Tousled-like kinase 2 targets ASF1 histone chaperones through client mimicry.

Tousled-like kinases (TLKs) are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants. A major function of TLKs is to phosphorylate the histone chaperone proteins ASF1a and ASF1b to facilitate DNA replication-coupled nucleosome assembly, but how TLKs selectively target these critical substrates is unknown. Here, we show that TLK2 selectivity towards ASF1 substrates is achieved in two ways. First, the TLK2 catalytic domain recognizes consensus phosphorylation site motifs in the ASF1 C-terminal tail. Second, a short sequence at the TLK2 N-terminus docks onto the ASF1a globular N-terminal domain in a manner that mimics its histone H3 client. Disrupting either catalytic or non-catalytic interactions through mutagenesis hampers ASF1 phosphorylation by TLK2 and cell growth. Our results suggest that the stringent selectivity of TLKs for ASF1 is enforced by an unusual interaction mode involving mutual recognition of a short sequence motifs by both kinase and substrate.

High-Efficiency Expression and Purification of DNAJB6b Based on the pH-Modulation of Solubility and Denaturant-Modulation of Size.

The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein "as is" with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.

Pathway of Hsp70 interactions at the ribosome.

In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb's ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.

RANBP2 mutation causing autosomal dominant acute necrotizing encephalopathy attenuates its interaction with COX11.

PURPOSE: Autosomal dominant acute necrotizing encephalopathy (ADANE) is caused by missense mutations in the gene encoding Ran-binding protein 2 (RANBP2), a nuclear pore protein regulating mitochondrial localization and function. Previous studies have found that RANBP2 binds to COX11 and suppresses its inhibitory activity over hexokinase1. To further elucidate mitochondrial dysfunction in ADANE, we analyzed the interaction between mutated RANBP2 and COX11. METHODS: We extracted cDNA from a patient and constructed pGEX wild-type or mutant-type vectors including RANBP2 c.1754C>T, the commonest variant in ADANE. We transformed E. coli competent cells with the vectors and had them express GST-RANBP2 recombinant protein, and conducted a pull-down assay of RANBP2 and COX11. RESULTS: The amount of COX11 bound to mutated RANBP2 was significantly smaller than that bound to the wild-type RANBP2. CONCLUSION: Mutated RANBP2 had an attenuated binding ability to COX11. Whether this change indeed decreases ATP production remains to be further explored.

Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations.

Many strategies have been pursued to trap and monitor single proteins over time to detect the molecular mechanisms of these essential nanomachines. Single-protein sensing with nanopores is particularly attractive because it allows label-free high-bandwidth detection on the basis of ion currents. Here we present the nanopore electro-osmotic trap (NEOtrap) that allows trapping and observing single proteins for hours with submillisecond time resolution. The NEOtrap is formed by docking a DNA-origami sphere onto a passivated solid-state nanopore, which seals off a nanocavity of a user-defined size and creates an electro-osmotic flow that traps nearby particles irrespective of their charge. We demonstrate the NEOtrap's ability to sensitively distinguish proteins on the basis of size and shape, and discriminate between nucleotide-dependent protein conformations, as exemplified by the chaperone protein Hsp90. Given the experimental simplicity and capacity for label-free single-protein detection over the broad bio-relevant time range, the NEOtrap opens new avenues to study the molecular kinetics underlying protein function.

Functional Expression, Purification and Identification of Interaction Partners of PACRG.

PACRG (Parkin co-regulated gene) shares a bi-directional promoter with the Parkinson's disease-associated gene Parkin, but the physiological roles of PACRG have not yet been fully elucidated. Recombinant expression methods are indispensable for protein structural and functional studies. In this study, the coding region of PACRG was cloned to a conventional vector pQE80L, as well as two cold-shock vectors pCold II and pCold-GST, respectively. The constructs were transformed into Escherichia coli (DE3), and the target proteins were overexpressed. The results showed that the cold-shock vectors are more suitable for PACRG expression. The soluble recombinant proteins were purified with Ni2+ chelating column, glutathione S-transferase (GST) affinity chromatography and gel filtration. His6 pull down assay and LC-MS/MS were carried out for identification of PACRG-binding proteins in HEK293T cell lysates, and a total number of 74 proteins were identified as potential interaction partners of PACRG. GO (Gene ontology) enrichment analysis (FunRich) of the 74 proteins revealed multiple molecular functions and biological processes. The highest proportion of the 74 proteins functioned as transcription regulator and transcription factor activity, suggesting that PACRG may play important roles in regulation of gene transcription.

