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J. Biol. Chem., Vol. 277, Issue 22, 19831-19838, May 31, 2002

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Characterization of a Brain-enriched Chaperone, MRJ, That Inhibits Huntingtin Aggregation and Toxicity Independently^*

Jen-Zen Chuang^§, Hui Zhou^§¶, Meicai Zhu, Shi-Hua Li^¶, Xiao-Jiang Li^¶, and Ching-Hwa Sung^**

From the  Department of Ophthalmology and ^** Department of Cell Biology and Anatomy, Weill Medical College of Cornell University, New York, New York 10012 and the ^¶ Department of Human Genetics, Emory University, Atlanta, Georgia 30322

Received for publication, October 4, 2001, and in revised form, March 12, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular chaperones are involved in a wide range of cellular events, such as protein folding and oligomeric protein complex assembly. DnaK- and DnaJ-like proteins are the two major classes of molecular chaperones in mammals. Recent studies have shown that DnaJ-like family proteins can inhibit polyglutamine aggregation, a hallmark of many neurodegenerative diseases, including Huntington's disease (HD). Although most DnaJ-like proteins studied are ubiquitously expressed, some have restricted expression, so it is possible that some specific chaperones may affect polyglutamine aggregation in specific neurons. In this report, we describe the isolation of a DnaJ-like protein MRJ and the characterization of its chaperone activity. Tissue distribution studies showed that MRJ is highly enriched in the central nervous system. In an in vitro cell model of HD, overexpressed MRJ effectively suppressed polyglutamine-dependent protein aggregation, caspase activity, and cellular toxicity. Collectively, these results suggest that MRJ has a relevant functional role in neurons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular chaperones (heat-shock proteins) were initially identified by their appearance under conditions of stress. During stress, these proteins prevent aggregation and assist in the refolding of misfolded proteins. Chaperones also play essential roles in a variety of cellular functions under normal conditions. These functions include assisting in the folding of newly translated proteins, guiding translocation proteins across organelle membranes, assembling and disassembling oligomeric protein complexes, and facilitating proteolytic degradation of unstable proteins (1).

In Escherichia coli, different classes of molecular chaperones (DnaK, DnaJ, GrpE) function in concert: DnaJ and GrpE act as co-factors for the ATPase activity catalyzed by DnaK (2). Mammalian homologues of DnaK (Hsp70)¹ and DnaJ have also been isolated, and, in contrast to the prokaryotes, mammals have many homologues of these genes. Although the Hsp70 family members share a high degree of sequence conservation, members of the DnaJ-like protein family are structurally diverse, containing different combinations of 1-3 conserved domains. All DnaJ-like proteins contain a characteristic J-domain, which is believed to mediate the interactions with Hsp70 that regulate ATPase activity (2, 3). Regions in the C terminus of the DnaJ-like protein are much less conserved and thought to mediate interactions with polypeptide substrates (4). In contrast to Hsp70, which is ubiquitously expressed, several DnaJ family proteins exhibit some tissue specificity (5, 6). Thus, DnaJ proteins may play a role in determining the specificity of functional engagement of Hsp70 in distinct cellular compartments or in different cell types (4).

A correlation has been observed between the expression levels of molecular chaperones and the ability of cells to survive stress. For example, heat shock treatment induces the overexpression of certain chaperones, which is correlated with the increasing protective effect against light damage in the retina (7) and against glutamate excitotoxicity in cortical neuron cultures (8, 9). Recently, the role of chaperones has been investigated in polyglutamine diseases, such as HD. A hallmark of polyglutamine diseases is the proteolytic production of an N-terminal fragment of huntingtin, containing the polyglutamine repeat, that forms aggregates (inclusions) in the nucleus and cytoplasm of affected neurons (10-16). Although the inclusions have not been shown to have a causal role in these diseases, their involvement in the pathogenesis of HD has been supported by the correlation between the number of inclusions in HD patients' cortex and the severity of their disease (17, 18). Moreover, inclusion formation precedes neurological dysfunction in the transgenic mice model of HD and is associated with predisposition to cell death in in vitro cell models of HD (19, 20).

The involvement of molecular chaperones in the polyglutamine expansion-induced diseases is best evidenced by the fact that Hsp70 and DnaJ-like protein Hsp40/HDJ1 were isolated as suppressors for polyglutamine-mediated neurodegeneration in the fly (21, 22). However, overexpression of different members of universally expressed DnaJ-like family proteins have had varied results in the cellular model of HD: Overexpression of HDJ-2/HSDJ increased polyglutamine-induced aggregation (23), whereas Hsp40/HDJ-1 overexpression inhibited polyglutamine-induced aggregation (24, 25).

