Efficient conversion of chemical energy into mechanical work by Hsp70 chaperones.

Hsp70 molecular chaperones are abundant ATP-dependent nanomachines that actively reshape non-native, misfolded proteins and assist a wide variety of essential cellular processes. Here, we combine complementary theoretical approaches to elucidate the structural and thermodynamic details of the chaperone-induced expansion of a substrate protein, with a particular emphasis on the critical role played by ATP hydrolysis. We first determine the conformational free-energy cost of the substrate expansion due to the binding of multiple chaperones using coarse-grained molecular simulations. We then exploit this result to implement a non-equilibrium rate model which estimates the degree of expansion as a function of the free energy provided by ATP hydrolysis. Our results are in quantitative agreement with recent single-molecule FRET experiments and highlight the stark non-equilibrium nature of the process, showing that Hsp70s are optimized to effectively convert chemical energy into mechanical work close to physiological conditions.

Cloning of three heat shock protein genes (HSP70, HSP90α and HSP90ß) and their expressions in response to thermal stress in loach (Misgurnus anguillicaudatus) fed with different levels of vitamin C.

Heat shock protein 70 (HSP70) and 90 (HSP90) are the most broadly studied proteins in HSP families. They play key roles in cells as molecular chaperones, in response to stress conditions such as thermal stress. In this study, full-length cDNA sequences of HSP70, HSP90α and HSP90ß from loach Misgurnus anguillicaudatus were cloned. The full-length cDNA of HSP70 in loach was 2332bp encoding 644 amino acids, while HSP90α and HSP90ß were 2586bp and 2678bp in length, encoding 729 and 727 amino acids, respectively. The deduced amino acid sequences of HSP70 in loach shared the highest identity with those of Megalobrama amblycephala and Cyprinus carpio. The deduced amino acid sequences of HSP90α and HSP90ß in loach both shared the highest identity with those of M. amblycephala. Their mRNA tissue expression results showed that the maximum expressions of HSP70, HSP90α and HSP90ß were respectively present in the intestine, brain and kidney of loach. Quantitative real-time PCR was employed to analyze the temporal expressions of HSP70, HSP90α and HSP90ß in livers of loaches fed with different levels of vitamin C under thermal stress. Expression levels of the three HSP genes in loach fed the diet without vitamin C supplemented at 0 h of thermal stress were significantly lower than those at 2 h, 6 h, 12 h and 24 h of thermal stress. It indicated that expressions of the three HSP genes were sensitive to thermal stress in loach. The three HSP genes in loaches fed with 1000 mg/kg vitamin C expressed significantly lower than other vitamin C groups at many time points of thermal stress, suggesting 1000 mg/kg dietary vitamin C might decrease the body damages caused by the thermal stress. This study will be of value for further studies into thermal stress tolerance in loach.

In Cell Interactome of Oleocanthal, an Extra Virgin Olive Oil Bioactive Component.

A copper-(I)-catalyzed variation of the Huisgen 1,3-dipolar cycloaddition has been applied to lead the in living-cell mass-spectrometry based identification of protein targets of oleocanthal, a natural metabolite daily ingested by millions of people. Chemical proteomics revealed heat-shock proteins, HSP70 and HSP90, as main oleocanthal interactors in living systems. These two proteins are involved in cancer development and, thus, our findings could have important outcomes for a deep evaluation of the bio-pharmacological significance of oleocanthal.

Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70.

Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.

ATP requirement for chloroplast protein import is set by the Km for ATP hydrolysis of stromal Hsp70 in Physcomitrella patens.

The 70-kD family of heat shock proteins (Hsp70s) is involved in a number of seemingly disparate cellular functions, including folding of nascent proteins, breakup of misfolded protein aggregates, and translocation of proteins across membranes. They act through the binding and release of substrate proteins, accompanied by hydrolysis of ATP. Chloroplast stromal Hsp70 plays a crucial role in the import of proteins into plastids. Mutations of an ATP binding domain Thr were previously reported to result in an increase in the Km for ATP and a decrease in the enzyme's kcat. To ask which chloroplast stromal chaperone, Hsp70 or Hsp93, both of which are ATPases, dominates the energetics of the motor responsible for protein import, we made transgenic moss (Physcomitrella patens) harboring the Km-altering mutation in the essential stromal Hsp70-2 and measured the effect on the amount of ATP required for protein import into chloroplasts. Here, we report that increasing the Km for ATP hydrolysis of Hsp70 translated into an increased Km for ATP usage by chloroplasts for protein import. This thus directly demonstrates that the ATP-derived energy long known to be required for chloroplast protein import is delivered via the Hsp70 chaperones and that the chaperone's ATPase activity dominates the energetics of the reaction.

