Reanalysis of Immunopeptidomics Datasets Provides Mechanistic Insight into TAPBPR-Mediated Peptide Editing on HLA-A, -B and -C Molecules.

Background: Major histocompatibility class I (MHC-I, human leukocyte antigen [HLA]-I in humans) molecules present small fragments of the proteome on the cell surface for immunosurveillance, which is pivotal to control infected and malignant cells. Immunogenic peptides are generated and selected in the MHC-I antigen processing and presentation pathway. In this pathway, two homologous molecules, tapasin and TAPBPR, optimise the MHC-I peptide repertoire that is ultimately presented at the plasma membrane. Peptide exchange on HLA-I by human TAPBPR involves the flexible loop region K22-D35, with the leucine at position 30 (L30) involved in mediating peptide dissociation. However, our understanding of the exact molecular mechanisms governing TAPBPR-mediated peptide exchange on HLA-I allotypes remains incomplete. Methods: Here, in-depth re-analyses of published immunopeptidomics datasets was used to further examine TAPBPR peptide editing activity and mechanism of action on HLA-I. The role of the TAPBPR editing loop in opening the HLA-I peptide binding groove was assessed using a molecular dynamics simulation. Results: We show that TAPBPR shapes the peptide repertoire on HLA-A, -B and -C allotypes. The TAPBPR editing loop was not essential to allow HLA-I to adopt an open state. L30 in the TAPBPR editing loop was typically sufficient to mediate peptide repertoire restriction on the three HLA-I allotypes expressed by HeLa cells. TAPBPR was also able to load peptides onto HLA-I in a loop-dependent manner. Conclusions: These results unify the previously hypothesised scoop loop and peptide trap mechanisms of TAPBPR-mediated peptide exchange, with the former involved in peptide filtering and the latter in peptide loading.

Major histocompatibility complex (MHC) class I molecules play an essential role in alerting the immune system to infection and cellular changes. They do this by displaying small fragments of proteins (peptides) from pathogen-infected cells and tumours on the cell surface to immune cells. When activated, immune cells can then destroy the target cell. In 2015, we discovered that a novel accessory protein, called TAPBPR, assists in the selection of peptides displayed on MHC class I molecules for immune surveillance. A specific region in the TAPBPR protein ­ the editing loop ­ is known to be involved in removing peptides from MHC class I. However, our understanding of the process of peptide selection on MHC class I molecules remains incomplete. Here, we show that TAPBPR is not only involved in removing peptides from MHC class I molecules but also assists in peptide loading. Additionally, we demonstrate that the TAPBPR editing loop is involved in both removing and loading of peptides. Our results suggest that TAPBPR fine-tunes the peptide repertoire displayed on three different types of MHC class I molecules. Developing our understanding of the mechanisms of peptide selection on MHC class I molecules has important implications in disease and the development of new therapies.

Get into the groove! The influence of TAPBPR on cargo selection.

Since the discovery of Transporter associated with antigen processing-binding protein-related (TAPBPR) over two decades ago, extensive studies have explored its function in the context of the major histocompatibility complex class-I (MHC-I) antigen processing and presentation pathway. As a chaperone and peptide editor, TAPBPR was recently revealed to have overlapping structural features when resolved with peptide-receptive MHC-I molecules compared with the two newly solved tapasin:MHC-I structures. Despite this, the two chaperones seem to have a unique criteria for loading high-affinity peptides on MHC-I molecules. Yet, the mechanism of action of how TAPBPR creates its distinct filter in cargo selection for peptide-receptive MHC-I molecules continues to be a subject of debate.

The glycosylation status of MHC class I molecules impacts their interactions with TAPBPR.

Glycosylation plays a crucial role in the folding, structure, quality control and trafficking of glycoproteins. Here, we explored whether the glycosylation status of MHC class I (MHC-I) molecules impacts their affinity for the peptide editor, TAPBPR. We demonstrate that the interaction between TAPBPR and MHC-I is stronger when MHC-I lacks a glycan. Subsequently, TAPBPR can dissociate peptides, even those of high affinity, more easily from non-glycosylated MHC-I compared to their glycosylated counterparts. In addition, TAPBPR is more resistant to peptide-mediated allosteric release from non-glycosylated MHC-I compared to species with a glycan attached. Consequently, we find the glycosylation status of HLA-A*68:02, -A*02:01 and -B*27:05 influences their ability to undergo TAPBPR-mediated peptide exchange. The discovery that the glycan attached to MHC-I significantly influences the affinity of their interactions with TAPBPR has important implications, on both an experimental level and in a biological context.

