A molecular chaperone has been defined as any interacting protein that promotes folding and assembly into functionally active conformations without being present in the final structure (Hartl 1996). Molecular chaperones are involved in every aspect of protein synthesis, maintenance, and degradation. Guarding of newly synthesized proteins begins when these emerge at the ribosome and ends when protein structures need to be unfolded for degradation.
Cellular protein biosynthesis
α-helices may form within nanoseconds in vitro. In vivo, the translational speed of ribosomes is considerably slower (~4 amino acids per second for eukaryotes, ~20 amino acids per second in bacteria; Hartl 2009). As soon as peptide bonds are formed, initial folding interactions begin within the ribosome. However, the limited space restricts growing polypeptides to α-helical conformations within the 10 nm long and 2 nm wide exit tunnel. Interactions with ribosomal RNA (rRNA) may already promote conformational compaction (Lu 2005, Bhushan 2010). However, structural rearrangements might occur once the N-terminus emerges from the exit tunnel (Bhushan 2005).
Incomplete chains usually do not achieve any stable fold or are prone to aggregation (Parker 1981).
Only complete proteins or domains offer the necessary information and thermodynamic energy to stabilize native conformations. Especially large β-sheet rich domains and complex α/β-folds contain long-distance interactions, and the respective interaction sites may not be available yet. Nascent chains therefore expose peptide segments lacking their native interaction partners, which makes them prone to participate in unspecific interactions. Especially in a highly crowded environment of a cell (300 mg/mL protein), cotranslational protection is highly necessary, which is provided by the first instance of chaperones.
Chaperones guarding the nascent chain
The ribosomal exit tunnel prevents non-native interactions of the emerging polypeptide chain.
Interestingly, in E. coli, ribosomes themselves have been suggested to exert a chaperon-like refolding activity, especially the 23S rRNA, as they promote folding and mediate refolding of denatured proteins (Kudlicki 1997). Emerging nascent chains need to be shielded from unfavorable intra- and intermolecular interactions. This is achieved by ribosome-associated chaperones such as trigger factor (TF) in prokaryotes and specialized Hsp70 and nascent chain-associated complexes (NAC) in mammals.
TF (~50 kDa) consists of a ribosome and a nascent chain binding domain, next to its peptidylprolyl cis/trans isomerase (PPIase). The PPIase recognizes stretches of eight amino acids enriched in basic and aromatic residues (Ferbitz 2004, Lakshmipathy 2007). Every domain of TF can take part in nascent chain binding, which enables TF to accommodate a wide range of polypeptides. TF primarily shields emerging hydrophobic chains from non-native interactions. Its subsequent release from the nascent chain promotes folding of the polypeptide and provides energy for this ATP-independent process. Then TF dimerizes, partially masking its substrate-binding regions (Kaiser 2006). TF does not exist in eukaryotes, but the heterodimeric α-/β-NAC complex (33/22 kDa) might fulfill a similar function. NAC associates with ribosomes and short nascent chains (del Alamo 2011, Preissler 2012) and most likely functions in parallel with MPP11/Hsp70L1 (Otto 2005, Jaiswal 2011). NAC knockouts in yeast have resulted in a strong upregulation of stress inducible chaperones (del Alamo 2011).
Combinatorial knockouts of NAC and Ssb strongly enhanced aggregation of newly synthesized proteins (Koplin 2010).
A subset of proteins is capable of autonomous folding. This is often true for small α-helical proteins, which straightforwardly fold into their native states. Chaperone dependence increases strongly with the length of polypeptide chains and the distance of long-range interactions. Chaperons ease folding for bacterial proteins with an average size of ~30 kDa in E. coli, while the average human protein reaches over 50 kDa (Wolff 2014).
Chaperone networks for folding, maintenance, and degradation
Downstream of the ribosome, the cellular chaperone systems form a complex network of factors, which guarantee proper protein folding, quality control, and maintenance of a healthy proteostasis (Hartl 2009). Already as the nascent chain emerges, Hsp70s and their Hsp40 cochaperones (DnaJ/DnaK in E. coli) cooperate with TF in shielding the polypeptide from harmful interactions.
Specific chaperone functions partially overlap, as single knockouts of e.g. TF or DnaK in E. coli are tolerated by cells under non-stress conditions, and the remaining chaperones widen their substrate spectrum (Deuerling 1999). However, under stress or upon multiple chaperon deletions (e.g. TF and DnaK), cells experience enhanced protein aggregation up to their inability to synthesize and fold new proteins (Calloni 2012).
