Samuel J. Landry

The ins and outs of a molecular chaperone machine

Genetic and biochemical work has highlighted the biological importance of the GroEL/GroES (Hsp60/Hsp10; cpn60/cpn10) chaperone machine in protein folding. GroELs donut-shaped structure has attracted the attention of structural biologists because of its elegance as well as the secrets (substrates) it can hide. The recent determination of the GroES and GroEL/GroES structures provides a glimpse of their plasticity, revealing dramatic conformational changes that point to an elaborate mechanism, coupling ATP hydrolysis to substrate release by GroEL.

Peptides Identified through Phage Display Direct Immunogenic Antigen to Dendritic Cells

Dendritic cells (DC) play a critical role in adaptive immunity by presenting Ag, thereby priming naive T cells. Specific DC-binding peptides were identified using a phage display peptide library. DC-peptides were fused to hepatitis C virus nonstructural protein 3 (NS3) while preserving DC targeting selectivity and Ag immunogenicity. The NS3-DC-peptide fusion protein was efficiently presented to CD4+ and CD8+ T cells derived from hepatitis C virus-positive blood cells, inducing their activation and proliferation. This immunogenic fusion protein was significantly more potent than NS3 control fusion protein or NS3 alone. In chimeric NOD-SCID mice transplanted with human cells, DC-targeted NS3 primed naive CD4+ and CD8+ T cells for potent NS3-specific proliferation and cytokine secretion. The capacity of peptides to specifically target immunogenic Ags to DC may establish a novel strategy for vaccine development.

The Importance of a Mobile Loop in Regulating Chaperonin/ Co-chaperonin Interaction HUMANS VERSUS ESCHERICHIA COLI

Chaperonins are universally conserved proteins that nonspecifically facilitate the folding of a wide spectrum of proteins. While bacterial GroEL is functionally promiscuous with various co-chaperonin partners, its human homologue, Hsp60 functions specifically with its co-chaperonin partner, Hsp10, and not with other co-chaperonins, such as the bacterial GroES or bacteriophage T4-encoded Gp31. Co-chaperonin interaction with chaperonin is mediated by the co-chaperonin mobile loop that folds into a β-hairpin conformation upon binding to the chaperonin. A delicate balance of flexibility and conformational preferences of the mobile loop determines co-chaperonin affinity for chaperonin. Here, we show that the ability of Hsp10, but not GroES, to interact specifically with Hsp60 lies within the mobile loop sequence. Using mutational analysis, we show that three substitutions in the GroES mobile loop are necessary and sufficient to acquire Hsp10-like specificity. Two of these substitutions are predicted to preorganize the β-hairpin turn and one to increase the hydrophobicity of the GroEL-binding site. Together, they result in a GroES that binds chaperonins with higher affinity. It seems likely that the single ring mitochondrial Hsp60 exhibits intrinsically lower affinity for the co-chaperonin that can be compensated for by a higher affinity mobile loop.

Recognition of nascent polypeptides for targeting and folding

A major difference between the refolding of proteins in vitro and the in vivo folding process, in which we include localization and assembly, is the need for additional factors in vivo, apart from the protein product itself. Thus, the amino acid sequence of a naturally selected protein contains not only the information specifying its three-dimensional structure, but also the information that enables these factors to recognize the nascent polypeptide. In this review, we consider how this latter information may be encoded and, in turn, interpreted by binding species.

Local protein instability predictive of helper T-cell epitopes

Although selectivity at the levels of peptide binding to major histocompatibility complex (MHC) class II and recognition by T cells may partially account for immunodominance patterns, it is clear that differential antigen processing also exerts a strong effect. Here, Sam Landry correlates immunodominant epitopes with nearby structurally unstable segments, as identified by hydrogen-deuterium exchange nuclear magnetic resonance (NMR), and suggests that epitope presentation is directed by preferential proteolytic cleavage at the unstable sites.

Tc1 effector diversity shows dissociated expression of granzyme B and interferon-γ in HIV infection

Objective: To examine antigen specific cytotoxic effector T cell diversity in HIV infected individuals. Design: We used a panel of previously defined HLA class I-restricted HIV peptides to stimulate CD8 cells in freshly isolated peripheral blood mononuclear cells of HIV infected patients, to determine cognate killing via the perforin–granzyme pathway and inflammation induced by secretion of interferon (IFN)-γ. Methods: ELISPOT assays were used to measure the secretion of granzyme B (GzB) and IFN-γ at single cell resolution. Results: In all nine patients only approximately 20% of the peptides triggered a canonical Tc1 response with simultaneous release of GzB and IFN-γ. The majority of these peptides (approximately 80%) that elicited recall responses fell into the ‘single positive’ category with induction of either GzB or IFN-γ alone. The GzB positive cells did not produce interleukin (IL)-4 or IL-5. Conclusion: The GzB positive but IFN-γ negative CD8 cells are programmed to induce apoptosis mediated killing without inflammation while the GzB negative and IFN-γ positive CD8 cells could mediate inflammation without killing. This Tc1 CD8 effector cell diversity and the understanding of these differentiation mechanisms may enable the precise implementation and fine-tuning of fundamentally different defense strategies against HIV and other infections.