In this paper, we describe the isolation and functional characterization of a DnaJ-like family protein, MRJ, which is highly enriched in the central nervous system. In HD cell models, overexpressed MRJ effectively suppressed the protein aggregation, caspase activity, and cellular toxicity induced by the mutant huntingtin, suggesting that MRJ may be a chaperone that acts on neuronal disease proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular Cloning of Bovine and Human mrj-- Partial cDNAs of bovine mrj were initially isolated in a yeast two-hybrid screen in which the second extracellular region of peripherin/rds was used as bait. To construct the bait plasmid, the coding sequence for peripherin/rds (Arg-123 to Ser-264) was PCR-amplified from the human retinal cDNA (forward primer, 5'-GGAATTCCATATGCGGGGCTCGCTGGAGAACACC; reverse primer, 5'-GTACCCATGGTTAGGAGTTCATGAGGCTGCTGTA) and inserted into NdeI/NcoI-digested pASII vector (CLONTECH, Palo Alto, CA). To obtain the full-length bovine mrj cDNA, 5'- and 3'-RACE were carried out using a Marathon cDNA amplification kit (CLONTECH). Briefly, first-strand cDNAs were synthesized on bovine retinal poly(A)⁺ RNA using gene-specific primers (5'-CTGGCCGTCTTCTTCAACTTCCACCC-3' for 5'-RACE; 5'-GGAGCCGAGGCACGGGCTCGTTC-3' for 3'-RACE). The second-strand cDNA was subsequently synthesized and ligated with an adapter primer. Finally, 5'-RACE and 3'-RACE products were PCR-amplified using adapter-ligated cDNAs as template. The forward primer used for the PCR was a modified adapter primer that contains an extra BamHI site, and the reverse primer is a nested gene-specific primer (5'-CATGCCATGGTGATCTTCCTGCTGTTCACCAC for 5'-RACE; 5'-CATGCCATGGAAGTCTGTTTCCTTCCTTTGATGC for 3'-RACE). The nested PCR products were then digested with NcoI and BamHI and inserted into NcoI/BamHI-digested pACTII vector (CLONTECH) for sequencing.

To isolate human mrj cDNA, full-length bovine mrj cDNA was radiolabeled with [-³²P]dCTP (Ready to Go kit, Amersham Biosciences, Inc., Piscataway, NJ) and used as a probe to screen a human retinal cDNA library (a kind gift from Dr. J. Nathans). The cDNA inserts were PCR-amplified from the positive isolates and cloned for sequencing. Several clones contained full-length human mrj, with an encoded sequence identical to the human mrj recorded in GenBank^TM (accession number NP_005485).

Constructs-- To generate a GST-MRJ fusion construct for antibody production, a BamHI/XhoI cDNA fragment containing the C-terminal 198 residues of bovine MRJ was excised from the pACTII vector isolated from the two-hybrid screen and inserted 3' of the GST open reading frame in pGEX-5X-2 vector. The same cDNA fragment was also subcloned into a BamHI/SalI-digested pMAL-cRI* vector (26) to produce an MBP-MRJ fusion for antibody purification. The coding sequence of full-length human MRJ and an N-terminal 41-amino acid-deleted mutant were also subcloned into a PRK5 vector for eukaryotic expression.

For the ATPase activity assay, a His₆ tag was fused to the C terminus of an ORF containing either the full-length (MRJ-(1-241)), the J plus G/F domain (MRJ-(1-109)), or the C-terminal domain (MRJ-(108-241)) of human MRJ in pET28a vector using standard PCR-cloning methods. All PCR-derived constructs were sequenced to confirm the absence of spurious mutations.

Northern Blotting-- Total RNA (20 µg) from various bovine tissues was purified and resolved by agarose gel electrophoresis under denaturing conditions and blotted as described previously (27). A -³²P-radiolabeled, bovine mrj cDNA fragment containing the region encoding the entire 3'-UTR region (nucleotides 811-1374) was hybridized in 5× SSC, 30% formamide, 2% SDS, 200 µg/ml denatured herring sperm DNA and 5× Denhardt's solution at 42 °C. Human poly(A)⁺ RNA tissue blots (CLONTECH) were similarly probed with radiolabeled cDNA fragment of the human mrj 3'-UTR region (nucleotides 730-1406). After hybridization, RNA blots were washed three times with 1× SSC and 0.1% SDS at room temperature before autoradiography.

ATPase Activity Assay-- BL21(DE3) harboring fusion constructs encoding His₆-tagged MRJ fragments were induced with isopropyl-1-thio--D-galactoside for 3 h. The cells were lysed, and postnuclear supernatant was purified on a nickel column according to the manufacturer's instructions (Novagen, Madison, WI). Fusion proteins were dialyzed against ATPase reaction buffer (20 mM Hepes-KOH, pH 7.4, 50 mM KCl, and 5 mM MgOAc). The protein concentration was determined using a protein assay (Bio-Rad, Hercules, CA), and the purity of the protein was examined by SDS-PAGE analysis followed by Coomassie Blue staining.