The multifunctional Hsp 70/Hsp40 system
“The constitutively expressed Hsc70 and stress-inducible forms of Hsp70 are central players in protein folding and proteostasis control” (Hartl 2011). They shield the cellular proteome from hazardous interactions, prevent misfolding and aggregation, and promote folding through cycles of binding and release. DnaK, the bacterial Hsp70, recognizes hydrophobic strands surrounded by positive charges, which are often buried in the core of folded proteins (Rüdiger 1997), but which become prone to aggregate when exposed. Hsp70s perform an ATP-dependent reaction cycle. ATP binding, hydrolysis, and ADP release in the nucleotide-binding domain of Hsp70 are allosterically coupled to substrate binding and release. ATP binding opens the peptide-binding pocket allowing substrates to enter. ATP hydrolysis leads to a closed binding pocket with low substrate on and off rates (Kim 2013, Hartl 2011).
Hsp70 is regulated by cochaperones and nucleotide exchange factors (more than 40 Hsp40s in humans; DnaJ, GrpE in E. coli), primarily through stimulating ATP hydrolysis that stabilizes Hsp70-substrate interactions (Qiu 2006). ADP release becomes accelerated by Hsp110 (Polier 2010) and Hsp170. A new cycle of ATP binding results in substrate release, which is either properly folded, or rebound to Hsp70 or further downstream chaperones (Mayer 2010).
Hsp40s bind with their conserved J-domains to the nucleotide-binding domain of Hsp70. Specialized Hsp40s function as chaperones themselves and recruit nonnative substrates to Hsp70. Mammalian DnaJB1 and yeast Sis1p mediate the transport of misfolded proteins into the nucleus, where they become degraded by the proteasome (Park 2013). This process currently seems to arise as an important cellular pathway for removal and degradation of aggregation prone structures from the cytosol.
Hsp70s are furthermore of major importance for protein targeting. Mitochondrial proteins encoded by the nuclear genome are synthesized in the cytoplasm tagged by a mitochondrial targeting signal.
Hsp70 guards (partially) unfolded mitochondrial precursor proteins through the cytoplasm to mitochondria. As the proteins become imported through the mitochondrial import complexes (TOM, TIM), they are immediately grasped by mtHsp70 with its open substrate binding site (Neupert 2007).
Chaperonins – single molecule folding chambers
Chaperonins (Hsp60s) are large double-ring complexes (7-9 subunits of ~60kDa) with a central cavity.
They offer substrate proteins an isolated chamber with a dynamic and active surface. Chaperonins create an optimal folding environment and allow proteins to fold independently from surrounding factors that risk their misfolding and aggregation.
GroEL from bacteria, Hsp60 in mitochondria, and Cpn60 in Chloroplasts are chaperonins that encapsulate proteins with lid-shaped cochaperones (GroES, Hsp10, Cpn10/20). The archaeal thermosome and the eukaryotic TRiC (TCP-1 Ring Complex) contain a built-in lid with an iris-like opening mechanism. The chaperonins offer a chamber to enclose proteins or domains of up to 60-70 kDa size. In case of TRiC, partial encapsulation of single domains with successful folding has been shown for proteins of >100 kDa (Rüßmann 2012).
Figure 10 | Overview of chaperones in the bacterial and eukaryotic cytoplasm, stabilizing the nascent chain and promoting folding of their substrates to the native state. The nascent chains interact with trigger factor (TF) or NAC in conjunction with specialized Hsp70 complexes. Members of the DnaJ/K or Hsp70/40 family mediate co- or posttranslational folding or distribute proteins to downstream chaperones. Depending on the sequence, the protein substrates are delivered to the chaperonins GroEL/ES or TRiC, or chaperones of the Hsp90 family. Numerous cofactors support the folding process (such as nucleotide exchange factors, NEFs) or mediate substrate recognition (such as the Hsp70-Hsp90 organizing protein, HOP). The numbers indicate the percentage of interacting proteins as part of the total proteome (figure from Hartl 2011).
The chaperonins interact with about 10% of newly synthesized proteins. Obligate GroEL substrates (around 85 E.coli proteins) are typically proteins with complex α/β-folds being kinetically trapped in folding intermediates that need to be resolved (Kerner 2005). Well-established TRiC substrates include the cytoskeletal actin and tubulin as well as proteins with β-propeller/WD40 domains (Dekker 2008, Yam 2008). Upon substrate binding, GroEL forms a hydrophobic folding chamber encapsulating mostly molten-globule like structures that partially lack native long-distance interactions. ATP-dependent GroES binding leads to a conformational transition, resulting in a hydrophilic, negatively net charged inner surface (Hartl 2011). The substrate protein has now ~10 sec for folding along an undisturbed, folding-optimized energy landscape. The chaperonins promote rather compact, native-like conformations (Brinker 2001, Chakraborty 2010). Rebinding of not yet folded structures allows multiple rounds of folding within the chaperonins, before a still misfolded protein might ultimately be delivered to degradation (Hartl 2011).
