Shingo Nishikori

Broad Ranges of Affinity and Specificity of Anti-Histone Antibodies Revealed by a Quantitative Peptide Immunoprecipitation Assay

Antibodies directed against histone posttranslational modifications (PTMs) are critical tools in epigenetics research, particularly in the widely used chromatin immunoprecipitation (ChIP) experiments. However, a lack of quantitative methods for characterizing such antibodies has been a major bottleneck in accurate and reproducible analysis of histone modifications. Here, we report a simple and sensitive method for quantitatively characterizing polyclonal and monoclonal antibodies for histone PTMs in a ChIP-like format. Importantly, it determines the apparent dissociation constants for the interactions of an antibody with peptides harboring cognate or off-target PTMs. Analyses of commercial antibodies revealed large ranges of affinity, specificity and binding capacity as well as substantial lot-to-lot variations, suggesting the importance of quantitatively characterizing each antibody intended to be used in ChIP experiments and optimizing experimental conditions accordingly. Furthermore, using this method, we identified additional factors potentially affecting the interpretation of ChIP experiments.

p97 homologs from Caenorhabditis elegans, CDC-48.1 and CDC-48.2, suppress the aggregate formation of huntingtin exon1 containing expanded polyQ repeat

Polyglutamine (polyQ)‐expanded proteins are associated with cytotoxicity in some neurodegenerative disorders such as Huntingtons disease. We have reported that the aggregation of the polyQ‐expanded protein is partially suppressed by co‐expression of either of two homologs of an AAA chaperone p97, CDC‐48.1 or CDC‐48.2, in Caenorhabditis elegans, but how p97 regulates the aggregation of polyQ‐expanded proteins remains unclear. Here we present direct evidence that CDC‐48.1 and CDC‐48.2 suppress the aggregation of a huntingtin (Htt) exon1 fragment containing an expanded polyQ repeat in vitro. CDC‐48.1 and CDC‐48.2 bound the Htt exon1 fragment directly, and suppressed the formation of SDS‐insoluble aggregates of Htt fragments containing 53 glutamine residues (HttQ53) independently of nucleotides. CDC‐48.1 and CDC‐48.2 also modulated the oligomeric states of HttQ53 during the aggregate formation. In the absence of CDC‐48.1 and CDC‐48.2, HttQ53 formed 70–150 kDa oligomers, whereas 300–500 kDa oligomers as well as 70–150 kDa oligomers accumulated in the presence of CDC‐48.1 and CDC‐48.2. Taken together, these results suggest that p97 plays a protective role in neurodegenerative disorders by directly suppressing the protein aggregation as a molecular chaperone.

Positive Cooperativity of the p97 AAA ATPase Is Critical for Essential Functions

p97 is composed of two conserved AAA (ATPases associated with diverse cellular activities) domains, which form a tandem hexameric ring. We characterized the ATP hydrolysis mechanism of CDC-48.1, a p97 homolog of Caenorhabditis elegans. The ATPase activity of the N-terminal AAA domain was very low at physiological temperature, whereas the C-terminal AAA domain showed high ATPase activity in a coordinated fashion with positive cooperativity. The cooperativity and coordination are generated by different mechanisms because a noncooperative mutant still showed the coordination. Interestingly, the growth speed of yeast cells strongly related to the positive cooperativity rather than the ATPase activity itself, suggesting that the positive cooperativity is critical for the essential functions of p97.

AAA+ Chaperone ClpX Regulates Dynamics of Prokaryotic Cytoskeletal Protein FtsZ

AAA+ chaperone ClpX has been suggested to be a modulator of prokaryotic cytoskeletal protein FtsZ, but the details of recognition and remodeling of FtsZ by ClpX are largely unknown. In this study, we have extensively investigated the nature of FtsZ polymers and mechanisms of ClpX-regulated FtsZ polymer dynamics. We found that FtsZ polymerization is inhibited by ClpX in an ATP-independent manner and that the N-terminal domain of ClpX plays a crucial role for the inhibition of FtsZ polymerization. Single molecule analysis with high speed atomic force microscopy directly revealed that FtsZ polymer is in a dynamic equilibrium between polymerization and depolymerization on a time scale of several seconds. ClpX disassembles FtsZ polymers presumably by blocking reassembly of FtsZ. Furthermore, Escherichia coli cells overproducing ClpX and N-terminal domain of ClpX show filamentous morphology with abnormal localization of FtsZ. These data together suggest that ClpX modulates FtsZ polymer dynamics in an ATP-independent fashion, which is achieved by interaction between the N-terminal domain of ClpX and FtsZ monomers or oligomers.

High-speed atomic force microscopic observation of ATP-dependent rotation of the AAA+ chaperone p97

p97 (also called VCP and CDC-48) is an AAA+ chaperone, which consists of a substrate/cofactor-binding N domain and two ATPase domains (D1 and D2), and forms a homo-hexameric ring. p97 plays crucial roles in a variety of cellular processes such as the ubiquitin-proteasome pathway, the endoplasmic reticulum-associated protein degradation, autophagy, and modulation of protein aggregates. Mutations in human p97 homolog VCP are linked to neurodegenerative diseases. The key mechanism of p97 in these various functions has been proposed to be the disassembly of protein complexes. To understand the molecular mechanism of p97, we studied the conformational changes of hexameric CDC-48.1, a Caenorhabditis elegans p97 homolog, using high-speed atomic force microscopy. In the presence of ATP, the N-D1 ring repeatedly rotates ~23 ± 8° clockwise and resets relative to the D2 ring. Mutational analysis reveals that this rotation is induced by ATP binding to the D2 domain.