The ATPase activity assay was performed as described previously (28) with minor modifications. Briefly, assays were carried out in duplicate, with two 50-µl reactions per sample. Each reaction contained 1 µM bovine Hsp70 (Hsc70, Sigma Chemical Co., St. Louis, MO), 50 µM ATP, 0.6 µM purified His₆-tagged MRJ fragment, and 2 µCi of [-³²P]ATP (>3000 Ci/mM, PerkinElmer Life Sciences, Wellesley, MA) in ATPase reaction buffer. Prior to the reaction, 7 µl of each sample was removed and used to assess the basal level of radioactive release. At different time intervals during the 30 °C incubation, 7-µl aliquots were taken out and transferred to 0.5 ml of suspension containing 50 mM HCl, 5 mM H₃PO₄, and 7% (w/v) activated charcoal (Amersham Biosciences, Inc.) to stop the reaction. The reactions were centrifuged and assayed by liquid scintillation counting. The cpm values were converted to moles of P_i release into the supernatant at each time point. Initial titration of MRJ proteins (0-6 µM) in the Hsp70 ATPase assay showed that the ATPase stimulation effect of MRJ is dosage-dependent and that components in the ratio of 1 mol of Hsp70 to 1-3 mol of full-length MRJ fragments were effective. Note that the His₆-tag has been found to have no adverse effect on the co-chaperone activity of Hsp40 (29).

Generation and Purification of Anti-MRJ Antibody-- Bovine GST-MRJ was produced according to the manufacturer's instructions (Amersham Pharmacia Biotech) with minor modifications. Briefly, the isopropyl-1-thio--D-galactoside-induced bacterial culture was centrifuged and the cell pellets were resuspended in breaking buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1 µg/ml lysozyme, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 0.7 µg/ml pepstatin) and sonicated. The sonicated samples were supplemented with NaCl to a final concentration of 0.5 M and then centrifuged at 14,000 rpm for 15 min. The resulting pellet was resuspended in 50 mM Tris, pH 8.0, sonicated, and recentrifuged. The final pellet was dissolved in 8 M urea, 10 mM dithiothreitol, 5 mM EDTA, and 50 mM Tris-Cl, pH 8.0, and subjected to SDS-PAGE. A gel slice containing GST-MRJ was excised, ground, and used for immunization (Cocalico, Reamstown, PA). The resulting antiserum was used in this report. MBP-MRJ fusion proteins, which are soluble in Triton X-100, were produced and purified according to manufacturer's instruction (New England BioLabs, Beverly, MA). To affinity-purify the immunized rabbit antiserum, crude serum was first passed through Sepharose columns conjugated with GST and MBP proteins, then a column conjugated with MBP-MRJ fusion protein. The antibodies were eluted with 50 mM glycine-HCl, pH 2.8, and neutralized with 1 M Tris-Cl, pH 9.5.

Cell Culture and Immunocytochemistry-- PRK vectors expressing HD exon 1 (1-67 amino acids) plus additional 150 glutamines, Hsp70, and red fluorescence protein have been described in a previous study (25). Transient transfection of 293 cells was performed with LipofectAMINE (Invitrogen, Carlsbad, CA). For co-transfection, same amounts of huntingtin cDNA and the PRK vectors were used for expression of huntingtin alone. At 24 or 48 h after transfection, cells grown in six-well plates were fixed in 4% paraformaldehyde and permeabilized by 0.2% Triton-100. Double labeling was carried out using rabbit anti-MRJ antibody and mouse monoclonal anti-huntingtin antibody. The latter one was generated against the human huntingtin N terminus (amino acids 1-256), the same antigen that has been used to generate rabbit antibody EM48 (30). Hoechst dye (1 µg/ml) was used to label the nuclei. A Zeiss fluorescence microscope (Axiovert 135) and video system (Dage-MTI Inc., Michigan City, IN) were used to capture images. The captured images were stored and processed using Adobe PhotoShop software.

Cell Viability and Caspase Activity Assays-- Cell viability was determined by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay (Cell Titer 96, Promega) as described in our previous study (25). Briefly, transfected 293 cells in six-well plates were transferred into 96-well plates at a density of 20,000 cells/well in serum-free medium, and 20 µl of MTS reagent (2.1 mg/ml) was added to each well. The cells were then incubated for 45-60 min at 37 °C in a 5% CO₂ incubator. The reaction was stopped by adding 25 µl of 10% SDS. The plates were read with a microplate reader (SPECTRAmax PLUS, Molecular Devices, Palo Alto, CA) at 490 nm. Each data point was obtained using a triplet assay.