Interestingly, the eukaryotic chaperonin TRiC has been shown to modify oligomeric species of polyQ expanded versions of the Huntingtin’s disease protein in cooperation with Hsp70, rendering them less toxic in a yeast model (Behrends 2006).
The Hsp90 chaperone family of activators and regulators
Hsp90 chaperones are a highly conserved essential chaperone family promoting the maturation of a wide range of substrate proteins. Hsp70s transfer many substrates to Hsp90 for the completion of folding and for functional activation with the help of HOP (Hsp90 organizing protein), which bridges the two chaperones with its TPR (tetratricopeptide repeat) domains (Scheuffler 2000). Substrates include a variety of proteins involved in signal transduction, protein trafficking, receptor maturation, especially kinases and receptor proteins, assigning Hsp90 chaperones the role of an important cellular regulator (Taipale 2010). Applying Hsp90 inhibitors simultaneously inhibited multiple cellular singling pathways important for cancer growth (Neckers 2007), however concurrently induced a general stress response (Labbadia 2015).
Hsp90 itself has been found to preferentially recognize intrinsically unstable kinases rather than binding to distinct sequence motives on their substrates. Its cochaperone CDC37 serves as an adaptor that binds to kinase folds and recruits Hsp90. Hsp90-kinase associations decrease after stabilization of the kinases by folding or by binding to stabilizing small molecules or natural ligands (Taipale 2012).
Disaggregases – chaperones reversing aggregation
Beyond promoting folding and refolding, certain chaperones are able to disassemble and remodel misfolded protein oligomers and aggregates.
Hsp104 is a hexameric ring AAA+ ATPase with a central pore, through which misfolded substrate proteins are (presumably) translocated and thereby disentangled. Hsp104 from yeast is driven by ATP hydrolysis and collaborates with Hsp70 and Hsp40 (Doyle 2009). The disaggregase complex resolves disordered aggregates as well as amyloids. Homologues of Hsp104 are found in bacteria, fungi, protozoa, chromista, and plants, however not in metazoan (animals). The bacterial homologue ClpB lacks the ability to disaggregate amyloid fibrils with stable “cross-β” cores (DeSantis 2012).
Reasons for the loss of Hsp104 from metazoan are unclear (Shorter 2011). A generally advanced
proteostasis network may have decreased the selective pressure to keep the disaggregase during evolution. Alternatively, the potential risk of producing soluble misfolded species by Hsp104 through disassembly of rather “safe” amyloid species might have been disadvantageous.
Recently Hsp110, so far known as a nuclear exchange factor (NEF) of Hsp70, has been found to disaggregate disordered aggregates (of Luciferase or GFP) in the mammalian cytoplasm and in vitro in collaboration with Hsp70 and Hsp40. However, this complex failed to remodel amyloid forms of α-synuclein or the yeast prion Sup35 (Shorter 2011). Hsp110 seems to accelerate Hsp70 kinetics by its NEF activity, and Hsp70 relies on numerous cofactors for efficient disaggregation. Putative substrate interactions of Hsp110 remain unclear (Rampelt 2012). Metazoan disaggregases are so far poorly understood, although their contribution to the proteostasis network would be highly interesting regarding the appearance of amyloid diseases.
Small heat shock proteins
Small heat shock proteins contain a highly conserved “α-crystallin” domain of 100 amino acids. This common core is flanked by extensions that mediate dynamic oligomerization and client recognition (Eyles 2010). Small heat shock proteins prevent aggregation through binding and/or release of unfolded or misfolded structures. These can then be transferred in a soluble state via other chaperones for functional reactivation or degradation. Small heat shock proteins do not require ATP hydrolysis, although they may be regulated by ATP binding (Bakthisaran 2015).
Many small heat shock proteins have been found in the deposits associated to protein misfolding diseases. Moreover, chaperones prevented nucleation and aggregation of amyloid fibril formation of Aβ peptides, polyglutamine sequences, and other aggregation prone proteins (see below;
Bakthisaran 2015). Overexpression of small heat shock proteins has been associated with stabilization of tumor cells (Bakthisaran 2015). This circumstance demonstrates the importance of a factor-specific fine-tuning and a proteostasis network-wide orchestration to create a healthy folding environment within a cell.