Mechanistic Insights into the Role of Molecular Chaperones in Protein Misfolding Diseases: From Molecular Recognition to Amyloid Disassembly.

Age-dependent alterations in the proteostasis network are crucial in the progress of prevalent neurodegenerative diseases, such as Alzheimer's, Parkinson's, or amyotrophic lateral sclerosis, which are characterized by the presence of insoluble protein deposits in degenerating neurons. Because molecular chaperones deter misfolded protein aggregation, regulate functional phase separation, and even dissolve noxious aggregates, they are considered major sentinels impeding the molecular processes that lead to cell damage in the course of these diseases. Indeed, members of the chaperome, such as molecular chaperones and co-chaperones, are increasingly recognized as therapeutic targets for the development of treatments against degenerative proteinopathies. Chaperones must recognize diverse toxic clients of different orders (soluble proteins, biomolecular condensates, organized protein aggregates). It is therefore critical to understand the basis of the selective chaperone recognition to discern the mechanisms of action of chaperones in protein conformational diseases. This review aimed to define the selective interplay between chaperones and toxic client proteins and the basis for the protective role of these interactions. The presence and availability of chaperone recognition motifs in soluble proteins and in insoluble aggregates, both functional and pathogenic, are discussed. Finally, the formation of aberrant (pro-toxic) chaperone complexes will also be disclosed.

Hsp27 chaperones FUS phase separation under the modulation of stress-induced phosphorylation.

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid-liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.

Highly Precise Protein Semisynthesis through Ligation-Desulfurization Chemistry in Combination with Phenacyl Protection of Native Cysteines.

Semisynthesis of proteins via expressed protein ligation is a powerful tool to furnish full-length proteins carrying site-specific (posttranslational) modifications. The development of various ß-mercapto amino acid building blocks coupled with ligation-desulfurization chemistry enabled further advances in this methodology by alleviating the need for cysteine residues at the desired ligation sites. However, this expansion in the availability of viable ligation sites is sometimes counterbalanced by the inadvertent desulfurization of unprotected native cysteines, which might be of structural and/or functional importance. Here, we provide a detailed protocol for using the cysteine-selective protecting group phenacyl (PAc) to achieve precise protein semisynthesis preserving native cysteine residues. The PAc group can be easily installed on cysteine(s) within recombinantly produced protein thioesters, withstands standard ligation, desulfurization and reversed phase HPLC conditions, and can be smoothly removed. We have previously demonstrated the utility of this protecting group through the semisynthesis of two model proteins, human small heat shock protein Hsp27 and Prion protein, in which one or two native cysteines, respectively, were maintained through the ligation-desulfurization sequence.

Mimicking cotranslational folding of prosubtilisin E in vitro.

Nascent polypeptides are synthesized on ribosomes starting at the N-terminus and simultaneously begin to fold during translation. We constructed N-terminal fragments of prosubtilisin E containing an intramolecular chaperone (IMC) at N-terminus to mimic cotranslational folding intermediates of prosubtilisin. The IMC-fragments of prosubtilisin exhibited progressive enhancement of their secondary structures and thermostabilities with increasing polypeptide length. However, even the largest IMC-fragment with 72 residues truncated from the C-terminus behaved as a molten globule, indicating the requirement of the C-terminal region to have a stable tertiary structure. Furthermore, truncation of the IMC in the IMC-fragments resulted in aggregation, suggesting that the IMC plays a crucial role to prevent misfolding and aggregation of cotranslational folding intermediates during translation of prosubtilisin polypeptide.

Medium-Throughput Detection of Hsp90/Cdc37 Protein-Protein Interaction Inhibitors Using a Split Renilla Luciferase-Based Assay.

The protein-folding chaperone Hsp90 enables the maturation and stability of various oncogenic signaling proteins and is thus pursued as a cancer drug target. Folding in particular of protein kinases is assisted by the co-chaperone Cdc37. Several inhibitors against the Hsp90 ATP-binding site have been developed. However, they displayed significant toxicity in clinical trials. By contrast, the natural product conglobatin A has an exceptionally low toxicity in mice. It targets the protein-protein interface (PPI) of Hsp90 and Cdc37, suggesting that interface inhibitors have an interesting drug development potential. In order to identify inhibitors of the Hsp90/Cdc37 PPI, we have established a mammalian cell lysate-based, medium-throughput amenable split Renilla luciferase assay. This assay employs N-terminal and C-terminal fragments of Renilla luciferase fused to full-length human Hsp90 and Cdc37, respectively. We expect that our assay will allow for the identification of novel Hsp90/Cdc37 interaction inhibitors. Such tool compounds will help to evaluate whether the toxicity profile of Hsp90/Cdc37 PPI inhibitors is in general more favorable than that of ATP-competitive Hsp90 inhibitors. Further development of such tool compounds may lead to new classes of Hsp90 inhibitors with applications in cancer and other diseases.