Fluorometric assays of caspase-3 activity (25, 31) were performed using kits obtained from Bio-Rad Laboratory (Hercules, CA). Cultured 293 cells were transiently transfected with mutant huntingtin 150Q or co-transfected with huntingtin and MRJ for 24 or 48 h in six-well plates. After washing with phosphate-buffered saline, the cells were lysed in the lysis buffer (10 mM Tris-HCl, 10 mM NaH₂PO₄/NaHPO₄, pH 7.5, 130 mM NaCl, 1% Triton X-100, 10 mM sodium pyrophosphate). To measure caspase activity, 200 µl of assay buffer (40 mM PIPES, pH 7.2, 200 mM NaCl, 20 mM dithiothreitol, 2 mM EDTA, 0.2% (w/v) CHAPS, 20% sucrose) was added to a tube, with a final concentration of 10 ng/µl peptide substrate (acetyl-Asp-Glu-Val-Asp-7-amido-4-(trifluoromethyl) for caspase-3. Cell lysates (200 µg of protein) were added to the tube to start the reaction. When the caspase inhibitor (Z-Val-Ala-Asp-fluoromethylketone) was used to measure the specificity of the assay, it was added to cell lysates at a concentration of 50 µM for 30 min before the addition of the specific caspase substrate. Background was obtained with the same assay buffer without cell lysates. The reaction was incubated at 37 °C for 1 h, followed by the measurement of the caspase activity with a fluorescence plate reader (Fluostar Galaxy, BMG Labtechnologies, Durham, NC) set at 390-nm excitation and 460-nm emission. For staurosporine treatment, transfected cells were grown in culture medium containing 1% fetal bovine serum and then incubated with staurosporine (2.5 µM, Sigma Chemical Co.) for 6 h before caspase-3 activity analysis. All values were obtained from three to five independent experiments and expressed as mean ± S.D. Statistical significance was assessed by using Student's t test. p < 0.05 was considered significant.

Immunoblotting-- Various tissues dissected from rats were homogenized and lysed in extraction buffer (1% Nonidet P-40, 0.5% deoxycholate, 10 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, and protease inhibitors). The supernatant prepared by centrifugation (40,000 × g for 1 h at 4 °C) was subjected to SDS-PAGE and blotted. Immunoblotting assays were performed by standard procedures. For signal detection, alkaline phosphatase-conjugated secondary antibody (Promega) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate were used. For the transfected cells, immunoblotting procedures were carried out as described previously (25).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular Cloning of Bovine and Human mrj-- During a two-hybrid screen for genes whose products interact with a retinal disease protein peripherin/rds (32, 33), a cDNA containing partial sequences with significant homology to several DnaJ-like family proteins was isolated. To obtain a full-length cDNA of this DnaJ-like protein, we produced 5'-RACE and 3'-RACE PCR products from bovine retinal RNA, and fused them together (see "Experimental Procedures"). The deduced amino acid sequence of this bovine cDNA (Fig. 1) was 94 and 90% identical to human and mouse MRJ, respectively (34, 35). Thus, our clones are the bovine orthologues of MRJ. This was further supported by the isolation of several full-length human mrj cDNA clones from a human retinal library probed with the bovine cDNA. Although human MRJ has 242 amino residues, both the bovine and mouse mrj encode an open reading frame of 241 amino acids with a predicted molecular mass of 26.9 kDa and a predicted pI of 7.4 (Table I). Hydropathy analysis revealed no transmembrane domains.

View larger version (73K): [in this window] [in a new window]   Fig. 1.   Sequence of the bovine mrj cDNA clone and its amino acid sequence. Nucleotide and deduced sequence of mrj containing the ORF, 5'-UTR, and 3'-UTR (GenBankTM accession number AF426743). The 729-bp ORF encodes a putative 242-amino acid protein. Note the Kozak sequence or ribosome binding sequence is locate upstream of the first ATG in the 5'-UTR. The asterisk represents the stop codon.

MRJ belongs to a growing list of DnaJ-like family proteins. It contains the signature J domain at the N terminus (amino acids 1-74), followed by a glycine/phenylalanine (G/F)-rich domain (amino acids 75-119), and unique C-terminal sequences. Sequence comparisons revealed that, among DnaJ-like family members, MRJ shares high overall identity to MSJ1 (70% identity (6)), HSJ-1b (61% identity (5)), Hsp40/HDJ-1 (41% identity (36, 37)), HDJ2/HSD-J (40% identity (38, 39)), and MTJ1 (31% identity (40)). However, the molecular masses and pI values of these proteins are rather diverse (Table I). On the other hand, the molecular mass of MRJ is similar to that of several small heat-shock proteins such as hsp20 (41), hsp25 (42), hsp27 (43), and crystallin (44, 45). However, there is no significant homology between MRJ and these other small heat-shock proteins.

Stimulatory Effect of MRJ on ATPase Activity of Hsp70-- One feature of E. coli DnaJ is its absolute requirement for interaction with DnaK to stimulate Hsp70 ATPase activity (46, 47). Although the mouse (34) and human MRJs (35, 48) have been previously isolated, the ability of MRJ to act as an Hsp70 co-chaperone has not yet been experimentally confirmed.