Development of a capillary electrophoresis platform for identifying inhibitors of protein-protein interactions.

Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 are separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound-to-free ratio. The method was used to screen a library of 3443 compounds, and the results were compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that were reconfirmed in subsequent testing, suggesting greater specificity. This finding was attributed to the use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens, but at the current stage of development it is attractive as a secondary screen to test hits found by higher-throughput methods.

Synthesis and initial evaluation of YM-08, a blood-brain barrier permeable derivative of the heat shock protein 70 (Hsp70) inhibitor MKT-077, which reduces tau levels.

The molecular chaperone, heat shock protein 70 (Hsp70), is an emerging drug target for treating neurodegenerative tauopathies. We recently found that one promising Hsp70 inhibitor, MKT-077, reduces tau levels in cellular models. However, MKT-077 does not penetrate the blood-brain barrier (BBB), limiting its use as either a clinical candidate or probe for exploring Hsp70 as a drug target in the central nervous system (CNS). We hypothesized that replacing the cationic pyridinium moiety in MKT-077 with a neutral pyridine might improve its clogP and enhance its BBB penetrance. To test this idea, we designed and synthesized YM-08, a neutral analogue of MKT-077. Like the parent compound, YM-08 bound to Hsp70 in vitro and reduced phosphorylated tau levels in cultured brain slices. Pharmacokinetic evaluation in CD1 mice showed that YM-08 crossed the BBB and maintained a brain/plasma (B/P) value of ∼0.25 for at least 18 h. Together, these studies suggest that YM-08 is a promising scaffold for the development of Hsp70 inhibitors suitable for use in the CNS.

Chemical proteomics reveals HSP70 1A as a target for the anticancer diterpene oridonin in Jurkat cells.

Oridonin, an ent-kaurane diterpene isolated from well known Chinese medicinal plant Isodon rubescens, has been shown to have multiple biological activities. Among them, the anticancer activity has been repeatedly reported by many research groups. The chemopreventive and antitumor effects of oridonin have been related to its ability to interfere with several pathways which are involved in cell proliferation, cell cycle arrest, apoptosis and/or autophagy. Despite the number of studies performed on this diterpene, the molecular mechanism underlying its cellular activity remains to be elucidated. Hence, we tried to mine target protein(s) of oridonin by employing a mass spectrometry-based chemical proteomics approach, providing evidences that oridonin is able to directly bind the multifunctional, stress-inducible heat shock protein 70 1A (HSP70 1A). Oridonin/HSP70 complex formation was confirmed in leukemia-derived Jurkat cells. The characterization of HSP70 inhibition by oridonin was performed using chemical and biological approaches. Moreover, the binding site of oridonin on the chaperone was identified by a mass-based approach combined with Molecular Dynamics simulations. BIOLOGICAL SIGNIFICANCE: Although natural products showed high efficiency and several of these agents have now entered in clinical trials, information concerning the mechanisms of action at a molecular level of many of them is very poor or completely missed. Nevertheless, the identification of the molecular target of a drug candidate has several advantages. The most significant is the ability to set up target-based assays and to allow structure-activity relationship studies to guide medicinal chemistry efforts towards lead optimization. The knowledge of drug targets can also facilitate the identification of potential toxicities or side effects, if there is any precedent of toxicities for the identified target. Achieving this in an effective, unbiased and efficient manner subsists as a significant challenge for the new era in drug discovery and optimization. In the present study, we used a chemical proteomic approach aimed to define the possible protein target of the ent-kaurane diterpene oridonin. This natural compound has drawn a rising attention for cancer biologists due to its remarkable anti-tumor activities: accumulating evidence has suggested that oridonin is able to hamper the progression of tumor, mitigate tumor burden and alleviate cancer syndrome, which may improve greatly the survival rates of cancer patients; however molecular mechanisms by which this compound exerts its anti-tumor activities still remained to be discovered. We identified the molecular chaperone HSP70 1A as an oridonin target in Jurkat cells, thus suggesting a mechanism of action for the diterpene consistent with the multiple biological activities described for it. HSP70 inhibition by oridonin might indeed simultaneously result in the impairment of some of client proteins, thus in turn affecting several molecular pathways. Shedding light on the molecular basis of the biological activity of oridonin, our findings may be relevant for possible therapeutic applications of oridonin, such as its use in combination and the design of new therapeutic approaches. In addition, this research demonstrates the effectiveness of chemical proteomic approaches in drug discovery studies and in orphan drug molecular target identification.