Compensatory Changes in GroEL/Gp31 Affinity as a Mechanism for Allele-specific Genetic Interaction

Previous work has shown that the GroEL-GroES interaction is primarily mediated by the GroES mobile loop. In bacteriophage T4 infection, GroES is substituted by the gene31-encoded cochaperonin, Gp31. Using a genetic selection scheme, we have identified a new set of mutations in gene31 that affect interaction with GroEL; all mutations result in changes in the mobile loop of Gp31. Biochemical analyses reveal that the mobile loop mutations alter the affinity between Gp31 and GroEL, most likely by modulating the stability of the GroEL-bound hairpin conformation of the mobile loop. Surprisingly, mutations ingroEL that display allele-specific interactions with mutations in gene 31 alter residues in the GroEL intermediate domain, distantly located from the mobile loop binding site. The observed patterns of genetic and biochemical interaction between GroES or Gp31 and GroEL point to a mechanism of genetic allele specificity based on compensatory changes in affinity of the protein-protein interaction. Mutations studied in this work indirectly alter affinity by modulating a folding transition in the Gp31 mobile loop or by modulating a hinged conformational change in GroEL.

Proximal Glycans Outside of the Epitopes Regulate the Presentation of HIV-1 Envelope gp120 Helper Epitopes

Glycosylation of HIV-1 envelope gp120 determines not only the proper structure, but also the immune responses against this Ag. Although glycans may be part of specific epitopes or shield other epitopes from T cells and Abs, this study provides evidence for a different immunomodulatory function of glycans associated with gp120 residues N230 and N448. These glycans are required for efficient MHC class II-restricted presentation of nearby CD4 T cell epitopes, even though they are not part of the epitopes. The glycans do not affect CD4 T cell recognition of more distant epitopes and are not essential for the proper folding and function of gp120. Data on CD4 T cell recognition of N448 mutants combined with proteolysis analyses and surface electrostatic potential calculation around residue N448 support the notion that N448 glycan near the epitope’s C terminus renders the site to be surface accessible and allows its efficient processing. In contrast, the N230 glycan contributes to the nearby epitope presentation at a step other than the proteolytic processing of the epitope. Hence, N-glycans can determine CD4 T cell recognition of nearby gp120 epitopes by regulating the different steps in the MHC class II processing and presentation pathway after APCs acquire the intact gp120 Ag exogenously. Modifications of amino acids bearing glycans at the C termini of gp120 helper epitopes may prove to be a useful strategy for enhancing the immunogenicity of HIV-1 envelope gp120.

Probing the interface in a human co-chaperonin heptamer: residues disrupting oligomeric unfolded state identified.

BackgroundThe co-chaperonin protein 10 (cpn10) assists cpn60 in the folding of nonnative polypeptides in a wide range of organisms. All known cpn10 molecules are heptamers of seven identical subunits that are linked together by β-strand interactions at a large and flexible interface. Unfolding of human mitochondrial cpn10 in urea results in an unfolded heptameric state whereas GuHCl additions result in unfolded monomers. To address the role of specific interface residues in the assembly of cpn10 we prepared two point-mutated variants, in each case removing a hydrophobic residue positioned at the subunit-subunit interface.ResultsReplacing valine-100 with a glycine (Val100Gly cpn10) results in a wild-type-like protein with seven-fold symmetry although the thermodynamic stability is decreased and the unfolding processes in urea and GuHCl both result in unfolded monomers. In sharp contrast, replacing phenylalanine-8 with a glycine (Phe8Gly cpn10) results in a protein that has lost the ability to assemble. Instead, this protein exists mostly as unfolded monomers.ConclusionsWe conclude that valine-100 is a residue important to adopt an oligomeric unfolded state but it does not affect the ability to assemble in the folded state. In contrast, phenylalanine-8 is required for both heptamer assembly and monomer folding and therefore this mutation results in unfolded monomers at physiological conditions. Despite the plasticity and large size of the cpn10 interface, our observations show that isolated interface residues can be crucial for both the retention of a heptameric unfolded structure and for subunit folding.

Influence of Disulfide-Stabilized Structure on the Specificity of Helper T-Cell and Antibody Responses to HIV Envelope Glycoprotein gp120

ABSTRACT CD4+ helper T cells specific for human immunodeficiency virus type 1 (HIV-1) are associated with control of viremia. Nevertheless, vaccines have had limited effectiveness thus far, in part because sequence variability and other structural features of the HIV envelope glycoprotein deflect the immune response. Previous studies indicated that CD4+ T-cell epitope dominance is controlled by antigen three-dimensional structure through its influence on antigen processing and presentation. In this work, three disulfide bonds in the outer domain of gp120 were individually deleted in order to destabilize the local three-dimensional structure and enhance the presentation of nearby weakly immunogenic epitopes. However, upon immunization of groups of BALB/c mice, the CD4+ T-cell response was broadly reduced for all three variants, and distinct epitope profiles emerged. For one variant, antibody titers were sharply increased, and the antibody exhibited significant CD4-blocking activity.