To test whether MRJ is a bona fide co-chaperone, recombinant His₆-tagged human MRJ was produced, purified, and subjected to an Hsp70 ATPase activity assay (Fig. 2). In agreement with a previous report (28), Hsp70 alone exhibited a weak ATPase activity. Under our experimental conditions, Hsp70 itself has a turnover rate of 0.025 mol of ATP hydrolyzed per minute (Fig. 2D). The addition of full-length MRJ fragment resulted in a significant stimulation of the Hsp70 ATPase; this stimulation was both dose-dependent (Fig. 2C) and time-dependent (Fig. 2D). In 20 min, MRJ increased the ATP hydrolysis rate ~8-fold relative to Hsp70 protein alone. Previously, it was shown that the region containing the J plus G/F domain of Hsp40, but not the J-domain alone, also had a stimulatory effect on Hsp70 ATPase activity (28). To compare the stimulatory effect of different domains of MRJ, the N terminus of MRJ-(1-109) containing both the J-domain and G/F domain and the C terminus of MRJ-(108-241) were subjected to Hsp70 ATPase assays. We showed that MRJ-(1-109) was capable of increasing Hsp70 ATPase activity by ~5.5-fold. Surprisingly, however, the C-terminal half of MRJ-(108-241) also displayed the ability to enhance the Hsp70-catalyzed ATPase activity ~4-fold. Previously, it has been shown that denatured protein substrates can also stimulate the ATPase activity catalyzed by Hsp70, however, only when they were added at the concentration of several hundred-fold molar excess to that of Hsp70 (27). In contrast, MRJ fragments are functionally active at 1- to 3-fold molar excess to Hsp70, indicating it acts as a co-chaperone, rather than as a substrate.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   ATPase catalyzed by Hsp70 can be stimulated by MRJ fragments. A, schematic diagram of the His6-tagged human MRJ fragments MRJ-(1-241), MRJ-(1-109), and MRJ-(108-241) used in Hsp70 ATPase assay (B) His6-tagged MRJ fragments were purified by nickel column, analyzed on SDS-PAGE, and stained by Coomassie Blue. The molecular weight markers are indicated. C, titration of stimulatory effect of full-length MRJ on Hsp70-catalyzed ATPase activity. Hsp70 (1 µM) and various concentrations of full-length MRJ (MRJ-(1-241)) were incubated with ATP in a 20-µl reaction at 30 °C for 30 min. The amount of [32P]Pi released into the supernatant was determined by liquid scintillation counting. The cpm data were converted to picomoles after correction for background release of radioactivity into the supernatant, counting efficiency, and specific activity of [-32P]ATP. D, Hsp70 (1 µM) without (diamond) or with 3 µM of His6-tagged MRJ-(1-241) (), MRJ-(1-109) (), or MRJ-(108-241) () were incubated with [-32P]ATP for 30 min at 30 °C. At each time point indicated, duplicate aliquots were removed and assayed. The results were obtained from three independent experiments.

Enrichment of MRJ in the Central Nervous System-- A Northern blot was performed to examine the tissue distribution of mrj. The initial attempt using the full-length bovine mrj cDNA as a probe detected two transcripts of ~3 and 1.7 kb in RNAs isolated from different tissues (data not shown). To rule out the possibility that multiple transcripts were derived from nonspecific cross-reactions of other DnaJ-like proteins, a probe containing only the 3'-UTR region of bovine mrj was then used (see "Experimental Procedures"). This probe detected a single ~1.7-kb transcript. The highest levels of the mrj messenger were found in retina and brain, whereas low levels of transcript were seen in other tissues (Fig. 3A). The conditions for blotting were very specific, as evidenced by the fact that the most closely related homologue, MSJ-1, which is testis-specific and has a transcript size of ~1.2 kb (Table I (6)), was not detected (data not shown). Similarly, a Northern blot of mRNAs isolated from various human tissues (CLONTECH) using a human mrj cDNA probe also revealed a brain-enriched distribution (Fig. 3B). Within brain, Northern blotting of mRNAs isolated from different brain regions showed that mrj was expressed at a higher level in hippocampus and thalamus and a lower level in amygdala, substantia nigra, corpus callosum, and caudate nucleus (Fig. 3C).

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Tissue expression patterns of MRJ determined by Northern blotting and immunoblotting assays. A, total RNAs isolated from the different bovine tissues as indicated were probed with radiolabeled bovine mrj cDNA fragment (top panel). Image of ethidium bromide-stained agarose demonstrates that a similar amount of each RNA was loaded in each sample (bottom panel). B, human tissue poly(A)+ RNA blot (CLONTECH) was probed with radiolabeled human mrj cDNA fragment. C, Northern blot of poly(A)+ RNAs isolated from different human brain regions probed with the same fragment used in B. A single band of ~1.7 kb mrj transcript was seen in both bovine and human RNAs. D, postnuclear supernatants of various rat tissues were electrophoresed on SDS-PAGE, transferred, and immunoblotted with the affinity-purified anti-MRJ antibody.

