Possible association of non-binding of HSP70 to HLA-DRB1 peptide sequences and protection from rheumatoid arthritis

The beta-chains of HLA-DR molecules associated with susceptibility to rheumatoid arthritis (RA) share a common amino acid sequence in their third hypervariable region at position 70-74. This shared epitope could either contribute to preferential binding of a given disease-associated peptide, be involved in disease-induction by molecular mimicry or, by binding to heat shock proteins, influence antigen presentation. It is known that the Escherichia coli M(r)70,000 heat shock protein DnaK can bind peptides from the shared epitope. Using a highly sensitive method, we show that peptides covering the third hypervariable region of associated, but also most of the non-associated HLA-DR alleles, bind to DnaK. Similar binding specificities could be found for the constitutively expressed mammalian M(r)70,000 heat shock protein Hsc73 and the inducible mammalian Hsp72. However, peptides containing the amino acid sequence DERAA, found in HLA-DR alleles and strongly associated with protection from RA, did not bind any HSP70. Thus, our results suggest a possible association of non-binding of HSP70 to HLA-DR molecules or its 70-74 fragments and protection from RA.     

Potential roles of protein oxidation and the immunoproteasome in MHC class I antigen presentation: the 'PrOxI' hypothesis

The major histocompatibility complex (MHC) class I (MHC-I) antigen presentation system is responsible for the cell-surface presentation of self-proteins and intracellular viral proteins. This pathway is important in screening between self, and non-self or infected cells. In this pathway, proteins are partially degraded to peptides in the cytosol and targeted to the cell surface bound to an MHC-I receptor protein. At the cell surface, T cells bypass cells displaying self-peptides but destroy others displaying foreign antigens. Cells contain several isoforms of the proteasome, but it is thought that the immunoproteasome is the major form involved in generating peptides for the MHC-I pathway. How all intracellular proteins are targeted for MHC-I processing is unclear. Oxidative stress is experienced by all cells, and all proteins are exposed to oxidation. We propose that oxidative modification makes proteins susceptible to degradation by the immunoproteasome. This could be called the protein oxidation and immunoproteasome or 'PrOxI' hypothesis of MHC-I antigen processing. Protein oxidation may, thus, be a universal mechanism for peptide generation and presentation in the MHC-I pathway.     

Bacterial heat shock proteins enhance class II MHC antigen processing and presentation of chaperoned peptides to CD4+ T cells

APCs process heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC molecules, but the ability of HSPs to contribute chaperoned peptides for class II MHC (MHC-II) Ag processing and presentation is unclear. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-II presentation, as detected by T hybridoma cells. Bacterial HSPs enhanced MHC-II presentation only if peptide was complexed to the HSP, suggesting that the key HSP function was enhanced delivery or processing of chaperoned peptide Ag rather than generalized enhancement of APC function. HSP-enhanced processing was intact in MyD88 knockout cells, which lack most TLR signaling, further suggesting the effect was not due to TLR-induced induction of accessory molecules. Bacterial HSPs enhanced uptake of peptide, which may contribute to increased MHC-II presentation. In addition, HSPs enhanced binding of peptide to MHC-II molecules at pH 5.0 (the pH of vacuolar compartments), but not at pH 7.4, indicating another mechanism for enhancement of MHC-II Ag processing. Bacterial HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD4+ T cells.     

Bacterial heat shock proteins promote CD91-dependent class I MHC cross-presentation of chaperoned peptide to CD8+ T cells by cytosolic mechanisms in dendritic cells versus vacuolar mechanisms in macrophages