Polymorphisms in MHC class I molecules influence their interactions with components of the antigen processing and presentation pathway.

MHC class I (MHC-I) molecules undergo an intricate folding process in order to pick up antigenic peptide to present to the immune system. In recent years, the discovery of a new peptide editor for MHC-I has added an extra level of complexity in our understanding of how peptide presentation is regulated. On top of this, the incredible diversity in MHC-I molecules leads to significant variation in the interaction between MHC-I and components of the antigen processing and presentation pathway. Here, we review our current understanding regarding how polymorphisms in human leukocyte antigen class I molecules influence their interactions with key components of the antigen processing and presentation pathway. A deeper understanding of this may offer new insights regarding how apparently subtle variation in MHC-I can have a significant impact on susceptibility to disease.

Why TAPBPR? Implications of an additional player in MHC class I peptide presentation.

The peptide editor TAPBPR is the newest member of the major histocompatibility complex class I (MHC-I) antigen processing and presentation pathway. Since 2013, studies have explored the functions and mechanisms of action of this tapasin homolog. Here, we review the key insights gained from structural studies of the TAPBPR:MHC-I complex and the involvement of the TAPBPR loop in peptide exchange. However, despite recent advances, the question still remains: why do we need TAPBPR? The recent appreciation that different MHC-I allotypes vary in their ability to interact with TAPBPR, together with a role for TAPBPR in alternative presentation pathways highlights that much remains unknown concerning the biological need for TAPBPR.

The Ins and Outs of TAPBPR.

Peptide presentation on MHC class I molecules (MHC-I) is central to mounting effective antiviral and antitumoral immune responses. The tapasin-related protein TAPBPR is an MHC-I peptide editor which shapes the final peptide repertoire displayed on the cell surface. Here, we review recent findings which further elucidate the mechanisms by which TAPBPR performs peptide editing on a molecular level, and how glycosylation on MHC-I influences the interaction with TAPBPR and the peptide loading complex. We also explore how the function of TAPBPR can be utilized to promote exogenous peptide loading directly onto plasma-membrane expressed MHC-I. This has led to the development of new assays to investigate TAPBPR-mediated peptide editing and uncovered translational opportunities of utilizing TAPBPR to treat human disease.

Distinct Polymorphisms in HLA Class I Molecules Govern Their Susceptibility to Peptide Editing by TAPBPR.

Understanding how peptide selection is controlled on different major histocompatibility complex class I (MHC I) molecules is pivotal for determining how variations in these proteins influence our predisposition to infectious diseases, cancer, and autoinflammatory conditions. Although the intracellular chaperone TAPBPR edits MHC I peptides, it is unclear which allotypes are subjected to TAPBPR-mediated peptide editing. Here, we examine the ability of 97 different human leukocyte antigen (HLA) class I allotypes to interact with TAPBPR. We reveal a striking preference of TAPBPR for HLA-A, particularly for supertypes A2 and A24, over HLA-B and -C molecules. We demonstrate that the increased propensity of these HLA-A molecules to undergo TAPBPR-mediated peptide editing is determined by molecular features of the HLA-A F pocket, specifically residues H114 and Y116. This work reveals that specific polymorphisms in MHC I strongly influence their susceptibility to chaperone-mediated peptide editing, which may play a significant role in disease predisposition.

Imbricated Coastal Boulder Deposits are Formed by Storm Waves, and Can Preserve a Long-Term Storminess Record.

Coastal boulder deposits (CBD) are archives of extreme wave events. They are emplaced well above high tide, and may include megagravel clasts weighing tens or even hundreds of tonnes. But do they represent storms or tsunami? Many are interpreted as tsunami deposits based simply on clast size and inferences about transport, despite the fact that there are no direct observations documenting formation of these inbricated boulder clusters and ridges. In this study, we use force-balanced, dynamically scaled wave-tank experiments to model storm wave interactions with boulders, and show that storm waves can produce all the features of imbricated CBD. This means that CBD, even when containing megagravel, cannot be used as de facto tsunami indicators. On the contrary, CBD should be evaluated for inclusion in long-term storminess analysis.

The (un)habitual geographies of Social Anxiety Disorder.

