Christopher V. Nicchitta

Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance

Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease and resists infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA (miRNA) composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic life cycle of P. falciparum, a subset of erythrocyte miRNAs translocate into the parasite. Two miRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes, and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite messenger RNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical miRNA activity, which may represent a unique host defense strategy against complex eukaryotic pathogens.

Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells

gp96 (GRP94) elicits antigen‐presenting cell (APC) activation and can direct peptides into the cross‐ presentation pathways of APC. These responses arise through interactions of gp96 with Toll‐like (APC activation) and endocytic (cross‐presentation) receptors of APC. Previously, CD91, the α2‐macroglobulin receptor, was identified as the heat shock/chaperone protein receptor of APC. Recent data indicates, however, that inhibition of CD91 ligand binding does not alter gp96 recognition and uptake. Furthermore, CD91 expression is not itself sufficient for gp96 binding and internalization. We now report that scavenger receptor class‐A (SR‐A), a prominent scavenger receptor of macrophages and dendritic cells, serves a primary role in gp96 and calreticulin recognition and internalization. gp96 internalization and peptide re‐presentation are inhibited by the SR‐A inhibitory ligand fucoidin, although fucoidin was without effect on α2‐macroglobulin binding or uptake. Ectopic expression of SR‐A in HEK 293 cells yielded gp96 recognition and uptake activity. In addition, macrophages derived from SR‐A−/− mice were substantially impaired in gp96 binding and uptake. These data identify new roles for SR‐A in the regulation of cellular responses to heat shock proteins.

Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation

The role of the lumenal contents (reticuloplasm) of the endoplasmic reticulum (ER) in protein translocation was determined by in vitro analysis. Depletion of the reticuloplasm from mammalian rough microsomes revealed two distinct stages of the translocation reaction. The first stage, translocation up to and including signal peptide cleavage, was insensitive to the loss of the reticuloplasm, whereas the second stage, net transfer of the nascent chain into the ER lumen, was reticuloplasm dependent. In reticuloplasm-depleted membranes, signal-cleaved and glycosylated translocation intermediates were observed to transit free from the translocation channel to the cis, or cytoplasmic, side of the membrane. This translocation defect was complemented by reconstitution of lumenal proteins into depleted membranes. We propose that lumenal proteins are necessary for unidirectional protein translocation in mammalian ER.

BIOCHEMICAL, CELL BIOLOGICAL AND IMMUNOLOGICAL ISSUES SURROUNDING THE ENDOPLASMIC RETICULUM CHAPERONE GRP94/GP96

The past year has born witness to compelling demonstrations of the utility of peptide complexes with glucose regulated protein 94 (GRP94, also known as gp96) in cancer immunotherapy. Insights into the structural basis of peptide binding to GRP94 have been obtained and the role of the transporter for antigen presentation in defining the GRP94-bound peptide composition has been determined.

Interaction of TLR2 and TLR4 Ligands with the N-terminal Domain of Gp96 Amplifies Innate and Adaptive Immune Responses

Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free (<0.5 enzyme units/mg). As described previously, innate immune cells are activated by Gp96 at high concentrations (≥50 μg/ml) but not at lower concentrations. However, preincubation of low amounts of Gp96 with TLR2 and TLR4 ligands at concentrations unable to activate dendritic cells by themselves results in the production of high levels of proinflammatory cytokines, up-regulation of activation markers, and amplification of T cell activation. Our results provide significant new insights into the mechanism of HSP-mediated dendritic cell activation and present a new function of HSPs in the amplification of dendritic cell activation by bacterial products and induction of adaptive immune responses.

