Cell Chaperones Keep Proteins Properly Folded

Imagine linking together a chain of 300 plastic shapes, some with magnets at various places.  Then let it go and see if you could get it to fold spontaneously into a teapot.  This is the challenge that cells face every minute: folding long chains of amino acids (polypeptides) into molecular machines and structures for the cell’s numerous tasks required for life.  DNA in the nucleus codes for these polypeptides.  They are assembled in ribosomes in single-file order.  How do they end up in complex folded shapes?  Some polypeptides will spontaneously collapse into their native folds, like the magnetic chain in our analogy.  Others, however, need help.  Fortunately, the cell provides an army of assistants, called chaperones, to monitor, coax, and repair unfolded proteins, to achieve “proteostasis” – a stable, working set of proteins.  That army is so well-organized and complex, scientists continue to try to figure out how it performs so well in the field.

Polypeptide chains don’t have magnets, but they have amino acids that produce other forces: side chains that are hydrophilic (water-loving) or hydrophobic (water-repelling), side chains that are acidic or basic, and side chains that are attracted chemically to certain other amino acids.  Let some of these chains go in a test tube and they will spontaneously fold properly because of the precise way they were coded by DNA.  Others require the help of chaperones to fold.  In a review article last week in Nature,¹ Hartl, Bracher and Hayer-Hartl from the Max Planck Institute surveyed what is currently known about protein chaperones.  The importance of proteostasis is evident in their first paragraph:

Most proteins must fold into defined three-dimensional structures to gain functional activity. But in the cellular environment, newly synthesized proteins are at great risk of aberrant folding and aggregation, potentially forming toxic species. To avoid these dangers, cells invest in a complex network of molecular chaperones, which use ingenious mechanisms to prevent aggregation and promote efficient folding. Because protein molecules are highly dynamic, constant chaperone surveillance is required to ensure protein homeostasis (proteostasis). Recent advances suggest that an age-related decline in proteostasis capacity allows the manifestation of various protein-aggregation diseases, including Alzheimer's disease and Parkinson's disease. Interventions in these and numerous other pathological states may spring from a detailed understanding of the pathways underlying proteome maintenance.

Our mammalian cells typically assemble 10,000 different types of proteins.  How they fold properly is “one of the most fundamental and medically relevant problems in biology,” the authors said.  The folded states, furthermore, must be loose enough in many cases to allow for movements (conformational changes) that are essential to their functions. 

Thus, protein quality control and the maintenance of proteome homeostasis (known as proteostasis) are crucial for cellular and organismal health.  Proteostasis is achieved by an integrated network of several hundred proteins, including, most prominently, molecular chaperones and their regulators, which assist in de novo folding or refolding, and the ubiquitin–proteasome system (UPS) and autophagy system, which mediate the timely removal of irreversibly misfolded and aggregated proteins.

Each polypeptide has to navigate a complex “folding-energy landscape” to find its native fold – something like a golf ball in a miniature golf game having to go up, down, and around a series of obstacles to land in the hole.  Simple polypeptides with simple landscapes can often find their fold in millionths of a second in a test tube environment.  Larger, complex ones can take minutes or even hours.  In the cell, it’s even more difficult, owing to the crowded environment of many kinds of molecules bouncing around.  Without the chaperone system, many of the contacts would lead to aggregates of useless or even toxic peptides, like golf balls accumulating in the wrong dip on the landscape, unable to get out.  Chaperones can nudge them out of their traps and back toward the hole.

What do the chaperones look like?  In the diagrams the authors provided, some look like clamps; in fact, the authors called HSP90 a “molecular clamp” that is able to free up stuck proteins and let them proceed to their native folds.  HSP90 requires ATP and a suite of cofactors and regulators to work.  That’s just one of hundreds of chaperone types.

When peptide chains emerge from the ribosome, there’s a risk they will start folding too early at the leading edge.  Folding needs to wait till the tail end gets out of the tunnel.  (Translation, the authors say, proceeds “relatively slow” at 4 to 20 amino acids per second in eukaryotes and bacteria, respectively.)  Premature folding is inhibited by ribosome complexes that arrange multiple ribosomes in ways that maximize the distance between nascent chains, and by ribosome-bound chaperones that monitor and protect the nascent chains till they fully exit the tunnel.

Another challenge cells face is organizing proteins that have multiple domains.  These domains may exit the ribosome separately, but need to be brought together for final assembly.  In such cases, whole chaperone complexes may be involved in post-translational assembly.  These systems are so finely tuned, they can take advantage of pauses at rare codons in the ribosome to achieve co-translational folding.  “Overall,” they remarked, “the eukaryotic translation and chaperone machinery has been highly optimized through evolution, ensuring efficient folding for the bulk of newly synthesized proteins.”  Further chaperone duty awaits at the endoplasmic reticulum: “The chaperone pathways operating in the endoplasmic reticulum (ER) follow analogous organizational principles, but specialized machinery is used in disulphide-bond formation and the glycosylation of many secretory proteins.”

