AB Vector - Multi-chaperone systems

  Multi-chaperone systems
  Chaperone control of protein folding does not occur randomly in cells, but is spatially compartmentalized in subcellular organelles and specialized microenvironments. Cell compartments contain specific ensembles of molecular chaperones (Altieri DC, Oncotarget, 2:347-51, 2011; Benyair et al., Int Rev Cell Mol Biol., 292:197-280, 2011; Baker et al., Cold Spring Harb Perspect Biol., 3(7). pii: a007559, 2011; Okiyoneda et al., Curr Opin Cell Biol., 23:483-91, 2011).

Some molecular chaperones are promiscuous and assist folding of a very broad range of proteins, while other chaperones are extremely specific and may assist folding of only one protein (Szolajska and Chroboczek, Cell Mol Life Sci., 68:3307-22, 2011). Additionally, some chaperones are in between these two categories and specialize in folding certain classes of proteins.

Of particular interest is Hsp90 molecular chaperone that assists folding of labile regulatory proteins, many of which are key drug targets involved in hormone signaling and cell cycle control (Scheibel and Buchner, Biochem Pharmacol., 56:675-82, 1998). Hsp90 does not act alone, but in ensemble with other molecular chaperones (multi-chaperone systems). Different sets of molecular chaperones could be necessary for chaperoning different classes of Hsp90 client proteins. For example, Hsp40, Hsp/Hsc70, Hop and p23 are required in addition to Hsp90 and are fully sufficient for maturation of steroid receptors (Pratt et al, J Biol Chem., 283:22885-9, 2008). However, chaperoning Hsp90-dependent protein kinases also requires another chaperone cdc37 (Caplan AJ, Trends Cell Biol., 17(2):87-92, 2007).

  Hsp90 multi-chaperone system
Hsp90 is a molecular chaperone that assists folding of a large number of therapeutically relevant proteins including:
  • steroid/nuclear receptors (Pratt et al., J Biol Chem. 283:22885-9, 2008)
  • protein kinases (Caplan et al., Trends Cell Biol., 17:87-92, 2007)
  • Hepatitis B, C, influenza and herpes virus polymerases (Hu et al., J Virol., 76:269-79, 2002; Miyamura et al., EMBO J., 25:5015-25, 2006, Naito et al., J Virol., 81:1339-49, 2007; Burch and Weller, J Virol., 79:10740-9, 2005)
  • REV1, a Y-family polymerase that plays a central role in tumor initiation and progression (Pozo et al., Mol Cell Biol., 31:3396-409, 2011)
  • mutant p53 that plays role in many forms of cancer (Li et al., Cell Death Differ., 18:1904-13, 2011)
  • survivin, an anti-apoptotic molecule playing role in survival of cancer cells (Chiosis G, Expert Opin Ther Targets., 10:37-50, 2006)
  • prostaglandin E synthase, a drug target for inflammation and cancer (Tanioka et al., Biochem Biophys Res Commun., 303:1018-23, 2003; Murakami and Kudo, Curr Pharm Des., 12:943-54, 2006)
  • inflammasome components (Mayor et al., Nature Immunology, 8:497-503, 2007). Inflammasomes are involved in pathogenesis of gout, atherosclerosis and Type 2 diabetes (Dunne A, Biochem Soc Trans., 39:669-73, 2011)
  • eNOS-endothelial nitric oxide synthase, a cardiovascular drugs target (Chatterjee and Catravas., Vascul Pharmacol., 49:134-40, 2008; Sawada and Liao, Expert Rev Neurother., 9:1171-86, 2009)
  • tau complexes in Alzheimer's disease (Salminen et al., Prog Neurobiol., 93:99-110, 2011)
  • mutant huntingtin in Huntington’s disease (Baldoet al., J Biol Chem., Nov 28. [Epub ahead of print], 2011)
  • PINK1 in Parkinson’s disease (Rakovic et al., Parkinsons Dis., 2011: 153979, 2011)
  • cystic fibrosis transmembrane conductance regulator (Wayne et al., Methods Mol Biol. 787:33-44, 2011)
A list of Hsp90 client proteins can be found on Prof. Picard's lab web site http://www.picard.ch/downloads/Hsp90interactors.pdf.

