Sunil Kumar Saini

Dipeptides promote folding and peptide binding of MHC class I molecules.

MHC class I molecules bind only those peptides with high affinity that conform to stringent length and sequence requirements. We have now investigated which peptides can aid the in vitro folding of class I molecules, and we find that the dipeptide glycyl-leucine efficiently supports the folding of HLA-A*02:01 and H-2Kb into a peptide-receptive conformation that rapidly binds high-affinity peptides. Treatment of cells with glycyl-leucine induces accumulation of peptide-receptive H-2Kb and HLA-A*02:01 at the surface of cells. Other dipeptides with a hydrophobic second amino acid show similar enhancement effects. Our data suggest that the dipeptides bind into the F pocket like the C-terminal amino acids of a high-affinity peptide.

Not all empty MHC class I molecules are molten globules: Tryptophan fluorescence reveals a two-step mechanism of thermal denaturation

When major histocompatibility complex (MHC) class I molecules bind peptide, they change their conformation and their dynamics. The structure and properties of the peptide-empty class I are still largely unknown. We have investigated the thermal denaturation of the murine class I allotypes H-2D(b) and H-2K(b) through the fluorescence of their intrinsic tryptophans, and we find that it occurs via an empty form that can also be produced by folding denatured recombinant class I molecules. It rapidly binds exogenous peptides. Our data demonstrate that the empty form of class I is a distinct conformational state with at least transient stability.

Peptide-independent stabilization of MHC class I molecules breaches cellular quality control

ABSTRACT The intracellular trafficking of major histocompatibility complex class I (MHC-I) proteins is directed by three quality control mechanisms that test for their structural integrity, which is correlated to the binding of high-affinity antigenic peptide ligands. To investigate which molecular features of MHC-I these quality control mechanisms detect, we have followed the hypothesis that suboptimally loaded MHC-I molecules are characterized by their conformational mobility in the F-pocket region of the peptide-binding site. We have created a novel variant of an MHC-I protein, Kb-Y84C, in which two &agr;-helices in this region are linked by a disulfide bond that mimics the conformational and dynamic effects of bound high-affinity peptide. Kb-Y84C shows a remarkable increase in the binding affinity to its light chain, beta-2 microglobulin (&bgr;2m), and bypasses all three cellular quality control steps. Our data demonstrate (1) that coupling between peptide and &bgr;2m binding to the MHC-I heavy chain is mediated by conformational dynamics; (2) that the folded conformation of MHC-I, supported by &bgr;2m, plays a decisive role in passing the ER-to-cell-surface transport quality controls; and (3) that &bgr;2m association is also tested by the cell surface quality control that leads to MHC-I endocytosis.

Dipeptides catalyze rapid peptide exchange on MHC class I molecules

Significance We have shown previously that dipeptides can support efficient in vitro folding of the MHC class I molecules. Here, we describe the discovery of dipeptides that catalyze the dissociation of low- and high-affinity peptides from class I and their replacement for exogenous peptides of interest, on both recombinant and cell surface HLA-A*02:01, HLA-B*27:05, and H-2Kb molecules. Understanding peptide exchange on class I will help us understand peptide optimization in live cells. We demonstrate that the peptide-exchange technology can be used to produce epitope-specific MHC tetramers much faster and more easily than possible with techniques currently available and to enhance peptide loading onto live cells for cancer immunotherapy. Further applications include allotype specific peptide epitope elution from tumor cells. Peptide ligand selection by MHC class I molecules, which occurs by iterative optimization, is the centerpiece of immunodominance in antiviral and antitumor immune responses. For its understanding, the molecular mechanisms of peptide binding and dissociation by class I molecules must be elucidated. To this end, we have investigated dipeptides that bind to the F pocket of class I molecules. We find that they accelerate the dissociation of prebound peptides of both low and high affinity, suggesting a mechanism of action for the peptide-exchange chaperone tapasin. Peptide exchange on class I molecules also has practical uses in epitope discovery and T-cell monitoring.

The Carboxy Terminus of the Ligand Peptide Determines the Stability of the MHC Class I Molecule H-2Kb: A Combined Molecular Dynamics and Experimental Study.

Major histocompatibility complex (MHC) class I molecules (proteins) bind peptides of eight to ten amino acids to present them at the cell surface to cytotoxic T cells. The class I binding groove binds the peptide via hydrogen bonds with the peptide termini and via diverse interactions with the anchor residue side chains of the peptide. To elucidate which of these interactions is most important for the thermodynamic and kinetic stability of the peptide-bound state, we have combined molecular dynamics simulations and experimental approaches in an investigation of the conformational dynamics and binding parameters of a murine class I molecule (H-2Kb) with optimal and truncated natural peptide epitopes. We show that the F pocket region dominates the conformational and thermodynamic properties of the binding groove, and that therefore the binding of the C terminus of the peptide to the F pocket region plays a crucial role in bringing about the peptide-bound state of MHC class I.

PrediQt-Cx: post treatment health related quality of life prediction model for cervical cancer patients.

Background Cervical cancer is the third largest cause of cancer mortality in India. The objectives of the study were to compare the pre and the post treatment quality of life in cervical cancer patients and to develop a prediction model to provide an insight into the possibilities in the treatment modules. Methodology/Principal Findings A total of 198 patients were assessed with two structured questionnaires of Health Related Quality of Life (The European Organisation for Research and Treatment of Cancer, EORTC QLQ C-30 and CX-24). The baseline observations were recorded when the patients first reported (T1) and second evaluation was done at 6 months post treatment (T2). The mean age of detection was 50.9 years with the literacy level being non-educated or less than high school. Majority of them were married/cohabiting 179 (90.4%). On histopathological examination (HPE) squamous cell carcinoma was found to be the most common cell type carcinoma 147 (74.2%) followed by Adenocarcinoma 31 (15.7%). Radical hysterectomy was the most common treatment modality 76 (38.4%), followed by Wertheims Hysterectomy 46 (23.2%) and Radiochemotherapy 59 (29.8%). The mean score of global health of cervical cancer patients post treatment was 77.90, which was significantly higher than the pre - treatment score (54.32). Mean “symptoms score” post treatment was 21.69 with an aggravation of 7.32 compared to pre treatment scores. Patients experienced substantial decrease in sexual activity post treatment. Conclusions/Significance The prediction model(PrediQt-Cx), based on Support Vector Machine(SVM) for predicting post treatment HRQoL in cervical cancer patients was developed and internally cross validated. After external validation PrediQt-Cx can be easily employed to support decision making by clinicians and patients from north India region, through openly made available for access at http://prediqt.org.