TAPBP

--Chicken-derived chaperone molecule, as well as human variant, enhanced the loading of peptides across HLA types, which could assist applications from cell therapy to vaccines— PHILADELPHIA, Feb. 24, 2023 /PRNewswire/ -- Researchers at Children's Hospital of Philadelphia (CHOP) have identified variants of a chaperone molecule that optimizes the binding and presentation of foreign antigens across the human population, which could open the door to numerous applications where robust presentation to the immune system is important, including cell therapy and immunization. The findings were published today in Science Advances. Class I major histocompatibility complex (MHC-I) proteins are found on the surface of cells from all jawed vertebrates and play an essential role in the immune system. The MHC-I displays peptide fragments of proteins from within the cell on the cell surface, effectively "presenting" them to the immune system, which is constantly scanning the body for foreign or toxic antigens. When foreign peptides are identified, they trigger a cascade that allows cytotoxic T cells to eliminate intruders. For a peptide to be presented to the immune system, it needs to be loaded on a folded MHC-I protein. Several molecules facilitate this process, including proteins known as molecular chaperones, which assist with MHC-I folding. Tapasin and a similar molecule known as TAPBPR are both molecular chaperones that facilitate MHC-I folding and peptide loading. Because TAPBR functions independently outside of the peptide-loading complex, it is well-suited for clinical applications that involve peptide exchange, such as loading immunogenic peptides on MHC-I molecules and generating libraries to detect T-cells that recognize peptides or antigens from infected or cancerous cells. However, TAPBPR-mediated peptide exchange has thus far only worked for a limited set of common allotypes of human MHC-I, known as human leukocyte antigen (HLA), which has limited wider use of these technologies in biomedical applications. Over time, HLA subtypes, which include HLA-A, HLA-B, and HLA-C, have evolved such that not all alleles interact equally well with TAPBPR. This has been a roadblock in developing and enhancing novel therapies with the help of molecular chaperones, as some HLA allotypes do not interact with these molecules. To solve this problem, the CHOP researchers analyzed three different TAPBPR proteins: one from humans, one from chickens, and one from mice. They found that unlike human TAPBPR, chicken TAPBPR co-evolved with its class I genes, so that it maintains high affinity across MHC-I allotypes. In their analysis, they found that chicken TAPBPR was able to react with multiple HLA allotypes, many of which were not able to bind to human TAPBPR. They also demonstrated that TAPBPR stabilizes the empty MHC-I groove in an "open" conformation, boosting its affinity for peptide loading. Simultaneously, in close collaboration with researchers at the University of Illinois led by Erik Procko, PhD, the research team used deep mutational scanning to characterize the effects from 100s of point mutations on human TAPBPR and found a variant that mimics the chicken sequence. Like the chicken TAPBPR, this variant enhanced peptide exchange across a broad range of HLA types. "Although the highly polymorphic nature of MHC-I molecules makes it challenging to engineer 'universal' chaperones, our research team demonstrated that both a chicken ortholog of TAPBPR and a human variant with minor adjustments could enhance peptide exchange across multiple disease-relevant HLAs," said senior author Nikolaos G. Sgourakis, PhD, Associate Professor in the Center for Computational and Genomic Medicine at Children's Hospital of Philadelphia. "These TAPBPR orthologs could be utilized in various cancer immunotherapeutic settings to narrow the peptide repertoire and increase immunogenicity. The knowledge gained by our studies can guide the design of engineered TAPBPR variants with tailored HLA specificity and catalytic efficiency for peptide exchange applications both in vitro and in vivo." This research was supported by funding from the National Institute of Allergy and Infectious Diseases (5R01AI143997) and National Institute of General Medical Sciences (5R35GM125034). Yi Sun, Georgia F. Papadaki, Christine A. Devlin, Julia N. Danon, Michael C. Young, Trenton J. Winters, George M. Burslem, Erik Procko, and Nikolaos G. Sgourakis. "Xeno-interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes," Science Advances, February 24, 2023, DOI: 10.1126/sciadv.ade7151 About Children's Hospital of Philadelphia: A non-profit, charitable organization, Children's Hospital of Philadelphia was founded in 1855 as the nation's first pediatric hospital. Through its long-standing commitment to providing exceptional patient care, training new generations of pediatric healthcare professionals, and pioneering major research initiatives, the 595-bed hospital has fostered many discoveries that have