Observation of unexpected molecular binding activity for Mu phage tail fibre chaperones.

In the history of viral research, one of the important biological features of bacteriophage Mu is the ability to expand its host range. For extending the host range, the Mu phage encodes two alternate tail fibre genes. Classical amber mutation experiments and genome sequence analysis of Mu phage suggested that gene products (gp) of geneS (gpS = gp49) and gene S' (gpS' = gp52) are tail fibres and that gene products of geneU (gpU = gp50) and geneU' (gpU' = gp51) work for tail fibre assembly or tail fibre chaperones. Depending on the gene orientation, a pair of genes 49-50 or 52-51 is expressed for producing different tail fibres that enable Mu phage to recognize different host cell surface. Since several fibrous proteins including some phage tail fibres employ their specific chaperone to facilitate folding and prevent aggregation, we expected that gp50 or gp51 would be a specific chaperone for gp49 and gp52, respectively. However, heterologous overexpression results for gp49 or gp52 (tail fibre subunit) together with gp51 and gp50, respectively, were also effective in producing soluble Mu tail fibres. Moreover, we successfully purified non-native gp49-gp51 and gp52-gp50 complexes. These facts showed that gp50 and gp51 were fungible and functional for both gp49 and gp52 each other.

Histone chaperone exploits intrinsic disorder to switch acetylation specificity.

Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.

Characterization of the 20S proteasome of the lepidopteran, Spodoptera frugiperda.

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.

Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME.

Dedicated chaperones protect newly synthesized ribosomal proteins (r-proteins) from aggregation and accompany them on their way to assembly into nascent ribosomes. Currently, only nine of the ∼80 eukaryotic r-proteins are known to be guarded by such chaperones. In search of new dedicated r-protein chaperones, we performed a tandem-affinity purification based screen and looked for factors co-enriched with individual small subunit r-proteins. We report the identification of Nap1 and Tsr4 as direct binding partners of Rps6 and Rps2, respectively. Both factors promote the solubility of their r-protein clients in vitro. While Tsr4 is specific for Rps2, Nap1 has several interaction partners including Rps6 and two other r-proteins. Tsr4 binds co-translationally to the essential, eukaryote-specific N-terminal extension of Rps2, whereas Nap1 interacts with a large, mostly eukaryote-specific binding surface of Rps6. Mutation of the essential Tsr4 and deletion of the non-essential Nap1 both enhance the 40S synthesis defects of the corresponding r-protein mutants. Our findings highlight that the acquisition of eukaryote-specific domains in r-proteins was accompanied by the co-evolution of proteins specialized to protect these domains and emphasize the critical role of r-protein chaperones for the synthesis of eukaryotic ribosomes.

In Vivo Trapping of Proteins Interacting with the Chloroplast CLPC1 Chaperone: Potential Substrates and Adaptors.

The chloroplast stromal CLP protease system is essential for growth and development. It consists of a proteolytic CLP core complex that likely dynamically interacts with oligomeric rings of CLPC1, CLPC2, or CLPD AAA+ chaperones. These ATP-dependent chaperones are predicted to bind and unfold CLP protease substrates, frequently aided by adaptors (recognins), and feed them into the proteolytic CLP core for degradation. To identify new substrates and possibly also new adaptors for the chloroplast CLP protease system, we generated an in vivo CLPC1 substrate trap with a C-terminal STREPII affinity tag in Arabidopsis thaliana by mutating critical glutamate residues (E374A and E718A) in the two Walker B domains of CLPC1 required for the hydrolysis of ATP (CLPC1-TRAP). On the basis of homology to nonplant CLPB/C chaperones, it is predicted that interacting substrates are unable to be released; that is, they are trapped. When expressed in the wild type, this CLPC1-TRAP induced a dominant visible phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered. Affinity purification of the CLPC1-TRAP resulted in a dozen proteins highly enriched compared with affinity-purified CLPC1 with a C-terminal STREPII affinity tag (CLPC1-WT). These enriched proteins likely represent CLP protease substrates or new adaptors. Several of these trapped proteins overaccumulated in clp mutants or were found as interactors for the adaptor CLPS1, supporting their functional relationship to CLP function. Importantly, the affinity purification of this CLPC1-TRAP also showed high enrichment of all CLPP, CLPR, and CLPT subunits, indicating the stabilization of the CLPC to CLP core interaction and providing direct support for their physical and functional interaction.