APCs process mammalian heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC (MHC-I) molecules to CD8(+) T cells. HSPs are also expressed in prokaryotes and chaperone microbial peptides, but the ability of prokaryotic HSPs to contribute chaperoned peptides for Ag presentation is unknown. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-I presentation by both murine macrophages and dendritic cells. HSP-enhanced MHC-I peptide presentation occurred only if peptide was complexed to the prokaryotic HSP and was dependent on CD91, establishing CD91 as a receptor for prokaryotic as well as mammalian HSPs. Inhibition of cytosolic processing mechanisms (e.g., by transporter for Ag presentation deficiency or brefeldin A) blocked HSP-enhanced peptide presentation in dendritic cells but not macrophages. Thus, prokaryotic HSPs deliver chaperoned peptide for alternate MHC-I Ag processing and cross-presentation via cytosolic mechanisms in dendritic cells and vacuolar mechanisms in macrophages. Prokaryotic HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD8(+) T cells.     

Identification of the peptide-binding site in the heat shock chaperone/tumor rejection antigen gp96 (Grp94)

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624-630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by alpha-helices and lies on a surface consisting of antiparallel beta-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.     

Identification of potential HLA class I and class II epitope precursors associated with heat shock protein 70 (HSPA)

Heat shock protein 70 (HSPA) is a molecular chaperone which has been suggested to shuttle human leukocyte antigen (HLA) epitope precursors from the proteasome to the transporter associated with antigen processing. Despite the reported observations that peptides chaperoned by HSPA are an effective source of antigens for cross-priming, little is known about the peptides involved in the process. In this study, we investigated the possible involvement of HSPA in HLA class I or class II antigen presentation and analysed the antigenic potential of the associated peptides. HSPA was purified from CCRF-CEM and K562 cell lines, and using mass spectrometry techniques, we identified 44 different peptides which were co-purified with HSPA. The affinity of the identified peptides to two HSPA isoforms, HSPA1A and HSPA8, was confirmed using a peptide array. Four of the HSPA-associated peptides were matched with 13 previously reported HLA epitopes. Of these 13 peptides, nine were HLA class I and four were HLA class II epitopes. These results demonstrate the association of HSPA with HLA class I and class II epitopes, therefore providing further evidence for the involvement of HSPA in the antigen presentation process.     

Loss of T cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1(27-35) tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1(27-35) antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide·MHC complex. These results help explain how the "anchor-fixing" strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.     

Mutational analysis reveals a complex interplay of peptide binding and multiple biological features of HLA-B27

Molecular polymorphism influences the strong association of HLA-B27 with ankylosing spondylitis through an unknown mechanism. Natural subtypes and site-directed mutants were used to analyze the effect of altering the peptide-binding site of this molecule on its stability, interaction with tapasin, folding, and export. The disease-associated subtypes B*2705, B*2702, and B*2704 showed higher thermostability at 50 °C than all other subtypes and mutants, except some mimicking B*2702 polymorphism. The lowest values were found among pocket B mutants, most of which interacted strongly with tapasin, but otherwise there was no correlation between thermostability and tapasin interaction. Mutants resulting in increased hydrophobicity frequently acquired their maximal thermostability faster than those with increased polarity, suggesting that this process is largely driven by the thermodynamics of peptide binding. Folding, export, and tendency to misfold were influenced by polymorphism all along the peptide-binding site and were not specifically dependent on any particular region or structural feature. Frequent uncoupling of thermostability, folding/misfolding, and export can be explained by the distinct effect of mutations on the acquisition of a folded conformation, the optimization rate of B27-peptide complexes, and their quality control in the endoplasmic reticulum, all of which largely depend on the ways in which mutations alter peptide binding, without excluding additional effects on interactions with tapasin or other proteins involved in folding and export. The similarity of the generally disease-associated B*2707 to nondisease-associated subtypes in all the features analyzed suggests that molecular properties other than antigen presentation may not currently explain the relationship between HLA-B27 polymorphism and ankylosing spondylitis.     