Hsp90 also plays a key role in:
  • translocation of extracellular antigens that are destined for presentation by major histocompatibility complex I (Imai et al., Proc Natl Acad Sci U S A., 108(39):16363-8, 2011)
  • assembly of large protein complexes, such as snoRNP, RNA polymerase II, phosphatidylinositol-3 kinase-related protein kinase (PIKK), telomere complex, kinetochore, RNA induced silencing complexes (RISC), and 26S proteasome (Makhnevych and Houry, Biochim Biophys Acta., [Epub ahead of print], 2011)
We provide all five molecular chaperones that are necessary for folding steroid receptors using a single recombinant baculovirus, FoldHelper™-905c. Chaperones are expressed to a high level and are visible as prominent bands on Coomassie-blue stained gels of crude extracts of insect cells infected with the FoldHelper™-905c (Fig. 1).

Another molecular chaperone, Cdc37 could be required in addition to Hsp90, Hsp40, Hsp70/Hsc70 and p23 for activation of Hsp90-dependent protein kinases (MacLean and Picard, Cell Stress Chaperones, 2003, 8:114-9). Cdc37 can be provided using a separate recombinant baculovirus FoldHelper™-37, which enables its expression to a high level.
  Fig. 1. Comparison of conventional and multi-chaperone baculovirus expression systems. A. Protein profiles and predominant steroid receptor folding events. In a conventional system, indigenous insect cell molecular chaperones are insufficient for folding a majority of the overexpressed recombinant receptor molecules. This results predominantly in the receptor misfolding. In the multi-chaperone system five human molecular chaperones are abundantly expressed and facilitate native folding of the receptor (details). The chaperones may either associate into a chaperone complex (foldosome), or act sequentially. For comparison of protein expression profiles Spodoptera frugiperda Sf9 cells were infected either with NC (Cat# C13), a baculovirus that does not express recombinant proteins, or FoldHelper™-905c a five-chaperone recombinant baculovirus. Cells were harvested at 60 h post infection, lysed in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, and the insoluble fraction was removed by centrifugation at 30,000g for 15 min. Soluble proteins were separated in reducing 4-12% SDS-PAGE in MES buffer and stained with Coomassie blue. B. Purified progesterone receptor ligand-binding domain (LBD) in complex with Hsp90 and other molecular chaperones. Proteins were separated in reducing 4-12% SDS-PAGE in MES buffer and stained with Coomassie blue. Hsp90 appears to associate with steroid receptors at about a 2:1 ratio, whereas Hsp70 and p23 typically are present at lesser ratios (Denis et al., J. Biol., Chem., 262, 11803-6, 1987; Iannotti et al., Arch. Biochem. Biophys., 264, 54-60, 1988; Renoir et al., Biochemistry, 23, 6016-23, 1984). Preparations of steroid receptors containing these chaperones are desirable since, in nature, all steroid receptors exist in complex with Hsp90 molecular chaperone, which is essential for the native ligand-binding properties of the receptors. We routinely use Fold-Helper™-905C for production of steroid receptors, i.e. insect cells are co-infected with two recombinant baculoviruses, one expressing a target protein, such as steroid/nuclear receptor, and another FoldHelper™-905C overexpressing the chaperones. Therefore the human receptor and five human molecular chaperones are co-synthesized in insect cells and molecular chaperones participate in co-translational folding of the receptor. C. Progesterone receptor LBD produced by the main competitor is not in complex with molecular chaperones as insect molecular chaperones are not as competent as human molecular chaperones in folding steroid receptors and forming the complexes (details). Furthermore, urea that was included by the competitor into protein formulation to prevent apo-Hsp90 receptor from aggregation during storage is not compatible with receptor/Hsp90 complexes.

  Co-infections of insect cells with recombinant baculoviruses expressing ligand binding domains (LBDs) of steroid receptors and FoldHelper™-905c result in a marked improvement in the production of receptors in soluble form and in a higher yield of purified proteins (Fig. 2A, B). Proteins expressed in the 5-chaperone system were still associated with human Hsp90 and Hsc70 even after extensive washes during the purification, whereas similar insect molecular chaperones either failed to associate with the LBDs or associated weakly and were removed during purification (Fig. 2B).