Interaction of Endoplasmic Reticulum Chaperone GRP94 with Peptide Substrates Is Adenine Nucleotide-independent

GRP94, the endoplasmic reticulum paralog of hsp90, has recently been identified as a peptide and adenine nucleotide-binding protein. To determine if adenine nucleotides directly contribute to the regulation of GRP94 peptide binding activity, an in vitro peptide binding assay was developed. Using purified GRP94, we observed specific, saturable, temperature-sensitive binding of the peptide VSV8, a known in vivo ligand. ATP was without effect on VSV8 binding to GRP94, whether present during or subsequent to peptide binding. To evaluate the interaction of GRP94 with adenine nucleotides, the ATP binding and hydrolysis activities were directly assayed. Only negligible binding of ATP to GRP94 was observed. In addition, analysis of the GRP94 adenine nucleotide content indicated that GRP94 did not copurify with bound adenine nucleotides. GRP94 preparations exhibited low ATPase and apparent autophosphorylation activities. Further purification, combined with inhibitor studies, indicated that both activities were the result of trace contamination (<0.1%) with casein kinase II. On the basis of these data, we propose that the peptide binding activity of GRP94 is adenine nucleotide-independent and that ATP binding and hydrolysis are not inherent properties of GRP94.

GRP94 (gp96) and GRP94 N-Terminal Geldanamycin Binding Domain Elicit Tissue Nonrestricted Tumor Suppression

In chemical carcinogenesis models, GRP94 (gp96) elicits tumor-specific protective immunity. The tumor specificity of this response is thought to reflect immune responses to GRP94-bound peptide antigens, the cohort of which uniquely identifies the GRP94 tissue of origin. In this study, we examined the apparent tissue restriction of GRP94-elicited protective immunity in a 4T1 mammary carcinoma model. We report that the vaccination of BALB/c mice with irradiated fibroblasts expressing a secretory form of GRP94 markedly suppressed 4T1 tumor growth and metastasis. In addition, vaccination with irradiated cells secreting the GRP94 NH2-terminal geldanamycin-binding domain (NTD), a region lacking canonical peptide-binding motifs, yielded a similar suppression of tumor growth and metastatic progression. Conditioned media from cultures of GRP94 or GRP94 NTD-secreting fibroblasts elicited the up-regulation of major histocompatibility complex class II and CD86 in dendritic cell cultures, consistent with a natural adjuvant function for GRP94 and the GRP94 NTD. Based on these findings, we propose that GRP94-elicited tumor suppression can occur independent of the GRP94 tissue of origin and suggest a primary role for GRP4 natural adjuvant function in antitumor immune responses.

Re-evaluating the role of heat-shock protein–peptide interactions in tumour immunity

Early investigations into the immune surveillance of chemically-induced sarcomas led to two important concepts in tumour immunobiology: one, tumour rejection can be elicited by immune recognition of tumour antigens; and two, tumours express unique sets of antigens, which are known as tumour-specific antigens. The pioneering studies of Srivastava and colleagues led to the proposal that heat-shock proteins (HSPs) function as ubiquitous tumour-specific antigens, with the specificity residing in a population of bound peptides that identify the tissue of origin of the HSP. However, recent findings, including new data on the cell biology of peptide generation and trafficking, have called into question the specificity of tumour rejection that is induced by HSPs.

Membrane topology mapping of vitamin K epoxide reductase by in vitro translation/cotranslocation

Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10–29, 101–123, and 127–149, and membrane-integration of residues 75–97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM.

Structure of the N-terminal Domain of GRP94 BASIS FOR LIGAND SPECIFICITY AND REGULATION

GRP94, the endoplasmic reticulum (ER) paralog of the chaperone Hsp90, plays an essential role in the structural maturation or secretion of a subset of proteins destined for transport to the cell surface, such as the Toll-like receptors 2 and 4, and IgG, respectively. GRP94 differs from cytoplasmic Hsp90 by exhibiting very weak ATP binding and hydrolysis activity. GRP94 also binds selectively to a series of substituted adenosine analogs. The high resolution crystal structures at 1.75–2.1 Å of the N-terminal and adjacent charged domains of GRP94 in complex with N-ethylcarboxamidoadenosine, radicicol, and 2-chlorodideoxyadenosine reveals a structural mechanism for ligand discrimination among hsp90 family members. The structures also identify a putative subdomain that may act as a ligand-responsive switch. The residues of the charged region fold into a disordered loop whose termini are ordered and continue the twisted beta sheet that forms the structural core of the N-domain. This continuation of the beta sheet past the charged domain suggests a structural basis for the association of the N-terminal and middle domains of the full-length chaperone.