[Cloning and expression analysis of HSP70 gene from Dendrobium officinale under low temperature stress].

OBJECTIVE: To investigate HSP70 gene expression from Dendrobium officinale under low temperature stress, which will provide the molecular biological foundation for breeding the low temperature resistant strain. METHOD: HSP70 gene full length cDNA was cloned by rapid amplification of cDNA ends (RACE) on the basis of HSP70 gene fragment sequences, and the structure and function of HSP70 gene were deduced. The expression of HSP70 under low temperature stress was detected by RT-PCR. RESULT: The full length of HSP70 gene cDNA was 2 296 bp containing a 1 944 bp open reading frame (ORF) that encoded a protein of 647 amino acids. Its amino acids sequence had typical HSP70 characteristics and high homology with other plant's HSP70. Cold stress expression analysis showed that expression of the HSP70 gene could be induced by low temperature. CONCLUSION: The HSP70 gene of D. officinale was successfully cloned and reported for the first time which proved that the expression could be induced by low temperature. The cloning of HSP70 gene provides a stable foundation for further study of D. officinale cultivation and the breeding of the cold resistance strains.

N-acetylglucosamine biofunctionalized CdSeTe quantum dots as fluorescence probe for specific protein recognition.

Hsp70 proteins are implicated in resistance to chemotherapy in cancers, the detection of which is important for cancer treatment and prognosis. In this work, we report the study on the detection of specific intracellular target protein in fixed cells using GlcNAc-conjugated CdSeTe QDs. The QDs were coupled with Con A via a carbodiimide reaction and then were further assembled with GlcNAc by lectin-carbohydrate interaction between Con A and GlcNAc. The obtained QDs-Con A-GlcNAc conjugates have an emission wavelength at 650 nm that is close to the near-infrared (NIR) regions and a specific recognition for Hsp70. These results show that the QDs-Con A-GlcNAc probe can be a promising tool for direct localization of the Hsp70 protein.

Chemical screens against a reconstituted multiprotein complex: myricetin blocks DnaJ regulation of DnaK through an allosteric mechanism.

DnaK is a molecular chaperone responsible for multiple aspects of bacterial proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors we screened plant-derived extracts against a reconstituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK's intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaK to DnaJ. Together, these results highlight a "gray box" screening approach, which might facilitate the identification of inhibitors of other protein-protein interactions.

Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus.

Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly upregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses.

The proteome of the developing tooth of the sea urchin, Lytechinus variegatus: mortalin is a constituent of the developing cell syncytium.

Echinoderm teeth are continuously growing calcite-mineralized tissues of complex structure. Two features are of special interest: (1) cell division takes place in a restricted aboral domain, the plumula, and the cells immediately merge into multinucleated syncytial layers; (2) the major part of the heavily mineralized tooth elongates and moves towards the adoral incisal tip continuously as the syncytial cells actively expand the syncytium and intermembrane mineral phase. As the first step to understanding the nature of the mineralization processes, we have isolated the proteins of the plumula and of the mature mineralized portions of the tooth, and begun their characterization. Peptide sequences were used to screen a plumula cDNA library by polymerase chain reaction. One primer set yielded a prominent amplified product which was cloned, and sequenced. Comparison with the nucleotide and protein data banks revealed the protein to be Mortalin, a member of the hsp-70 family, with >75% of its sequences identical to that of human mortalin. Immunocytochemical localization of mortalin within the plumula, using Anti-human Grp75, showed staining of the odontoblast cytosol and matrix at the point where syncytial formation was occurring. The cytosol of the syncytial layers was weakly stained. The nuclei within the syncytia were stained at their periphery. In the mature part of the tooth, the perinuclear staining of the nuclei was more prominent. We conclude that mortalin is involved in syncytium formation and maintenance. The urchin mortalin has a distinctive aspartic acid and serine-rich C-terminal domain that may link it to the mineralization process.

Hyperthermia enhances CTL cross-priming.

