The AAA؉ molecular chaperone Hsp104 mediates the extraction of proteins from aggregates by unfolding and threading them through its axial channel in an ATP-driven process. An Hsp104-binding peptide selected from solid phase arrays enhanced the refolding of a firefly luciferase-peptide fusion protein. Analysis of peptide binding using tryptophan fluorescence revealed two distinct binding sites, one in each AAA؉ module of Hsp104. As a further indication of the relevance of peptide binding to the Hsp104 mechanism, we found that it competes with the binding of a model unfolded protein, reduced carboxymethylated ␣-lactalbumin. Inactivation of the pore loops in either AAA؉ module prevented stable peptide and protein binding. However, when the loop in the first AAA؉ was inactivated, stimulation of ATPase turnover in the second AAA؉ module of this mutant was abolished. Drawing on these data, we propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA؉ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA؉ module.Hsp104 is a AAAϩ protein disaggregase that functions in yeast in the resolubilization and reactivation of thermally denatured and aggregated proteins (1, 2). In unstressed cells, Hsp104 is critical to the mitotic stability of the yeast prions [, and [URE3] (3-5). Hsp104 and its bacterial orthologue ClpB are members of the Hsp100/Clp family of proteins (6). Other Hsp100s, such as ClpA, ClpX, and ClpY (HslU), unfold and unidirectionally translocate polypeptides through a central axial channel (7-11). Crystal structures of HslU (12, 13) and cryoelectron microscopic reconstructions of ClpB (14) reveal that the diameter of the axial channel is regulated by flexible loops whose conformation is regulated by the nucleotide status of the nucleotide binding domain of each AAAϩ module. Modification of these loops impairs protein translocation and/or degradation implying that these loops play critical roles in translocation (15-18). Likewise, mutation of the flexible loops of Hsp104 and ClpB results in refolding defects suggesting that all Hsp100s employ a similar unfolding/threading mechanism to process substrates whether they are ultimately degraded or refolded (16,19,20). Despite the growing body of knowledge regarding the unfolding and translocation mechanism of Hsp104, the determinants of the initial stage of the unfolding process, substrate recognition and binding, remain unclear.In other Hsp100s, recognition of specific peptide sequences initiates unfolding and translocation. Protein substrates of ClpXP generally contain recognition signals of roughly 10 -15 residues that can be located either at the N or C termini (21). The SsrA tag, an 11-amino acid peptide (AANDENYALAA) that is appended to the C terminus of polypeptides by the action of transfer-messenger RNA on stalled ribosomes (22), is a particularly well studied example of an Hsp10...
An unusual "densely methylated island" (DMI), in which all cytosine residues are methylated on both strands for 127-516 base pairs, has been reported at mammalian origins of DNA replication. This report had far-reaching implications in understanding of DNA methylation and DNA replication. For example, since this DMI appeared in about 90% of proliferating cells, but not in stationary cells, it may regulate origin activation. In an effort to confirm and extend these observations, the DMI at the well characterized ori- locus 17 kilobases downstream of the dhfr gene in chromosomes of Chinese hamster ovary cells was checked for methylated cytosines in genomic DNA. The methylation status of this region was examined in randomly proliferating and stationary cells and in cell populations enriched in the G 1 , S, or G 2 ؉ M phases of their cell division cycle. DNA was subjected to 1) cleavage by methylation-sensitive restriction endonucleases, 2) hydrazine modification of cytosines followed by piperidine cleavage, and 3) permanganate modification of 5-methylcytosines ( m C) followed by piperidine cleavage. The permanganate reaction is a novel method for direct detection of m C residues that complements the more commonly used hydrazine method. These methods were capable of detecting m C in 2% of the cells. At the region of the proposed DMI, only one mC at a CpG site was detected. However, the ori- DMI was not detected in any of these cell populations using any of these methods.DNA methylation at CpG dinucleotides in mammalian cells has been implicated as an important component of such pivotal processes as transcription (1), imprinting (2), recombination (3), carcinogenesis (4), development (5), and replication timing (6). CpG methylation is carried out by a methyltransferase that is associated with replication foci in the nucleus (7). In general, this enzyme methylates specifically hemimethylated CpG dinucleotides, although de novo methylation of unmethylated CpG dinucleotides can occur but at a low rate (8). In addition, this enzyme occasionally methylates cytosines in sequences other than CpG (9, 10). However, given the specificity of the mammalian methyltransferase, this non-target methylation should be of little significance in vivo because the pattern of non-CpG methylation will not be maintained during the subsequent round of DNA replication.Recently, an unusual form of DNA methylation has been implicated as an important component of mammalian origins of DNA replication. A densely methylated island (DMI) 1 has been reported at three different mammalian origins of DNA replication (11, 12). These DMIs were found only in proliferating cell populations and consisted of a 127-, 258-, or 516-bp stretch of DNA in which all cytosines were methylated, regardless of their sequence context (12). The discovery of DMIs has several important implications. First, this unprecedented methylation pattern suggested that a second (or modified) methylation enzyme exists in mammalian cells. Interestingly, limited proteolysis of the mamm...
Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum (ER) whose folding-promoting functions are directed predominantly toward aspargine-linked glycoproteins. This is a consequence of calnexin and calreticulin being lectins with specificity for the early oligosaccharide (OS)-processing intermediate, Glc1Man9GlcNAc2. In addition, they interact with non-native conformers of glycoprotein polypeptide chains to prevent aggregation and recruit the thiol oxidoreductase ERp57 to catalyze glycoprotein disulfide formation/isomerization. In vitro assays of these functions have contributed greatly to our understanding of how calnexin and calreticulin promote glycoprotein folding. This chapter describes the isolation of Glc1Man9GlcNAc2 OS, as well as the assay used to measure OS binding. Furthermore, details are provided of assays that detect ERp57 binding by calnexin and calreticulin, as well as the abilities of these chaperones to suppress the aggregation of non-native protein substrates.
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