Quantitative membrane proteomics reveals new cellular targets of viral immune modulators.
Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from Kaposi sarcoma-associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif-deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins.
Bartee
Eric
E
Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, United States of America.
McCormack
Ashley
A
Früh
Klaus
K
D015703:Antigens, CD / Q000032:analysis* / Q000502:physiology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000502:physiology; D015816:Cell Adhesion Molecules, Neuronal / Q000032:analysis*; D004721:Endoplasmic Reticulum / Q000737:chemistry; D005326:Fetal Proteins / Q000032:analysis*; D058851:GPI-Linked Proteins; D006056:Golgi Apparatus / Q000737:chemistry; D006367:HeLa Cells; D006566:Herpesviridae Infections / Q000276:immunology*; D019288:Herpesvirus 8, Human / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000502:physiology*; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D040901:Proteomics; D050765:Qa-SNARE Proteins / Q000032:analysis*; D020410:Activated-Leukocyte Cell Adhesion Molecule; D000074010:Bone Marrow Stromal Antigen 2
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D015816:Cell Adhesion Molecules, Neuronal; D005326:Fetal Proteins; D058851:GPI-Linked Proteins; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; C105613:ALCAM protein, human; D020410:Activated-Leukocyte Cell Adhesion Molecule; C094572:BST2 protein, human; D000074010:Bone Marrow Stromal Antigen 2; C062279:CD6 antigen; C405867:K5 protein, Human herpesvirus 8