  Fig. 2. Yields of steroid receptors produced in a conventional versus 5-chaperone baculovirus system. A. Densitometry data. B. 10% SDS-PAGE, Coomassie blue staining. Ligand binding domains (LBDs) of mineralocorticoid (MR), glucocorticoid (GR), estrogen alpha (ERα) and progesterone (PR) receptors were expressed as GST fusion proteins using recombinant baculoviruses. Insect cells either were infected with recombinant baculoviruses expressing steroid receptors without human chaperones (conventional system), (-), or co- infected with the same recombinant baculoviruses and FoldHelper™-905c (5-chaperone expression system), (+). GST-LBD fusion proteins were purified from the soluble protein fraction using affinity chromatography on glutathione beads. At the applied conditions molecular chaperones were largely stripped from receptors. Proteins were separated in 10% SDS-PAGE, stained with Coomassie blue, and bands corresponding to the steroid receptor's LBDs were quantitated using densitometry. Hsp90 and Hsc70 molecular chaperones co-purified with the steroid receptors are marked by triangles. The difference in the yield of soluble receptors produced with or without human molecular chaperones is even more drastic if receptors are purified at native conditions which do not result in disruption of receptor/molecular chaperones complexes (details).

  Improvement in the production of soluble proteins typically correlates with an improvement in the production of proteins in biologically active form. More importantly, appropriately produced receptors are likely to exhibit native ligand-binding properties, which could allow for making fine distinctions between similar ligands and similar receptors, and overcome the notorious poor selectivity of drugs targeting steroid receptors.

  Improving production of difficult proteins
At AB Vector we apply multi-chaperone systems internally for production of drug targets. Evidently, otherwise some prominent drug targets can’t be produced in their native, biologically-relevant form, allowing for advanced protein structure-based drug design, compound library screening, lead-optimization and development of protein or antibody-based therapeutics.

Among drug targets, GPCRs, steroid/nuclear receptors and protein kinases comprise the most important categories. The current market for protein kinase inhibitors is estimated to exceed $7 billion, for drugs targeting steroid/nuclear receptors about $27.5 billion, and for drugs targeting GPCRs over $50 billion (Lundstrom K, Curr Protein Pept Sci., 7:465-70, 2006; Insight Pharma Reports. Nuclear receptors: The Pipeline Outlook Report. September 2010).

Some of the key GPCRs, steroid receptors, and protein kinases may never have been available in a form suitable for adequate drug screening, due to the inability to produce them in native form or in sufficient quantities. AB Vector's technology will enable partners to unlock the potential of such promising but "difficult to produce" targets.

Steroid receptors are difficult to produce in mammalian, baculovirus, yeast, and E.coli systems (Srinivasan, Mol Endocrinol., 1992, 6:857-60). So far, all commercially available steroid receptors and the overwhelming majority of protein kinases have been produced using conventional recombinant baculoviruses. Recombinant baculoviruses are also a system of choice for producing GPCRs and could be preferable to other systems (Akermoun et al., Protein Expr Purif. 2005, 44:65-74).

AB Vector's multi-chaperone systems are the next generation of baculovirus systems, which are specifically designed to produce certain classes of target proteins. Multi-chaperone systems outperform traditional baculovirus systems due to overexpression of specific sets of human molecular chaperones and protein folding partners.

  Multi-chaperone systems for G-protein coupled receptors (GPCRs)
Multi-chaperone expression systems specifically designed for production of certain GPCR classes are currently in development. Such systems will include major molecular chaperones as well as certain protein folding partners specific to GPCRs. Meanwhile, you can follow our recommendations for expression of Transmembrane proteins.

AB Vector provides recombinant baculovirus stocks for expression of all Histamine receptors, most of the Serotonin and Adrenergic receptors as well as selected Dopamine, Muscarinic and Angiotensin receptors of prime importance. Recombinant baculoviruses expressing GPCRs were constructed using ProEasy™ technology which ensures generation of 100% pure baculovirus stocks.