This article investigates experiences of Social Anxiety Disorder ('social anxiety') with reference to recent geographical debates on habit. It considers how habit simultaneously captures (un)reflective modes of being in the world and the foreboding disruptive capacity of uncertainty as people attempt to adapt to, negotiate and manage everyday life with social anxiety. Drawing on lived accounts from online questionnaires and online interviews with people diagnosed, or self-diagnosing, with social anxiety, it uncovers the relational and embodied practices-and the inherent spatialities of such practices-that enable individuals to (re)gain control of their socio-spatial surroundings. It also considers the capacity for habits to become disrupted and displaced through pervasive anxieties and persistent rumination and anticipation, situated within the context of participants' everyday lives. This analysis highlights the social, spatial and temporal dimensions of socially anxious experiences. Overall, by interpreting lived experience in this way, this article introduces a socio-spatial dynamic to otherwise extremely limited accounts of social anxiety found outside of the dominant biomedical framework.

Preferential interaction of MHC class I with TAPBPR in the absence of glycosylation.

We recently discovered that TAPBPR promotes reglucosylation of the N-linked glycan on MHC class I molecules, a modification that restores their recognition by calreticulin and reincorporation into the peptide-loading complex. We wondered whether TAPBPR displayed some degree of glycan specificity, as is known to be the case for tapasin via its interaction with calreticulin & ERp57, or whether its interaction with MHC class I was glycan independent. Here, we explored this by comparing the ability of TAPBPR to bind to MHC class I containing either an intact or disrupted NxS/T glycosylation consensus sequence. In contrast to tapasin, TAPBPR bound strongly to MHC class I molecules that lacked N-linked glycosylation, suggesting that the TAPBPR:MHC class I interaction is glycan independent. Furthermore, we found that glycosylated HLA-A2 preferentially interacts with tapasin rather than TAPBPR, possibly explaining, in part, why MHC class I molecules bind efficiently to tapasin in the face of an alternative chaperone. The distinction in glycan specificity between the two peptide editors suggests that TAPBPR may bind to MHC class I molecules that are associated with a broader diversity of oligosaccharides attached compared with tapasin. This may explain, to some extent, the ability of TAPBPR to interact with MHC class I molecules outside of the ER.

TAPBPR mediates peptide dissociation from MHC class I using a leucine lever.

Tapasin and TAPBPR are known to perform peptide editing on major histocompatibility complex class I (MHC I) molecules; however, the precise molecular mechanism(s) involved in this process remain largely enigmatic. Here, using immunopeptidomics in combination with novel cell-based assays that assess TAPBPR-mediated peptide exchange, we reveal a critical role for the K22-D35 loop of TAPBPR in mediating peptide exchange on MHC I. We identify a specific leucine within this loop that enables TAPBPR to facilitate peptide dissociation from MHC I. Moreover, we delineate the molecular features of the MHC I F pocket required for TAPBPR to promote peptide dissociation in a loop-dependent manner. These data reveal that chaperone-mediated peptide editing on MHC I can occur by different mechanisms dependent on the C-terminal residue that the MHC I accommodates in its F pocket and provide novel insights that may inform the therapeutic potential of TAPBPR manipulation to increase tumour immunogenicity.

Utilizing TAPBPR to promote exogenous peptide loading onto cell surface MHC I molecules.

The repertoire of peptides displayed at the cell surface by MHC I molecules is shaped by two intracellular peptide editors, tapasin and TAPBPR. While cell-free assays have proven extremely useful in identifying the function of both of these proteins, here we explored whether a more physiological system could be developed to assess TAPBPR-mediated peptide editing on MHC I. We reveal that membrane-associated TAPBPR targeted to the plasma membrane retains its ability to function as a peptide editor and efficiently catalyzes peptide exchange on surface-expressed MHC I molecules. Additionally, we show that soluble TAPBPR, consisting of the luminal domain alone, added to intact cells, also functions as an effective peptide editor on surface MHC I molecules. Thus, we have established two systems in which TAPBPR-mediated peptide exchange on MHC class I can be interrogated. Furthermore, we could use both plasma membrane-targeted and exogenous soluble TAPBPR to display immunogenic peptides on surface MHC I molecules and consequently induce T cell receptor engagement, IFN-γ secretion, and T cell-mediated killing of target cells. Thus, we have developed an efficient way to by-pass the natural antigen presentation pathway of cells and load immunogenic peptides of choice onto cells. Our findings highlight a potential therapeutic use for TAPBPR in increasing the immunogenicity of tumors in the future.

Damnum versus Quaestus.

Not only is bioethics fundamental to determining or guiding how we live and die, its role as the key interconnecting strand between various disciplines, the public, and decision makers is unique. The works featured here are from a collection entitled "Damnum versus Quaestus" (loss versus gain). They are informed by the lived experience of being with someone (described here as "the patient") as he or she lives through the process of dying.

Properties of the tapasin homologue TAPBPR.

