RESUMO
Cytolytic CD8(+) T cells (CTLs) kill virally infected cells, tumor cells, or other potentially autoreactive T cells in a calcium-dependent manner. To date, the molecular mechanism that leads to calcium intake during CTL differentiation and function has remained unresolved. We demonstrate that desmoyokin (AHNAK1) is expressed in mature CTLs, but not in naive CD8(+) T cells, and is critical for calcium entry required for their proper function during immune response. We show that mature AHNAK1-deficient CTLs exhibit reduced Ca(v)1.1 alpha1 subunit expression (also referred to as L-type calcium channels or alpha1S pore-forming subunits), which recently were suggested to play a role in calcium entry into CD4(+) T cells. AHNAK1-deficient CTLs show marked reduction in granzyme-B production, cytolytic activity, and IFN-gamma secretion after T cell receptor stimulation. Our results demonstrate an AHNAK1-dependent mechanism controlling calcium entry during CTL effector function.
Assuntos
Sinalização do Cálcio/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Linfócitos T/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Interferon gama/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase , Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Engagement of the T cell antigen receptor (TCR) during antigen presentation initiates a coordinated action of a large number of signaling proteins and ion channels. AHNAK1 is a scaffold protein, highly expressed by CD4+ T cells, and is a critical component for calcium signaling. We showed that AHNAK1-deficient mice were highly susceptible to Leishmania major infection. AHNAK1-deficient CD4+ T cells responded poorly to TCR stimulation in vitro with low proliferation and low Interleukin-2 production. Furthermore, AHNAK1 deficiency resulted in a reduced calcium influx upon TCR crosslinking and subsequent poor activation of the transcription factor NFAT. AHNAK1 was required for plasma membrane expression of L-type calcium channels alpha 1S (Cav1.1), probably through its interaction with the beta regulatory subunit. Thus, AHNAK1 plays an essential role in T cell Ca2+ signaling through Cav1 channels, triggered via TCR activation; therefore, AHNAK1 is a potential target for therapeutic intervention.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Sinalização do Cálcio/imunologia , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/microbiologia , Caveolina 1/metabolismo , Proliferação de Células , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Leishmaniose/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Alzheimer's disease is a complex neurodegenerative process that is believed to be due to the accumulation of short, hydrophobic peptides derived from amyloid precursor proteins by proteolytic cleavage. It is widely believed that these Abeta peptides are secreted into the extracellular spaces of the CNS, where they assemble into toxic oligomers that kill neurons and eventually form deposits of senile plaques. This essay explores the possibility that a fraction of these Abeta peptides never leave the membrane lipid bilayer after they are generated, but instead exert their toxic effects by competing with and compromising the functions of intramembranous segments of membrane-bound proteins that serve many critical functions. Based on the presence of shared amino acid sequences containing GxxG motifs, I speculate that accumulations of intramembranous Abeta peptides might affect the functions of amyloid precursor protein itself and the assembly of the PS1, Aph1, Pen 2, Nicastrin complex.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Membrana Celular/metabolismo , Sistema Nervoso Central , Dimerização , Endopeptidases , Humanos , Bicamadas Lipídicas/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Neurônios/metabolismo , Peptídeo Hidrolases , Peptídeos/química , Presenilina-1 , Ligação Proteica , Homologia de Sequência de Aminoácidos , Dodecilsulfato de Sódio/química , TemperaturaRESUMO
To explore the function of the giant AHNAK molecule, first described in 1992 [Shtivelman, E., Cohen, F. E. & Bishop, J. M. (1992) Proc. Natl. Acad. Sci. USA 89, 5472-5476], we created AHNAK null mice by homologous recombination. Homozygous knockouts showed no obvious phenotype, but revealed instead a second AHNAK-like molecule, provisionally designated AHNAK2. Like the original AHNAK, AHNAK2 is a 600-kDa protein composed of a large number of highly conserved repeat segments. Structural predictions suggest that the repeat segments of both AHNAKs may have as their basic framework a series of linked, antiparallel beta-strands similar to those found in beta-propeller proteins. Both AHNAKs appear to localize to Z-band regions of mouse cardiomyocytes and cosediment with membrane vesicles containing the dihydropyridine receptor, which is consistent with earlier reports that the AHNAKs are linked to L-type calcium channels and can be phosphorylated by protein kinase A. The localization of the AHNAKs in close proximity to transverse tubule membranes and Z-band regions of cardiac sarcomeres raise the possibility that they might be involved in regulating excitation/contraction coupling of cardiomyocytes, but other studies indicate that the association of AHNAKs with calcium channel proteins is more widespread. AHNAK2 is predicted to have a PDZ domain within its N-terminal, nonrepeating domain, which may mediate these interactions.