Dendritic cells (DCs) loaded with killed allogeneic melanoma cells can cross-prime naive CD8(+) T cells to differentiate into melanoma-specific CTLs in 3-wk cultures. In this study we show that DCs loaded with killed melanoma cells that were heated to 42 degrees C before killing are more efficient in cross-priming of naive CD8(+) T cells than DCs loaded with unheated killed melanoma cells. The enhanced cross-priming was demonstrated by several parameters: 1) induction of naive CD8(+) T cell differentiation in 2-wk cultures, 2) enhanced killing of melanoma peptide-pulsed T2 cells, 3) enhanced killing of HLA-A*0201(+) melanoma cells in a standard 4-h chromium release assay, and 4) enhanced capacity to prevent tumor growth in vitro in a tumor regression assay. Two mechanisms might explain the hyperthermia-induced enhanced cross-priming. First, heat-treated melanoma cells expressed increased levels of 70-kDa heat shock protein (HSP70), and enhanced cross-priming could be reproduced by overexpression of HSP70 in melanoma cells transduced with HSP70 encoding lentiviral vector. Second, hyperthermia resulted in the increased transcription of several tumor Ag-associated Ags, including MAGE-B3, -B4, -A8, and -A10. Thus, heat treatment of tumor cells permits enhanced cross-priming, possibly via up-regulation of both HSPs and tumor Ag expression.

Diversity in domain architectures of Ser/Thr kinases and their homologues in prokaryotes.

BACKGROUND: Ser/Thr/Tyr kinases (STYKs) commonly found in eukaryotes have been recently reported in many bacterial species. Recent studies elucidating their cellular functions have established their roles in bacterial growth and development. However functions of a large number of bacterial STYKs still remain elusive. The organisation of domains in a large dataset of bacterial STYKs has been investigated here in order to recognise variety in domain combinations which determine functions of bacterial STYKs. RESULTS: Using sensitive sequence and profile search methods, domain organisation of over 600 STYKs from 125 prokaryotic genomes have been examined. Kinase catalytic domains of STYKs tethered to a wide range of enzymatic domains such as phosphatases, HSP70, peptidyl prolyl isomerases, pectin esterases and glycoproteases have been identified. Such distinct preferences for domain combinations are not known to be present in either the Histidine kinase or the eukaryotic STYK families. Domain organisation of STYKs specific to certain groups of bacteria has also been noted in the current anlaysis. For example, Hydrophobin like domains in Mycobacterial STYK and penicillin binding domains in few STYKs of Gram-positive organisms and FHA domains in cyanobacterial STYKs. Homologues of characterised substrates of prokaryotic STYKs have also been identified. CONCLUSION: The domains and domain architectures of most of the bacterial STYKs identified are very different from the known domain organisation in STYKs of eukaryotes. This observation highlights distinct biological roles of bacterial STYKs compared to eukaryotic STYKs. Bacterial STYKs reveal high diversity in domain organisation. Some of the modular organisations conserved across diverse bacterial species suggests their central role in bacterial physiology. Unique domain architectures of few other groups of STYKs reveal recruitment of functions specific to the species.

Characterization of the G alpha(s) regulator cysteine string protein.

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP(1-198) (full-length), CSP(1-112), and CSP(1-82) on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with G alpha(s) and increases steady-state GTP hydrolysis. CSP(1-198) modulation of G alpha(s) was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP(1-112) was Hsc70-SGT-independent. CSP(1-112) preferentially associated with the inactive GDP-bound conformation of G alpha(s). Consistent with the stimulation of GTP hydrolysis, CSP(1-112) increased guanine nucleotide exchange of G alpha(s). The interaction of native G alpha(s) and CSP was confirmed by coimmunoprecipitation and showed that G alpha(s) associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the beta2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of G alpha(s) and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.

A hairpin-like structure within an AU-rich mRNA-destabilizing element regulates trans-factor binding selectivity and mRNA decay kinetics.

In mammals, rapid mRNA turnover directed by AU-rich elements (AREs) is mediated by selective association of cellular ARE-binding proteins. These trans-acting factors display overlapping RNA substrate specificities and may act to either stabilize or destabilize targeted transcripts; however, the mechanistic features of AREs that promote preferential binding of one trans-factor over another are not well understood. Here, we describe a hairpin-like structure adopted by the ARE from tumor necrosis factor alpha (TNFalpha) mRNA that modulates its affinity for selected ARE-binding proteins. In particular, association of the mRNA-destabilizing factor p37(AUF1) was strongly inhibited by adoption of the higher order ARE structure, whereas binding of the inducible heat shock protein Hsp70 was less severely compromised. By contrast, association of the mRNA-stabilizing protein HuR was only minimally affected by changes in ARE folding. Consistent with the inverse relationship between p37(AUF1) binding affinity and the stability of ARE folding, mutations that stabilized the ARE hairpin also inhibited its ability to direct rapid mRNA turnover in transfected cells. Finally, phylogenetic analyses and structural modeling indicate that TNFalpha mRNA sequences flanking the ARE are highly conserved and may stabilize the hairpin fold in vivo. Taken together, these data suggest that local higher order structures involving AREs may function as potent regulators of mRNA turnover in mammalian cells by modulating trans-factor binding selectivity.

