Viruses have evolved a multitude of mechanisms, which allow immune evasion in both initial and persistent infection. Understanding the intricacies of these pathways is essential to our future ability to combat primary and reactive viral infections. The murine gamma-2 herpesvirus 68 (γHV68) encodes a protein mK3, which targets Major Histocompatibility Complex (MHC) class I heavy chains for ubiquitin-dependent proteasome degradation. MK3 is able to target and ubiquitinate MHC class I by binding to Endoplasmic Reticulum (ER) resident proteins tapasin, Transporter associated with antigen processing (TAP) 1 and TAP2 that are subunits in the complex known as the peptide-loading complex (PLC). The aforementioned characteristics of mK3 make this novel protein an excellent vehicle to study MHC class I assembly, immune evasion, and ER associated degradation (ERAD). Deepening our understanding of class I assembly and viral immune evasion will impact both the fields of immunology and virology. The homology between γHV68 and many of the human γ-herpesviruses makes this an indispensable model to clarify mechanisms that can then be applied to a broader spectrum of viruses. ERAD, an emerging field of study, is known to play a key role in numerous cellular housekeeping pathways as well as a number of disease states. Illuminating the mechanisms implicated in the mK3-mediated ubiquitination of MHC class I, specifically requirements for substrate recognition and degradation, will yield an increased understanding of cellular pathways involved in ERAD. The studies in this dissertation aim to expand our understanding of the relationship between mK3 and adapter proteins TAP/tapasin as well as mK3 and mK3-targeted substrates. The results show that TAP/tapasin act as adapter proteins by recruiting substrates for mK3. Further, mK3 ubiquitinates TAP/tapasin-associated substrates as long as the substrates have a tail greater than 6aa in length and the tail possesses an ubiquitin acceptor residue (lysine, serine or threonine). These studies also confirm that location of a protein within the PLC will determine the substrate’s susceptibility to mK3-mediated degradation. In the field of ubiquitin ligases and ERAD, these studies lend support to the concept of adapter mediated substrate recruitment.

Somatic cell nuclear transfer (SCNT), or cloning, is a form of artificial reproductive technology that can be used to improve economic traits of domestic animals. However, extreme inefficiency of producing viable offspring via this method is a major limitation. An aggressive immune response at the maternal-fetal interface is an important reason for SCNT pregnancy loss. The goal of this project was to investigate the molecular mechanisms of immune-mediated miscarriage in cloned cattle pregnancies. Many publications hint that immune-mediated miscarriage is associated with abnormal MHC-I expression in the placenta. The regulation of bovine MHC-I genes was systematically studied to identify the cause of abnormal MHC-I expression during immune-mediated miscarriage. We also produced cloned pregnancies to study immune- mediated pregnancy loss. MHC-I and cytokines involved in proinflammatory responses were highly expressed in the placental trophoblast cells of cloned fetuses and in the uterine endometrium of recipients carrying MHC-I incompatible fetuses, respectively, suggesting that MHC-I compatibility between fetus and surrogate mother is important for the success of animal cloning. The results from this research not only reveal the cause of high pregnancy loss in cloned animals but also provide molecular clues to prevent immune-mediated miscarriage in cattle and potentially in human clinics.

The major histocompatibility complex (MHC) is a genomic region containing genes of immunomodulatory importance. MHC class I genes encode cell-surface glycoproteins that present peptides to circulating T cells, playing a key role in recognition of self and non-self. Studies of MHC loci in vertebrates have examined levels of polymorphism and molecular evolutionary processes generating diversity. The bovine MHC (BoLA) has been associated with disease susceptibility, resistance and progression. To delineate mechanisms by which MHC class I genes evolved to function optimally in a species like cattle, it is necessary to study genomic organization of BoLA to define gene content, and investigate characteristics of expressed class I molecules. This study describes development of a physical map of BoLA class I region derived from screening two BAC libraries, isolating positive clones and confirming gene content, order and chromosomal location through PCR, novel BAC end sequencing techniques, and selected BAC shotgun cloning and/or sequencing and FISH analysis. To date, this is the most complete ordered BAC array encompassing the BoLA class I region from the class III boundary to the extended class I region. Characterization of a frameshift allele exhibiting trans-species polymorphism in Bos and Bison by flow cytometry, real-time RT-PCR, 1D and 2D gel analysis is also described. This frameshift allele encodes an early termination signal within the antigen recognition site (ARS) of exon 3 of the BoLA BSA-Adel class I gene predicting a truncated class I protein that is soluble. An ability to assess MHC diversity in populations and provision of animals with defined MHC haplotypes and genetic content for experimental research is necessary in developing a basis upon which to build functional studies to elucidate associations between haplotype and disease in bovids. The BoLA class I region is immunologically important for disease association studies in an economically important species. This study provides knowledge of gene content and organization within the class I MHC region in cattle, providing a template for more detailed analysis and elucidation of complex disease associations through functional genomics and comparative analysis, as well as evolution of the MHC in bovids to optimize a population’s immune response.

