little is known about its cellular roles. To reveal its involvement in specific pathways, we sought to identify the binding partners of Drosophila myosin VI. We used affinity chromatography and mass spectrometry to discover interacting proteins, which we tested for direct binding. Using this approach, we found that the microtubule-associated protein Cornetto bound myosin VI, and we demonstrated a role for both in secretion of the lipidated morphogen Hedgehog. We also identified a number of other binding proteins, and further characterization of their interactions with myosin VI will advance our understanding of the roles of these complexes in cellular and developmental processes. Thus, our method has provided us the means to gain valuable insight into the multifaceted roles of a motor protein in vivo.