Once proteins are folded properly, the chaperone army’s work is not done.  Protein surveillance machines monitor the proteome and deal with proteins that start to unfold or otherwise go awry.  “Although it is generally accepted that the chaperone machinery is required for initial protein folding,” they said, “we are only beginning to appreciate the extent to which many proteins depend on macromolecular assistance throughout their cellular lifetime to maintain or regain their functionally active conformations.”  This is especially true in eukaryotes, which have a “much greater number and diversity of multidomain proteins.”  Think of all that can go wrong:

In the dynamic cellular environment, these proteins constantly face numerous challenges to their folded states; these result from post-translational modifications (phosphorylation and acetylation), changes in cell physiology and alterations in the composition and concentration of small-molecule ligands that may influence protein stability. Moreover, 20-30% of all proteins in mammalian cells are intrinsically unstructured; that is, they may adopt defined three-dimensional conformations only after binding to other macromolecules or membrane surfaces. Such proteins probably require assistance to avoid aberrant interactions and aggregation, particularly when their concentration is increased and they are not in complexes with partner molecules.

To maintain proteostasis, the cell employs some 200 chaperones and co-chaperones and 600 other machines concerned with trash collection and recycling.  The barrel-shaped chaperone Gro-EL/Gro-ES in bacteria, which provides a protein “dressing room” with lid, has an even more complex counterpart in eukaryotes called TRiC with an “iris-like, built in lid” for privacy, allowing even more time for the encaged protein to try to fold properly.  “TRiC interacts with approximately 10% of newly synthesized cytosolic proteins, including actin and tubulins,” they said.  “Interestingly, TRiC also functions in preventing the accumulation of toxic aggregates by the Huntington's disease protein.” 

Another family are the heat-shock proteins (HSP) that are up-regulated in times of cellular stress.  “They are involved in a multitude of proteome-maintenance functions, including de novo folding, refolding of stress-denatured proteins, oligomeric assembly, protein trafficking and assistance in proteolytic degradation.”  HSP90, the one mentioned earlier, is a “proteostasis hub” with multiple jobs: “cell-cycle progression, telomere maintenance, apoptosis, mitotic signal transduction, vesicle-mediated transport, innate immunity and targeted protein degradation.”

The authors concluded by discussing some of the diseases that occur when chaperones fail, and how ageing itself might be a result of decreasing chaperone function that leads to aggregation of useless protein fragments.  There is still a great deal to learn about chaperones.  “Key questions include determining how certain aberrantly folding proteins aggregate into toxic species whereas others are degraded, how the composition of the proteosome changes during ageing, what the signature of a youthful proteome is, and how we can find ways to maintain it for longer as we age.”

How did this elaborate quality-control system arise?  The authors mentioned evolution six times, but not once did they explain a plausible pathway from early life without chaperones to life with them (which is true in all three kingdoms of life, archaea, bacteria and eukaryotes).  They merely assumed evolution invented these machines because cells needed them: “It seems likely, therefore, that the fundamental requirement for molecular chaperones arose very early during the evolution of densely crowded cells, owing to the need to minimize protein aggregation during folding and maintain proteins in soluble, yet conformationally dynamic states.” 

They suggested that chaperones might aid evolution: “Moreover, as mutations often disrupt the ability of a protein to adopt a stable fold, it follows that the chaperone system provides a crucial buffer, allowing the evolution of new protein functions and phenotypic traits.”  No examples of this were provided; just vague suggestions: “the evolution and maintenance of these functional networks is thought to depend on the ability of HSP90 to buffer the effects of structurally destabilizing mutations in the underlying protein complexes, thereby allowing the acquisition of new traits.”  Thus, it might act as a kind of “evolutionary capacitor in protecting mutated protein variants from degradation.”  This seems an odd suggestion, since the role of chaperones is to maintain proteostasis.  Nevertheless, they piled on more suggestions: “Sequential domain folding during translation, which is highly efficient on eukaryotic ribosomes, probably promoted the explosive evolution of complex multidomain proteins in eukaryotes.”

In short, though, they could not deny that “the eukaryotic translation and chaperone machinery has been highly optimized through evolution, ensuring efficient folding for the bulk of newly synthesized proteins.”  Everyone can agree on the optimization without necessarily agreeing on the mechanism of evolution.

1.  F. Ulrich Hartl, Andreas Bracher, and Manajit Hayer-Hartl, “Molecular chaperones in protein folding and proteostasis,” Nature 475  (21 July 2011), pp. 324–332, doi:10.1038/nature10317.

This is a fascinating review paper worth reading for the marvelous realities revealed that go on every minute of every day, silently, inside our bodies.  Just hold your nose at the occasional evolutionary stories and you will be blessed.