The class I myosins are an ubiquitous family of non-filamentous, single-headed actin-binding motor proteins. The objective of this study was to identify the light chain composition of the short-tailed Dictyostelium class I myosins, MyoIA and MyoIE. Flag-tagged MyoIA head-neck and MyoIE head-neck constructs were generated and expressed in Dicyostelium discoidem. The MyoIA and MyoIE head-neck constructs both co-purified with a 17-kDa protein that reacted with an anti-calmodulin antibody and exhibited a mobility shift on SDS gels in the presence of calcium. Mass spectrometry analysis confirmed that the light chain bound to MyoIA and MyoIE was calmodulin. The finding that the short-tailed class I Dictyostelium myosins use the generic calcium-binding protein calmodulin as a light chain contrasts with previous work showing that the long-tailed Dictyostelium class I myosins MyoIB, MyoIC, and MyoID each bind a unique, specialized light chain called MlcB, MlcC, and MlcD, respectively. Despite having a calmodulin light chain, calcium did not affect the actin-activated Mg-ATPase activities of MyoIA or MyoIE. The p21-activated kinases (PAKs) are serine-threonine protein kinases that are activated by the small GTPases Cdc42 and Rac. PAKs phosphorylate a site in the motor domain of Dictyostelium class I myosins that is required for myosin activity. Studies were carried out to determine whether Dictyostelium RacB, which is known to bind to and activate Dictyostelium PAKs, promotes the phosphorylation of MyoID in vivo. A vector that expresses a constitutively active RacB under the control of a doxycycline-inducible promoter was created and transformed into Dictyostelium cells. Immunostaining demonstrated that the constitutively active RacB increased actin filament formation in AX3 cells by ~3-fold but by only ~1.5-fold in PakB-null cells. A rabbit polyclonal antibody against the MyoID tail was made. An anti-phospho antibody raised against a phosphorylated peptide corresponding to the MyoID TEDS site was tested and found to specifically recognize purified phosphorylation MyoIA and MyoID. The anti-phospho antibody did not detect phosphorylated MyoIA or MyoID in crude Dictyostelium cell extracts or in immunoprecipitates prepared using the anti-MyoID antibody. Further work is needed to improve the specificity of the anti-phospho MyoID antibody. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-24 19:51:55.032

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "December 1996." Typescript. Vita. Includes bibliographical references (leaves [128]-155). Also available on the Internet.

Thesis (M.S.)--University of Michigan, 1958.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / "May 1999." Typescript. Vita. Includes bibliographical references (leaves 199-250). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