Comparative analysis of the two-step reaction catalyzed by prokaryotic and eukaryotic phytochelatin synthase by an ion-pair liquid chromatography assay

Genes encoding phytochelatin (PC) synthase have been found in higher plants, fission yeast and worm. Recently, kinetic and mutagenic analyses of recombinant PC synthase have been revealing the molecular mechanisms underlying PC synthesis, however, a conclusive model has not been established. To clarify the mechanism of PC synthase found in eukaryotes, we have compared the two-step reactions catalyzed by the prokaryotic Nostoc PC synthase (NsPCS) and the eukaryotic Arabidopsis PC synthase (AtPCS1). Comparative analysis shows that in the first step of PC synthesis corresponding to the cleavage of γ-glutamylcysteine (γ-EC) from glutathione (GSH), free GSH or PCs acts as a donor molecule to supply a γ-EC unit for elongation of the PC chain, and heavy metal ions are required to carry out the cleavage. Furthermore, functional analyses of various mutants of NsPCS and AtPCS1, selected by comparing the sequences of NsPCS and AtPCS1, indicate that the N-terminal region (residues 1–221) in AtPCS1 is the catalytic domain, and in this region, the Cys56 residue is associated with the PC synthesis reaction. These results enable us to propose an advanced model of PC synthesis, describing substrate specificity, heavy metal requirement, and the active site in the enzyme.

High Temperature Increases the Refolding Yield of Reduced Lysozyme: Implication for the Productive Process for Folding

Misfolding poses a serious problem in the biotechnological field in obtaining the active protein from inclusion bodies. Here we show that high temperature increases the refolding yield of reduced lyosyzme by a simple dilution method. The refolding yields at 98 °C were three times higher than those at 20 °C in the solutions tested, which is related to the fact that the thermally unfolded state of lysozyme is a more productive form for folding than the denaturant‐induced fully unfolded state. The thermal‐assisted refolding could be used for various reduced and denatured proteins as a result of its simplicity and low cost.

Microtubule Severing by Katanin p60 AAA+ ATPase Requires the C-terminal Acidic Tails of Both α- and β-Tubulins and Basic Amino Acid Residues in the AAA+ Ring Pore

Background: Katanin p60 is a protein that actively severs microtubules. Results: Mutations within the AAA+ pore of katanin p60 and in the C-terminal regions of tubulins perturb efficient microtubule severing. Conclusion: Interactions between the conserved residues in the katanin p60 pore and the acidic tails of both tubulins may be important. Significance: Both tubulin molecules are essential for microtubule severing by katanin. The microtubule (MT) network is highly dynamic and undergoes dramatic reorganizations during the cell cycle. Dimers of α- and β-tubulins rapidly polymerize to and depolymerize from the end of MT fibrils in an intrinsic GTP-dependent manner. MT severing by ATP-driven enzymes such as katanin and spastin contributes significantly to microtubule dynamics, and it has been shown that katanin p60, a AAA+ family protein, has ATPase and MT-severing activities. The mechanism of MT severing by katanin p60 is poorly understood, and the residues in katanin p60 and tubulins important for severing activity were therefore explored in this study. MT-severing activity, but not ATPase activity, was inhibited by mutations of the conserved aromatic residue and the flanking basic residues in the pore region of the katanin p60 hexameric ring. When the acidic residue-rich C-terminal unstructured segment of either α- or β-tubulin was removed, polymerized MTs were resistant to katanin p60 treatment. Interactions between katanin p60 and the mutant MTs, on the other hand, were unaffected. Taken together, these findings led us to propose that the interactions between the positively charged residues of katanin p60 and the acidic tails of both tubulins are essential for efficient severing of MTs.

FtsH cleavage of non-native conformations of proteins

FtsH is a peculiar prokaryotic protease with low unfoldase activity. Different reports have proposed that FtsH substrates could be either tagged proteins or proteins of low stability. We show here that FtsH degradation of 31 point mutants of Anabaena apoflavodoxin is inversely proportional to their conformational stabilities, and that the same applies to other substrate proteins. In contrast, highly stable proteins such as GST and holoflavodoxin are not degraded at all. Attempts to identify sequence tags signaling for degradation in apoflavodoxin fragments have been unsuccessful. Apoflavodoxin adopts three conformations: native, partly unfolded and fully unfolded. It is revealing that degradation of the 31 variants is proportional to the molar fraction of fully unfolded molecules and inversely proportional to the fraction of stable apoflavodoxin molecules. This indicates that FtsH, rather than unfolding the protein, acts on the fraction that is already unfolded.

Role of C-terminal Cys-rich region of phytochelatin synthase in tolerance to cadmium ion toxicity.

Phytochelatins (PCs) are Cys-rich peptides, synthesized by PC synthase in response to heavy metal ions. The C-terminal Cys-rich region of the PC synthase has homology with functional domains of metallochaperone, metallothionein and thioredoxin. To test the possibility that the C-terminal Cys-rich region of PC synthase has a role in regulating PC synthesis, we introduced point mutations into the PC synthase, replacing Cys358, Cys359 Cys363 and Cys366 residues with Ala. The mutant PC synthase had a lower PC synthesis ability than the wild-type enzyme. Further, oxidative conditions severely damaged mutant PC synthase whilst the wild-type enzyme suffered less damage, suggesting that the Cys-rich region of PC synthase may play an important role in anti-oxidation activity. Although the C-terminal of PC synthase is not conserved, our studies support the possibility that this region performs several important biological functions.