Mycobacterial and mouse HSP70 have immuno-modulatory effects on dendritic cells

Previously, it has been shown that heat shock protein 70 (HSP70) can prevent inflammatory damage in experimental autoimmune disease models. Various possible underlying working mechanisms have been proposed. One possibility is that HSP70 induces a tolerogenic phenotype in dendritic cells (DCs) as a result of the direct interaction of the antigen with the DC. Tolerogenic DCs can induce antigen-specific regulatory T cells and dampen pathogenic T cell responses. We show that treatment of murine DCs with either mycobacterial (Mt) or mouse HSP70 and pulsed with the disease-inducing antigen induced suppression of proteoglycan-induced arthritis (PGIA), although mouse HSP70-treated DCs could ameliorate PGIA to a greater extent. In addition, while murine DCs treated with Mt- or mouse HSP70 had no significantly altered phenotype as compared to untreated DCs, HSP70-treated DCs pulsed with pOVA (ovalbumin peptide 323–339) induced a significantly increased production of IL-10 in pOVA-specific T cells. IL-10-producing T cells were earlier shown to be involved in Mt HSP70-induced suppression of PGIA. In conclusion, this study indicates that Mt- and mouse HSP70-treated BMDC can suppress PGIA via an IL-10-producing T cell-dependent manner.     

Enhanced immunogenicity of heat shock protein 70 peptide complexes from dendritic cell-tumor fusion cells

We have developed a molecular chaperone-based tumor vaccine that reverses the immune tolerance of cancer cells. Heat shock protein (HSP) 70 extracted from fusions of dendritic (DC) and tumor cells (HSP70.PC-F) possess superior properties such as stimulation of DC maturation and T cell proliferation over its counterpart from tumor cells. More importantly, immunization of mice with HSP70.PC-F resulted in a T cell-mediated immune response including significant increase of CD8 T cells and induction of the effector and memory T cells that was able to break T cell unresponsiveness to a nonmutated tumor Ag and provide protection of mice against challenge with tumor cells. By contrast, the immune response to vaccination with HSP70-PC derived from tumor cells is muted against such nonmutated tumor Ag. HSP70.PC-F complexes differed from those derived from tumor cells in a number of key manners, most notably, enhanced association with immunologic peptides. In addition, the molecular chaperone HSP90 was found to be associated with HSP70.PC-F as indicated by coimmunoprecipitation, suggesting ability to carry an increased repertoire of antigenic peptides by the two chaperones. Significantly, activation of DC by HSP70.PC-F was dependent on the presence of an intact MyD88 gene, suggesting a role for TLR signaling in DC activation and T cell stimulation. These experiments indicate that HSP70-peptide complexes (PC) derived from DC-tumor fusion cells have increased their immunogenicity and therefore constitute an improved formulation of chaperone protein-based tumor vaccine.     

Ionizing Radiation Drives Key Regulators of Antigen Presentation and a Global Expansion of the Immunopeptidome

Little is known about the pathways regulating MHC antigen presentation and the identity of treatment-specific T cell antigens induced by ionizing radiation. For this reason, we investigated the radiation-specific changes in the colorectal tumor cell proteome. We found an increase in DDX58 and ZBP1 protein expression, two nucleic acid sensing molecules likely involved in induction of the dominant interferon response signature observed after genotoxic insult. We further observed treatment-induced changes in key regulators and effector proteins of the antigen processing and presentation machinery. Differential regulation of MHC allele expression was further driving the presentation of a significantly broader MHC-associated peptidome postirradiation, defining a radiation-specific peptide repertoire. Interestingly, treatment-induced peptides originated predominantly from proteins involved in catecholamine synthesis and metabolic pathways. A nuanced relationship between protein expression and antigen presentation was observed where radiation-induced changes in proteins do not correlate with increased presentation of associated peptides. Finally, we detected an increase in the presentation of a tumor-specific neoantigen derived from Mtch1. This study provides new insights into how radiation enhances antigen processing and presentation that could be suitable for the development of combinatorial therapies. Data are available via ProteomeXchange with identifier PXD032003.     