The presentation of antigenic peptides by MHC class I molecules plays a vital role in generating T cell responses against infection and cancer. Over the last two decades the central role of tapasin as a peptide editor that influences the loading and optimisation of peptides onto MHC class I molecules has been extensively characterised. Recently, it has become evident that the tapasin-related protein, TAPBPR, functions as a second peptide editor which influences the peptides displayed by MHC class I molecules. Here, we review the discovery of TAPBPR and current understanding of this novel protein in relation to its closest homologue tapasin.

TAPBPR bridges UDP-glucose:glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway.

Recently, we revealed that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules. Here, we asked whether any other co-factors associate with TAPBPR, which would explain its effect on peptide selection. We identify an interaction between TAPBPR and UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a folding sensor in the calnexin/calreticulin quality control cycle that is known to regenerate the Glc1Man9GlcNAc2 moiety on glycoproteins. Our results suggest the formation of a multimeric complex, dependent on a conserved cysteine at position 94 in TAPBPR, in which TAPBPR promotes the association of UGT1 with peptide-receptive MHC class I molecules. We reveal that the interaction between TAPBPR and UGT1 facilities the reglucosylation of the glycan on MHC class I molecules, promoting their recognition by calreticulin. Our results suggest that in addition to being a peptide editor, TAPBPR improves peptide optimisation by promoting peptide-receptive MHC class I molecules to associate with the peptide-loading complex.

Examining Intervention Design: Lessons from the Development of Eight Related Malaria Health Care Intervention Studies.

Abstract-Rigorous evidence of "what works" to improve health care is in demand, but methods for the development of interventions have not been scrutinized in the same ways as methods for evaluation. This article presents and examines intervention development processes of eight malaria health care interventions in East and West Africa. A case study approach was used to draw out experiences and insights from multidisciplinary teams who undertook to design and evaluate these studies. Four steps appeared necessary for intervention design: (1) definition of scope, with reference to evaluation possibilities; (2) research to inform design, including evidence and theory reviews and empirical formative research; (3) intervention design, including consideration and selection of approaches and development of activities and materials; and (4) refining and finalizing the intervention, incorporating piloting and pretesting. Alongside these steps, projects produced theories, explicitly or implicitly, about (1) intended pathways of change and (2) how their intervention would be implemented.The work required to design interventions that meet and contribute to current standards of evidence should not be underestimated. Furthermore, the process should be recognized not only as technical but as the result of micro and macro social, political, and economic contexts, which should be acknowledged and documented in order to infer generalizability. Reporting of interventions should go beyond descriptions of final intervention components or techniques to encompass the development process. The role that evaluation possibilities play in intervention design should be brought to the fore in debates over health care improvement.

TAPBPR alters MHC class I peptide presentation by functioning as a peptide exchange catalyst.

Our understanding of the antigen presentation pathway has recently been enhanced with the identification that the tapasin-related protein TAPBPR is a second major histocompatibility complex (MHC) class I-specific chaperone. We sought to determine whether, like tapasin, TAPBPR can also influence MHC class I peptide selection by functioning as a peptide exchange catalyst. We show that TAPBPR can catalyse the dissociation of peptides from peptide-MHC I complexes, enhance the loading of peptide-receptive MHC I molecules, and discriminate between peptides based on affinity in vitro. In cells, the depletion of TAPBPR increased the diversity of peptides presented on MHC I molecules, suggesting that TAPBPR is involved in restricting peptide presentation. Our results suggest TAPBPR binds to MHC I in a peptide-receptive state and, like tapasin, works to enhance peptide optimisation. It is now clear there are two MHC class I specific peptide editors, tapasin and TAPBPR, intimately involved in controlling peptide presentation to the immune system.

F pocket flexibility influences the tapasin dependence of two differentially disease-associated MHC Class I proteins.

The human MHC class I protein HLA-B*27:05 is statistically associated with ankylosing spondylitis, unlike HLA-B*27:09, which differs in a single amino acid in the F pocket of the peptide-binding groove. To understand how this unique amino acid difference leads to a different behavior of the proteins in the cell, we have investigated the conformational stability of both proteins using a combination of in silico and experimental approaches. Here, we show that the binding site of B*27:05 is conformationally disordered in the absence of peptide due to a charge repulsion at the bottom of the F pocket. In agreement with this, B*27:05 requires the chaperone protein tapasin to a greater extent than the conformationally stable B*27:09 in order to remain structured and to bind peptide. Taken together, our data demonstrate a method to predict tapasin dependence and physiological behavior from the sequence and crystal structure of a particular class I allotype. Also watch the Video Abstract.