The tissue expression pattern of MRJ was also examined by immunoblotting. A single band with an estimated molecular mass of ~28 kDa was recognized by the affinity-purified anti-MRJ antibody in a few tissues (Fig. 3D). Consistent with the distribution of the mrj transcript, the highest amount of MRJ was detected in brain and retina among the tissues examined.

Reductions in HD Aggregation and Cellular Toxicity-- The evidence that MRJ is a co-factor of Hsp70 and is preferentially expressed in brain prompted us to examine whether MRJ can reduce polyglutamine protein aggregation associated with neurodegenerative diseases, such as HD. In the HD brain, N-terminal fragments of mutant huntingtin form inclusions in both the cytoplasm and nucleus (15, 29). Similar inclusions are also observed in tissue culture cells transfected with N-terminal huntingtin fragments (19, 20, 49). Using human embryonic kidney 293 cells transfected with mutant huntingtin N terminus as a model, we found that cells transfected with huntingtin alone exhibited puncta or aggregates distributed throughout the cytoplasm and nucleus (Fig. 4A). At 24 h post-transfection, 61.8% of these cells displayed aggregates. In contrast, cells where MRJ was co-expressed had diffusely distributed huntingtin and fewer aggregates; only ~11.2% of them had aggregates (Fig. 4B). To confirm this observation biochemically, we analyzed huntingtin aggregation by Western blot. At 24 h, co-expression of MRJ dramatically decreased the high molecular weight huntingtin aggregates (Fig. 4D, bracket) and increased the amount of huntingtin monomers (Fig. 4D, arrow). The reduction in huntingtin aggregation was not due to a decrease in the expression of transfected proteins, because double immunostaining showed that transfected cells expressed both MRJ and mutant huntingtin at relatively high levels. We found that the MRJ lacking the partial J-domain (first 41 residues) also displayed a similar ability in inhibiting huntingtin aggregation (data not shown), consistent with the C-terminal chaperone activity observed in the ATPase assays.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Inhibition of aggregation of mutant huntingtin by MRJ. A, low magnification (×100) of 293 cells transfected with the HD exon1 protein containing 150 glutamines (150Q) alone or 150Q and MRJ combined. Transfected cells were stained with mouse antibody to huntingtin and rabbit antibody to MRJ 24 h after transfection. B, quantitative assessment of transfected cells containing huntingtin aggregates. Approximately 61.8% of cells transfected with huntingtin alone contain aggregates (n = 462) whereas only about 11.2% of cells transfected with both huntingtin and MRJ contain aggregates (n = 583). C, high magnification of (×400) of 293 cells co-transfected with MRJ and 150Q at 24 or 48 h. Arrows indicate huntingtin inclusions that are also MRJ immunoreactive. D, immunoblotting analysis of aggregated huntingtin in 293 cells co-transfected with 150Q alone () or with (+) MRJ after 24 and 48 h. High molecular weight huntingtin aggregates and monomeric huntingtin are indicated by bracket and arrow, respectively.

Consistent with the previous report (25), the amount of huntingtin aggregation increased over time after transfection. Almost all cells exhibited huntingtin aggregates at 48 h after transfection (Fig. 4C). This was supported by the increase of high molecular weight aggregates detected in the Western blot assays (Fig. 4D). In contrast to the 24-h time point, the inhibitory effect of MRJ on huntingtin aggregation was remarkably reduced. As shown in Fig. 4C (arrows), many MRJ co-transfected cells also had aggregates, and these aggregates were immunoreactive for MRJ. Consistently, MRJ showed little or no effect on the removal of the high molecular weight aggregates in the Western blots (Fig. 4D).

Mutant huntingtin is toxic to transfected 293 cells (50). To examine whether MRJ also protects against huntingtin excitotoxicity, we measured the cell viability of transfected cells using an MTS assay. Cells transfected with huntingtin alone exhibited about 55% cell viability relative to the mock-transfected control cells (Fig. 5A). A similar degree of decreased viability was seen at both 24 and 48 h after transfection, despite the greater aggregation at the 48-h time point. Co-expression of MRJ significantly improved the viability of the huntingtin-transfected cells to 75-82% of the mock transfected control cells (Fig. 5A). The protective effect of MRJ was similar to that of Hsp70. However, the aggregation level was different between the 24- and 48-h transfected cells, indicating that cell viability may not be critically dependent on the amount of huntingtin aggregation. Overexpression of controls, either red fluorescence protein or huntingtin 20Q, had no effect on cell viability (Fig. 5A).