Recombinant expression and purification of a functional bacterial metallo-chaperone PbrD-fusion construct as a potential biosorbent for Pb(II).

PbrD is a lead (II) binding protein encoded by the pbr lead resistance operon found exclusively in Cupriavidus metallidurans CH34. Its ability to sequester Pb(II) shows potential for it to be developed as a biosorbent for Pb in the bioremediation of contaminated wastewaters. In this study the pbrD gene from C. metallidurans CH34 was transformed and overexpressed in Escherichia coli BL21 (DE3) using the pET32 Xa/Lic vector. Optimal expression of recombinant (r)PbrD (∼50 kDa) was achieved post-induction with IPTG within inclusion bodies (IBs). Inclusion bodies were solubilised by denaturation and purified by Ni-NTA affinity chromatography. The purified denatured protein containing the N-terminal Trx•Tag™, His•Tag® and S®Tag™ was refolded in vitro via dialysis to a biologically functional form. Circular dichroism spectra of refolded rPbrD-fusion protein indicated a high degree of turns, ß-sheets and 310 helices content and tryptophan fluorescence showed a structural conformational change in the presence of Pb(II). Refolded rPbrD-fusion protein bound 99.7% of Pb(II) when mixed with lead nitrate in ten-fold increasing concentrations. Adsorption isotherms including Langmuir, Freundlich, Temkin and Dubinin-Radushkevich models were applied to determine the biosorption mechanism. A biologically functional rPbrD-fusion protein has potential application in the development of a biosorbent for remediation of Pb(II) from wastewater.

Nucleotide triphosphatase and RNA chaperone activities of murine norovirus NS3.

Modulation of RNA structure is essential in the life cycle of RNA viruses. Immediate replication upon infection requires RNA unwinding to ensure that RNA templates are not in intra- or intermolecular duplex forms. The calicivirus NS3, one of the highly conserved nonstructural (NS) proteins, has conserved motifs common to helicase superfamily 3 among six genogroups. However, its biological functions are not fully understood. In this study we report the oligomeric state and the nucleotide triphosphatase (NTPase) and RNA chaperone activities of the recombinant full-length NS3 derived from murine norovirus (MNV). The MNV NS3 has an Mg2+-dependent NTPase activity, and site-directed mutagenesis of the conserved NTPase motifs blocked enzyme activity and viral replication in cells. Further, the NS3 was found via fluorescence resonance energy transfer (FRET)-based assays to destabilize double-stranded RNA in the presence of Mg2+ or Mn2+ in an NTP-independent manner. However, the RNA destabilization activity was not affected by mutagenesis of the conserved motifs of NTPase. These results reveal that the MNV NS3 has an NTPase-independent RNA chaperone-like activity, and that a FRET-based RNA destabilization assay has the potential to identify new antiviral drugs targeting NS3.

Biophysical and structural characterization of the putative nickel chaperone CooT from Carboxydothermus hydrogenoformans.

Carboxydothermus hydrogenoformans is a model microorganism for the study of [NiFe]-CODH, a key enzyme of carbon cycle in anaerobic microorganisms. The enzyme possesses a unique active site (C-cluster), constituted of a distorted [NiFe3S4] cubane linked to a mononuclear Fe(II) center. Both the biogenesis of the C-cluster and the activation of CODH by nickel insertion remain unclear. Among the three accessory proteins thought to play a role in this latter step (CooC, CooJ, and CooT), CooT is identified as a nickel chaperone involved in CODH maturation in Rhodospirillum rubrum. Here, we structurally and biophysically characterized a putative CooT protein present in C. hydrogenoformans (pChCooT). Despite the low sequence homologies between CooT from R. rubrum (RrCooT) and pChCooT (19% sequence identity), the two proteins share several similarities, such as their overall structure and a solvent-exposed Ni(II)-binding site at the dimer interface. Moreover, the X-ray structure of pChCooT reveals the proximity between the histidine 55, a potential nickel-coordinating residue, and the cysteine 2, a highly conserved key residue in Ni(II)-binding.

Controlled Assembly of the Filamentous Chaperone Gamma-Prefoldin into Defined Nanostructures.

Self-assembling protein templates have enormous potential for the fabrication of multifunctional nanostructures that require precise positioning of individual molecules, such as enzymes and inorganic moieties, in regular patterns. A recently described approach uses ultrastable filaments composed of the gamma-prefoldin (γPFD) protein and engineered connector proteins to construct novel architectures useful for basic research and practical applications in nanobiotechnology. Here we describe the production of the γPFD and connector proteins from E. coli, and the assembly of γPFD with connector proteins into macromolecular structures with defined shapes.