CD8 T cells recognize complexes of MHC class I and peptide on the surface of target cells. MHC class I antigen presentation is a long pathway, in which proteins are degraded by proteasomes to generating oligopeptides, which may be further trimmed by aminopeptidases in the cytosol. Peptides are transported into the ER, where they may be further trimmed by ER lumenal aminopeptidases and bind to newly-synthesized MHC class I complexes. Proteins degraded by the proteasome are generally tagged with ubiquitin by a combination of ubiquitin-conjugating enzymes and ubiquitin ligases. UBC9 is one ubiquitin conjugating enzyme, which does not conjugate ubiquitin, but instead conjugates small ubiquitin-like molecules (SUMO) to target protein. UBC9 has been found to regulate the functions of many proteins in vivo, most importantly by modifying nuclear transportation and function. Curing [During] my thesis work, I studied the function of UBC9 in MHC class I antigen presentation. UBC9 over-expression in COS cells co-expressing ovalbumin markedly increased presentation SIINFEKL (the immunodominant epitope from ovalbumin in the context of H-2Kb), and UBC9 overexpression increased cell surface H-2Kbin general, suggesting that Ubc9 increased MHC class I antigen presentation by increasing peptide supply. UBC9 did not increase synthesis or degradation of ovalbumin. In transient transfection experiments, Ubc9 increased presentation of SIINFEKL precursors that did, and that did not, depend on proteasomes for processing, as well as SIINFEKL precursors targeted to the ER, bypassing cytosolic processing altogether. However, a C-terminal extended precursor of SIINFEKL, which requires only proteasomal processing before presentation, was the most markedly affected by UBC9 overexpression. This suggested that UBC9 was affecting the pattern of cleavages made by proteasomes in ways that enhance the generation of the C-terminus of SIINFEKL. Because presentation of SIINFEKL itself (which requires no further proteolytic processing) was also enhanced, UBC9 must also affect steps in the class I pathway that occur after the generation of the mature epitopes. UBC9 did not affect the rate of peptide degradation in cytosolic extracts or in intact cells. These findings suggested that UBC9 might have multiple effects on the MHC class I antigen presentation pathway. Immunofluorescent microscopy demonstrated that UBC9 increased the expression of the beta subunits of immunoproteasomes (LMP2, LMP7, and MECL1) as well as of TAP1 and tapasin. In contrast, UBC9 expression did not increase levels of calnexin, calreticulin, ERp57, or Protein disulfide isomerase (PDI). Similarly, levels of leucine aminopeptidase were not increased in UBC9-transfected cells. Therefore, UBC9 overexpression increases the levels of some but not all components of the class I pathway. UBC9 overexpression increased protein levels of MECL1, LMP2 or LMP7 that were under the control of viral promoters, and levels of MECL1 mRNA were similar in control vector and UBC9 transfected cells. Therefore, UBC9 did not increase the level of expression of these subunits through increased transcription. Pulse-chase experiments showed that UBC9 overexpression reduced the degradation of MECL1. Therefore, UBC9 increases the levels of at least some of these components of the MHC class I antigen presentation pathway by increasing their stability. To know the biological significance of UBC9 in MHC class I antigen presentation, I used small interfering RNA (siRNA) to knock down UBC9. Though UBC9 can be successfully knocked down by siRNA, the UBC9-negative cells became very sick, and were not suitable for the study of MHC class I antigen presentation. There are three forms of SUMO molecules in mammalian cells: SUMO-1, SUMO-2 and SUMO-3. My study suggested that SUMO-2 may be involved in UBC9's regulation of MHC class I antigen presentation, since mutant SUMO-2 blocked UBC9's ability to increase H-2Kb-SIINFEKL levels on the cell surface after the cells were loaded with ovalbumin. To further study the function of UBC9, I mutated the active amino acid Cys 93 of UBC9 to Ser (UBC9OH). Unexpectedly, this mutant form (UBC9OH) has very similar effects as wild-type UBC9, increasing Kb-SIINFEKL levels at the cells surface. This suggested that UBC9 protein regulates MHC class I antigen presentation pathway proteins by direct or indirect protein interaction, rather than (or as well as) by SUMO conjugation. Taking account of SUMO-2 results, I propose that wild-type UBC9 (either transfected or endogenous) conjugates SUMO-2 to its substrates, and then UBC9 (wild-type or mutant) interacts with its sumoylated targets, thus affecting protein functions. I also studied heat shock protein Hsp27, which is known to be a substrate for UBC9 in vivo. Hsp27 is expressed in a variety of tissues in the absence of stress, and may regulate actin dynamics. Hsp27 overexpression decreased generation of H-2Kb-SIINFEKL complexes from SIINFEKL precursors that did, and did not, require proteasomes for processing, or that were targeted to the ER. Hsp27 over-expression did not affect protein synthesis, and globally decreased cell surface H2-Kb and H2-Dblevels, but did not affect HLA-A0302 level. Hsp27 overexpression inhibits the presentation of ER-localized SIINFEKL. Taken together, my data suggested that HSP27 may inhibit MHC class I antigen presentation by affecting MHC class I molecules itself rather than peptide supply. After Hsp27 was eliminated with siRNA, the effects were very similar to those seen with Hsp27 overexpression. Levels of H-2Kb-SIINFEKL decreased, and overall cell surface H-2Kb and H-2Db levels decreased. It is possible that when Hsp27 is over-expressed, it acts as a dominant negative form, conferring a similar phenotype to Hsp27 knockdown. These observations suggest that Hsp27 plays an important role in MHC class I antigen presentation.