View larger version (48K): [in this window] [in a new window]   Fig. 5.   Inhibition of huntingtin toxicity by MRJ. A, cell viability was detected using MTS assay. Transfection of 150Q alone or with control red fluorescence protein (150Q+RFP) for either 24 or 48 h resulted in a similar decrease in cell viability. Co-expression of MRJ (150Q+MRJ) or Hsp70 (150Q+Hsp70) for 24 or 48 h significantly increased the viability of cells that were transfected with 150Q. 20Q or Hsp70 transfection alone did not significantly affect viability. The viability of cells transfected with empty PRK vector is considered to be 100%. **, p < 0.01 compared with 20Q transfection alone. B, MRJ inhibits caspase-3 activity in huntingtin-transfected cells. Co-expression of MRJ significantly inhibited the increase of caspase-3 activity mediated by 150Q at 24 as well as 48 h after transfection. 20Q or Hsp70 transfection alone did not significantly affect caspase-3 activity. The increase is compared with that of cells transfected with PRK vector alone. C, staurosporine treatment of 293 cells (STS, 2.5 µM) for 6 h markedly increased caspase-3 activity. This increase was reduced by MRJ overexpression (+MRJ) for either 24 or 48 h. Values are expressed as a percentage of control (PRK vector transfection alone). **, p < 0.01 compared with cells without MRJ.

Our recent studies suggested that chaperones protect against polyglutamine toxicity via inhibition of caspase activity (25). To examine whether MRJ also inhibits caspase activation induced by mutant huntingtin, we measured caspase-3 activity in transfected 293 cells. Expanded huntingtin (150Q), but not 20Q, increased caspase-3 activity (Fig. 5B). Like Hsp70, MRJ also significantly reduced the caspase-3 activity induced by mutant huntingtin. Co-expression of control red fluorescence protein did not prevent the increase in caspase-3 activity. The inhibitory effect of MRJ on caspase-3 activity was not much different between the 24- and 48-h time points. Finally, MRJ also inhibited the caspase-3 activity augmented by the apoptotic reagent staurosporine (Fig. 5C). This finding suggests that MRJ is capable of reducing caspase-3 activity mediated by not only mutant huntingtin but also another insult.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This report describes the isolation and functional characterization of a neuronal-enriched DnaJ-like protein, MRJ. In vitro, MRJ is capable of stimulating Hsp70 ATPase activity. In the cell model of HD, MRJ can inhibit huntingtin aggregation, caspase activity, and cellular toxicity. Many of these functions have been assigned to other co-chaperones and/or chaperones (25, 51-55), arguing that MRJ acts like a functional chaperone. These abilities of MRJ, taken together with its neuronal-enriched distribution, suggested that MRJ could be an endogenous molecular chaperone for neuronal proteins, including the HD disease protein.

The E. coli genome encodes only one DnaK, one DnaJ, and two or three additional J-domain containing molecules (56). In contrast, at least 11 Hsp70 (57) and 40 DnaJ-like proteins are identified in the mammal expressed sequence tag sequences (34), suggesting that chaperone functions are more specific and highly regulated in mammals. One possible mechanism to regulate chaperone function is the consideration of specific DnaJ family members with the protein substrates and/or specific members of Hsp70. This hypothesis has been supported by the unique tissue expression patterns of multiple members of the DnaJ-like family. At both the mRNA and protein levels, we showed a remarkable enrichment of MRJ in the brain of adult animals. This is consistent with the in situ studies in the mouse embryo (E12.5), where high levels of MRJ expression were seen in retina and several regions of the brain (trigeminal ganglia, diencephalon, and midbrain), whereas little or no expression of this molecule was detected in other organs (34). Moreover, the same report showed that the defective placental development caused by the loss of MRJ expression cannot be compensated for by the other two DnaJ-like family proteins, suggesting that different members of DnaJ proteins may have specific and unique cellular functions.

In both prokaryotes and eukaryotes, DnaJ-like proteins are believed to act as co-chaperones with the Hsp70 protein. The J-domain of DnaJ proteins is thought to regulate the ATPase activity of Hsp70, whereas the C-terminal domain binds to misfolded polypeptides and modulates the folding process (1). Similar to the results described for mammalian Hsp40 (29), we found that the J plus G/F domain of MRJ is functionally active in stimulating Hsp70 ATPase activity. For both Hsp40 and MRJ, this region itself yields more than half of the stimulatory activity of the full-length protein. Interestingly, our results suggest that the unique MRJ C terminus also was able to regulate Hsp70 ATPase activity. The C terminus activity was slightly weaker than that of the J plus G/F domain. Lower but substantial ATPase stimulatory activity has also been seen in the C terminus of E. coli DnaJ (58), but it has yet been described in mammalian DnaJ. In agreement with the in vitro ATPase assays, the MRJ protein with a partially deleted J-domain also retains some ability to inhibit huntingtin aggregation.