Defining the properties of the nonhelical tail domain in type II keratin 5: insight from a bullous disease-causing mutation.

Inherited mutations in the intermediate filament (IF) proteins keratin 5 (K5) or keratin 14 (K14) cause epidermolysis bullosa simplex (EBS), in which basal layer keratinocytes rupture upon trauma to the epidermis. Most mutations are missense alleles affecting amino acids located in the central alpha-helical rod domain of K5 and K14. Here, we study the properties of an unusual EBS-causing mutation in which a nucleotide deletion (1649delG) alters the last 41 amino acids and adds 35 residues to the C terminus of K5. Relative to wild type, filaments coassembled in vitro from purified K5-1649delG and K14 proteins are shorter and exhibit weak viscoelastic properties when placed under strain. Loss of the C-terminal 41 residues contributes to these alterations. When transfected in cultured epithelial cells, K5-1649delG incorporates into preexisting keratin IFs and also forms multiple small aggregates that often colocalize with hsp70 in the cytoplasm. Aggregation is purely a function of the K5-1649delG tail domain; in contrast, the cloned 109 residue-long tail domain from wild type K5 is distributed throughout the cytoplasm and colocalizes partly with keratin IFs. These data provide a mechanistic basis for the cell fragility seen in individuals bearing the K5-1649delG allele, and point to the role of the C-terminal 41 residues in determining K5's assembly properties.

The sulfogalactose moiety of sulfoglycosphingolipids serves as a mimic of tyrosine phosphate in many recognition processes. Prediction and demonstration of Src homology 2 domain/sulfogalactose binding.

Multiple ligand co-recognition of 3'-sulfogalactosylceramide (SGC) and sulfotyrosine initiated the comparison of SGC and sulfotyrosine and, subsequently, phosphotyrosine (pY) binding. SGC is a receptor for ligands involved in cell adhesion/microbial pathology. pY forms a Src homology domain 2 recognition motif in intracellular signaling. Using hsp70, anti-SGC, and anti-pY antibodies, ligand binding is retained following phosphate/sulfate and tyrosine/galactose substitution in SGC and sulfate/phosphate exchange in pY. Remarkable lipid-dependent binding to phosphatidylethanolamine-conjugated sulfotyrosine suggests "microenvironmental" modulation of sulfotyrosine-containing receptors, similar to glycosphingolipids. Based on an aryl substrate-bound co-crystal of arylsulfatase A, a sulfogalactose and phosphotyrosine esterase, modeling provides a solvation basis for co-recognition. c-Src/Src homology domain 2:SGC/phosphogalactosylceramide binding confirms our hypothesis, heralding a carbohydrate-based approach to regulation of phosphotyrosine-mediated recognition.

SREC-I, a type F scavenger receptor, is an endocytic receptor for calreticulin.

Calreticulin and gp96 (GRP94) traffic associated peptides into the major histocompatibility complex class-I cross-presentation pathway of antigen-presenting cells (APCs). Efficient accession of the cross-presentation pathway requires APC receptor-mediated endocytosis of the chaperone/peptide complexes. Previously, scavenger receptor class-A (SRA) was shown to play a substantial role in trafficking gp96 and calreticulin into macrophages, accounting for half of total receptor-mediated uptake. However, the scavenger receptor ligand fucoidin competed the chaperone uptake beyond that accounted for by SRA, indicating that another scavenger receptor(s) may also contribute. Consistent with this hypothesis, we showed that the residual calreticulin uptake into SRA(-/-) macrophages is competed by the scavenger receptor ligand acetylated low density lipoprotein (LDL). We now report that an additional scavenger receptor, SREC-I (scavenger receptor expressed by endothelial cell-I), mediates the endocytosis of calreticulin and gp96. Ectopic expression of SREC-I in Chinese hamster ovary cells yielded chaperone recognition and uptake, and these processes were competed by the inhibitory ligands fucoidin and acetylated (Ac)LDL. Although AcLDL competes for the chaperone interactions with SRA and SREC, we showed that not all of the scavenger receptors, which bind AcLDL, bind calreticulin or gp96. The overexpression of SREC-I in macrophages increased chaperone endocytosis, indicating that SREC-I functions in APCs and that the cytosolic components necessary for the endocytosis of SREC-I and its cargo are present and not limiting in APCs. These data identify a novel class of ligands for SREC-I and provide insight into the mechanisms by which APCs and potentially endothelial cells traffic chaperone/antigen complexes.