benefited children worldwide. Its pediatric research program is among the largest in the country. The institution has a well-established history of providing advanced pediatric care close to home through its CHOP Care Network, which includes more than 50 primary care practices, specialty care and surgical centers, urgent care centers, and community hospital alliances throughout Pennsylvania and New Jersey, as well as a new inpatient hospital with a dedicated pediatric emergency department in King of Prussia. In addition, its unique family-centered care and public service programs have brought Children's Hospital of Philadelphia recognition as a leading advocate for children and adolescents. For more information, visit . Contact: Dana Bate Children's Hospital of Philadelphia (267) 426-6055 [email protected] SOURCE Children's Hospital of Philadelphia

For an adequate immune response, it is essential that T lymphocytes recognize infected or degenerated cells. They do so by means of antigenic peptides, which these cells present with the help of specialized surface molecules (MHC I molecules). Using X-ray structure analysis, a research team has now been able to show how the MHC I molecules are loaded with peptides and how suitable peptides are selected for this purpose. As task forces of the adaptive immune system, T lymphocytes are responsible for attacking and killing infected or cancerous cells. Such cells, like almost all cells in the human body, present on their surface fragments of all the proteins they produce inside. If these include peptides that a T lymphocyte recognises as foreign, the lymphocyte is activated and kills the cell in question. It is therefore important for a robust T-cell response that suitable protein fragments are presented to the T lymphocyte. The research team led by Simon Trowitzsch and Robert Tampé from the Institute of Biochemistry at Goethe University Frankfurt has now shed light on how the cell selects these protein fragments or peptides. Peptide presentation takes place on so-called major histocompatibility complex class I molecules (MHC I). MHC I molecules are a group of very diverse surface proteins that can bind myriads of different peptides. They are anchored in the cell membrane and form a peptide-binding pocket with their outward-facing part. Like all surface proteins, MHC I molecules take the so-called secretory pathway: they are synthesised into the cell's cavity system (endoplasmic reticulum (ER) and Golgi apparatus) and folded there. Small vesicles then bud off from the cavity system, migrate to the cell membrane and fuse with it. The maturation process of the MHC I molecules is very strictly controlled: in the ER, proteins known as "chaperones" help them fold. The chaperone tapasin is essential for peptide loading in this process. "When an MHC I molecule has bound a peptide, tapasin checks how tight the binding is," says Trowitzsch, explaining the chaperone's task. "If the bond is unstable, the peptide is removed and replaced by a tightly binding one." However, it has not yet been possible to clarify how exactly tapasin performs this task -- especially because the loading process is extremely fast. The biochemists and structural biologists from Goethe University Frankfurt have now succeeded for the first time in visualising the short-lived interaction between chaperone and MHC I molecule by means of X-ray structure analysis. To do this, they produced variants of the two interaction partners that were no longer embedded in the membrane, purified them and brought them together. A trick helped to capture the loading complex in action for crystallisation: first, the research team loaded the MHC I molecule with a high-affinity peptide so that a stable complex was created. A light signal triggered cleavage of the peptide, which greatly reduced its ability to bind the MHC I molecule. Immediately, tapasin entered the scene and remained bound to the MHC I molecule that lacks its peptide. "The photo-induced cleavage of the peptide was pivotal to the success of our experiment," says Tampé. "With the help of this optochemical biology, we can now systematically reproduce complex cellular processes one by one." X-ray structure analysis of the crystals revealed how tapasin widens the peptide-binding pocket of the MHC I molecule, thereby testing the strength of the peptide bond. For this purpose, the interaction partners form a large contact area; for stabilisation, a loop of tapasin sits on top of the widened binding pocket. "This is the first time we have shown the process of loading at high resolution," Tampé is pleased to report. The images also reveal how a single chaperone can interact with the enormous diversity of MHC I molecules, says the biochemist: "Tapasin binds precisely the non-variable regions of the MHC I molecules." However, the new structure not only improves our understanding of the complex processes involved in loading MHC I molecules. It should also help select suitable candidates for vaccine development.