Previously, two MRJ homologues, Hsp40/HDJ-1 and HDJ-2/HSDJ, have been shown to be able to reduce polyglutamine aggregation in different cell models (23-25, 59). Although these chaperones share ~40% overall amino acid identity with MRJ, their C-terminal sequences are rather diverged. Interestingly, it has been noted that, like MRJ, truncated HDJ-2/HSDJ lacking the J-domain also can effectively suppress aggregation caused by the mutant ataxin-3 in vitro (60). It is thus probable that the J-domain, the specific C terminus, or both could be involved in the chaperone activity of DnaJ proteins in cells. These findings indicate that molecular chaperones have a broad substrate specificity, and thus the physiological roles of these chaperones are more likely to be determined by their expression level and tissue localization. At present, the only other known neuron-enriched DnaJ-like family protein is HSJ1, which was initially isolated by screening an expression library with an antibody against a putative structural component of neurofibrillary tangles associated with Alzheimer's disease (5). Interestingly, HSJ1b and MRJ are phylogenetically closely related: They share 61% identity overall. The endogenous substrate for HSJ1 has not yet been identified.

The ability of MRJ to reduce huntingtin aggregation and the co-localization of MRJ with huntingtin aggregates suggested that huntingtin could be the endogenous substrate of MRJ-mediated chaperone activity. Currently, we do not know how MRJ inhibits the formation of huntingtin protein aggregates. It is possible that MRJ constantly corrects the misfolding of mutant huntingtin during its protein synthesis and thus improves its solubility. Another possibility is that MRJ is involved in the rapid degradation of unstable proteins soon after their synthesis, a function that has been described for several DnaJ-like proteins in yeast (61, 62). The increase of huntingtin degradation products was indeed noted in the MRJ transfected cell lysates (Fig. 4D).

Although the pathological role of huntingtin inclusions is still controversial (56), the formation of polyglutamine aggregates is apparently correlated with disease progression in HD animal models (16, 17). Moreover, chaperones appear to be able to prevent neurodegeneration in polyglutamine disease models of the fly (21, 22). Nevertheless, the exact molecular mechanism by which chaperones prevent cell death is poorly understood. The present study showed that decreases in huntingtin aggregation in MRJ-overexpressing cells were not always kinetically associated with a decrease in cellular toxicity. Namely, aggregation was severe at 48 h even in the presence of MRJ, whereas cell death remained low in the MRJ-transfected cells. Although there are several possibilities, the simplest explanation is that the ability of MRJ to inhibit polyglutamine aggregation is independent of cellular toxicity. Interestingly, the caspase-3 activity inhibition exhibited by MRJ correlates well with the cell viability. Our previous study has shown that, although chaperones, Hsp70 and N-ethylmaleimide-sensitive factor, cannot inhibit huntingtin aggregation, they are able to inhibit cellular toxicity as well as caspase activity (25). Furthermore, MRJ is able to inhibit staurosporine-induced cell death, which is irrelevant to huntingtin aggregation. It is thus conceivable that the MRJ-mediated cell rescue occurs through an aggregation-independent pathway.

If the above hypothesis is true, the ability of MRJ to inhibit huntingtin toxicity suggests that MRJ may also be able to reduce the cellular toxicity caused by the expansion of the polyglutamine tracts responsible for other types of neuronal degenerative diseases, or even other kinds of diseases. These other neuronal polyglutamine diseases include dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia type 1 (SCA1), SCA3, and SCA7. One common feature of these polyglutamine diseases is the striking specificity of cell death. For example, although huntingtin is ubiquitously expressed in neuronal and non-neuronal cells, neurons in the striatum are primarily affected in HD patients (63-66). The high expression level of MRJ in neurons suggests that MRJ could act as a surveillance chaperone for regulating neuronal protein aggregation and inhibition of polyglutamine toxicity in vivo. Impairment of these processes could be particularly related to the neuropathology of the diseases. Further investigation of the temporal and spatial regulation of MRJ expression in brain, and its correlation with the vulnerable neurons will be important to reveal the putative role of chaperones in these diseases and may also provide novel therapeutic approaches for HD or other neuronal degenerative diseases.

	ACKNOWLEDGEMENTS

We thank Ramee Lee, Kai Xu, and Fengli Cao for expert technical assistance during the initial phase of this work.

	FOOTNOTES

^* This work was supported by a career development award, by the Dolley Green Special Scholar Award (Research To Prevent Blindness), by The Foundation Fighting Blindness, and by National Institutes of Health Grants EY11307 (to C.-H. S.), AG19206, and NS41449 (to X.-J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF426743.

^§ Both authors contributed equally to this work.

Present address: Center of Molecular Biology, Air Force General Hospital, Beijing 100036, China.

To whom correspondence should be addressed: The Margaret M. Dyson Vision Research Institute, Weill Medical College of Cornell University, 1300 York Ave., LC313, New York, NY 10021. Tel.: 212-746-2291; Fax: 212-746-6670; E-mail: chsung@mail.med.cornell.edu.

Published, JBC Papers in Press, March 14, 2002, DOI 10.1074/jbc.M109613200

	ABBREVIATIONS

The abbreviations used are: Hsp70, heat-shock protein 70; HD, Huntington's disease; RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; ORF, open reading frame; UTR, untranslated repeat; MTS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; SCA1, spinocerebellar ataxia type 1.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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