Targeting tumour cells with defects in the MHC Class I antigen processing pathway with CD8+ T cells specific for hydrophobic TAP- and Tapasin-independent peptides: the requirement for directed access into the ER

It is becoming increasingly apparent that the majority of tumours display defects in the MHC class I antigen processing pathway, particularly low levels of the transporters-associated with antigen processing (TAP) and tapasin. Thus, immunotherapy approaches targeting such tumours with CD8+ cytotoxic T lymphocytes (CTL) requires strategies to overcome these defects. Previously we had identified an antigen processing pathway by which cytosolically derived hydrophobic peptides could be presented in the absence of TAP. Here we show in the tapasin-negative cell line 721.220 that a number of these hydrophobic TAP-independent peptides can also be presented in a tapasin-independent manner. Yet when these experiments were extended to tumour cell lines derived from small cell lung cancer (SCLC), which we show to be tapasin deficient in addition to TAP-negative, the TAP-, tapasin-independent peptides were not presented. This lack of presentation could be rectified by pre-treatment of SCLC cells with IFNgamma. Alternatively, by directing the TAP-, tapasin-independent peptides into the endoplasmic reticulum (ER) via an ER signal sequence, these peptides were presented efficiently by SCLC cells. We infer from this data that the TAP-independent pathway for presentation of hydrophobic peptides generates a low concentration of peptide in the ER and, for tumour cells which also lack tapasin, this concentration of antigenic peptide is insufficient to load onto MHC class I molecules. Thus, for immunotherapeutic approaches to target SCLC and other tumours with defects in the MHC class I antigen processing pathway it will be important to consider strategies that address tapasin-defects.     

Analyses of Conformational States of the Transporter Associated with Antigen Processing (TAP) Protein in a Native Cellular Membrane Environment

The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.     

Investigation on the effect of peptides mixture from tumor cells inducing anti-tumor specific immune response

The peptides mixture was prepared from tumor cells by freezing-thawing cells, precipitation by heating, followed by acidification of the solution. The activation and proliferation of mouse splenocytes by HSP70-peptide complex, formed by the binding of HSP70 and peptides in vitro, were observed, so was the specific cytotoxicity of the proliferative lymphocytes to tumor cells. The phenotypes of the proliferative lymphocytes were analyzed by a flow cytometer. BALB/c mice inoculated with H22 hepatocarcinoma cells in peritoneal cavity or hind thigh were immunized by injection with HSP70-peptides complex to observe the inhibitory effect of the immunization on tumor and lifetime of tumor-bearing mice. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney. The results showed that the peptides mixture from tumor cells contained tumor-specific antigen peptides which could be presented by HSP70 to activate lymphocytes in vitro, the proliferative lymphocyt     

The study of complex formation of human recombinant HSP70 with tumor-associated peptides

Molecular chaperones of the HSP70 family (70 kDa heat shock proteins) are involved in presentation of exogenous antigenic peptides by antigen-presenting cells (APC). HSP70 complexes with tumor-associated peptides are powerful immunotherapeutic agents, inducing an adaptive cytotoxic T-cell mediated immune response. Several clinical trials have shown that HSP-based autological anticancer vaccines possess high efficiency and safety. However, sometimes it is impossible to isolate sufficient amount of such vaccines and so human recombinant HSP are used for in vitro loading with tumor-associated peptides. In this study we have investigated binding of two human recombinant proteins HSP70_HYB and HSC70 with antigenic peptides of different origin and optimized conditions for complex formation. The proposed method for complex formation increases the repertoire of HSP70 bound peptides compared with in vivo formed complexes.     

Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells

Our study demonstrates that tumor-derived heat shock protein (HSP)70 chaperones a tyrosinase peptide and mediates its transfer to human immature dendritic cells (DCs) by receptor-dependent uptake. Human tumor-derived HSP70 peptide complexes (HSP70-PC) thus have the immunogenic potential to instruct DCs to cross-present endogenously expressed, nonmutated, and tumor antigenic peptides that are shared among tumors of the melanocytic lineage for T cell recognition. T cell stimulation by HSP70-instructed DCs is dependent on the Ag bound to HSP70 in that only DCs incubated with HSP70-PC purified from tyrosinase-positive (HSP70-PC/tyr(+)) but not from tyrosinase-negative (HSP70-PC/tyr(-)) melanoma cells resulted in the specific activation of the HLA-A*0201-restricted tyrosinase peptide-specific cytotoxic T cell clone. HSP70-PC-mediated T cell stimulation is very efficient, delivering the tyrosinase peptide at concentrations as low as 30 ng/ml of HSP70-PC for T cell recognition. Receptor-dependent binding of HSP70-PC and active cell metabolism are prerequisites for MHC class I-restricted cross-presentation and T cell stimulation. T cell stimulation does not require external DC maturation signals (e.g., exogenously added TNF-alpha), suggesting that signaling DC maturation is an intrinsic property of the HSP70-PC itself and related to receptor-mediated binding. The cross-presentation of a shared human tumor Ag together with the exquisite efficacy are important new aspects for HSP70-based immunotherapy in clinical anti-cancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting.     

Investigation on the effect of peptides mixture from tumor cells inducing anti-tumor specific immune response

The peptides mixture was prepared from tumor cells by freezingthawing cells, precipitation by heating, followed by acidification of the solution. The activation and proliferation of mouse splenocytes by HSP70-peptide complex, formed by the binding of HSP70 and peptides in vitro, were observed, so was the specific cytotoxicity of the proliferative lymphocytes to tumor cells. The phenotypes of the proliferative lymphocytes were analyzed by a flow cytometer. BALB/c mice inoculated with H22 hepatocarcinoma cells in peritoneal cavity or hind thigh were immunized by injection with HSP70-peptides complex to observe the inhibitory effect of the immunization on tumor and lifetime of tumor-bearing mice. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney. The results showed that the peptides mixture from tumor cells contained tumor-specific antigen peptides which could be presented by HSP70 to activate lymphocytes in vitro, the proliferative lymphocytes were T cells which were specifically cytotoxic to tumor cells, the in vivo growth of both ascitic and solid carcinoma could be suppressed by immunization with HSP70-peptides and the lifetime of tumor-bearing mice was prolonged, the in vivo immunization with HSP70-H22-peptides had no impact on the function of mouse liver and kidney, suggesting that there was no occurrence of autoimmunity in vivo after immunization.     

Phage display biopanning identifies the translation initiation and elongation factors (IF1α-3 and eIF-3) as components of Hsp70–peptide complexes in breast tumour cells

The heat shock protein, HSP70, is over-expressed in many tumours and acts at the crossroads of key intracellular processes in its role as a molecular chaperone. HSP70 associates with a vast array of peptides, some of which are antigenic and can mount adaptive immune responses against the tumour from which they are derived. The pool of peptides associated with HSP70 represents a unique barcode of protein metabolism in tumour cells. With a view to identifying unique protein targets that may be developed as tumour biomarkers, we used purified HSP70 and its associated peptide pool (HSP70–peptide complexes, HSP70-PCs) from different human breast tumour cell lines as targets for phage display biopanning. Our results show that HSP70-PCs from each cell line interact with unique sets of peptides within the phage display library. One of the peptides, termed IST, enriched in the biopanning process, was used in a ‘pull-down’ assay to identify the original protein from which the HSP70-associated peptides may have been derived. The eukaryotic translation initiation factor 3 (eIF-3), a member of the elongation factor EF1α family, and the HSP GRP78, were pulled down by the IST peptide. All of these proteins are known to be up-regulated in cancer cells. Immunohistochemical staining of tumour tissue microarrays showed that the peptide co-localised with HSP70 in breast tumour tissue. The data indicate that the reservoir of peptides associated with HSP70 can act as a unique indicator of cellular protein activity and a novel source of potential tumour biomarkers.     

A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter

Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.