Targeting γ-herpesvirus 68 Bcl-2-mediated down-regulation of autophagy.
γ-herpesviruses (γHVs) are common human pathogens that encode homologs of the anti-apoptotic cellular Bcl-2 proteins, which are critical to viral reactivation and oncogenic transformation. The murine γHV68 provides a tractable in vivo model for understanding general features of these important human pathogens. Bcl-XL, a cellular Bcl-2 homolog, and the murine γHV68 Bcl-2 homolog, M11, both bind to a BH3 domain within the key autophagy effector Beclin 1 with comparable affinities, resulting in the down-regulation of Beclin 1-mediated autophagy. Despite this similarity, differences in residues lining the binding site of M11 and Bcl-XL dictate varying affinities for the different BH3 domain-containing proteins. Here we delineate Beclin 1 differential specificity determinants for binding to M11 or Bcl-XL by quantifying autophagy levels in cells expressing different Beclin 1 mutants and either M11 or Bcl-XL, and we show that a G120E/D121A Beclin 1 mutant selectively prevents down-regulation of Beclin 1-mediated autophagy by Bcl-XL, but not by M11. We use isothermal titration calorimetry to identify a Beclin 1 BH3 domain-derived peptide that selectively binds to M11, but not to Bcl-XL. The x-ray crystal structure of this peptide bound to M11 reveals the mechanism by which the M11 BH3 domain-binding groove accommodates this M11-specific peptide. This information was used to develop a cell-permeable peptide inhibitor that selectively inhibits M11-mediated, but not Bcl-XL-mediated, down-regulation of autophagy.
From the Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58108-6050.
Minfei
M
Su
Yang
Y
Mei
Ruslan
R
Sanishvili
Beth
B
Levine
Christopher L
CL
Colbert
Sangita
S
Sinha
D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D015536:Down-Regulation / Q000187:drug effects*; D018142:Gammaherpesvirinae / Q000737:chemistry / Q000502:physiology*; D006566:Herpesviridae Infections / Q000188:drug therapy / Q000378:metabolism; D054884:Host-Pathogen Interactions / Q000187:drug effects*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D060066:Protein Interaction Maps; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D051020:bcl-X Protein / Q000737:chemistry / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D014764:Viral Proteins; D051020:bcl-X Protein
Autophagy; Bcl-2 Family Proteins; Crystal Structure; Herpesvirus; Host Defense; Host-Pathogen Interactions; Protein-Protein Interactions