Elaborate Quality Control Governs the Cells Protein-Folding Factory
If it weren’t for quality control in our cells, we’d be dead. That’s the gist of an amazing Insight article in the Dec. 18 issue of Nature.1 “Aberrant proteins are extremely harmful to cells,” the authors begin. How harmful? Here is a short list of diseases that can result from improperly folded proteins or failures in the quality control systems that direct their formation: Creutzfeldt-Jakob disease, Alzheimer’s disease, and other degenerative diseases, scurvy, cystic fibrosis and more. In fact, serious defects in protein assembly are probably never seen, because they could prevent an organism from getting past the first cell division in the embryo. The only way a cell can live and grow is with the assistance of a host of traffic controllers, regulators, monitors, ushers, transporters, inspectors, security guards and emergency technicians maintaining the complex processes of protein assembly. Success must be ensured constantly, 24 x 7, that despite a flurry of activity, must maintain a state of dynamic equilibrium, called homeostasis.
Each cell in the body is like a city of interrelated factories made up of protein machines and structures operating under strict regulations, built on coded instructions. One of the most important factories is the protein folding system, which ensures that newly-sequenced proteins coming out of ribosomes are folded into their correct (native) shapes. Proteins are made up of amino acids, usually hundreds of them, that are first sequentially assembled in ribosomes, based on templates sent from the DNA code. Then, they are folded into specific, complex three-dimensional shapes that perform numerous and diverse functions in the cell (see 06/13/02 and 05/31/02 headlines.) Protein folding is assisted by enzymes whimsically called chaperones (see 05/05/03 headline) but is also checked and rechecked by numerous other quality control systems (see 09/09/02 headline).
In the current paper in Nature, the authors have unveiled more of the complexity in the quality controls governing protein folding. Some of the folding occurs in networked subway tunnels that run throughout the cell, called the endoplasmic reticulum (ER). Before getting into the ER, some proteins already begin their folding with assistance from certain chaperones. The authors explain, “In mammalian cells, proteins are translocated into the ER … where they start to fold co-translationally [i.e., while they are en route into the ER]. Folding is completed post-translationally, and, generally, individual subunits have folded before assembly and oligomerization [the joining together of multiple chains] take place. Sequential interactions with distinct chaperones are required for each of these steps.” (Emphasis added in all quotations.) The job is completed inside the ER, and the finished protein “tool” is then sent on its way to work. But that is just the tip of a huge iceberg made up of a multitude of processes – hardware and software – that work together to ensure success.
In the following examples from the article, entitled “Quality control in the endoplasmic reticulum protein factory,” don’t worry about unfamiliar technical terms. Just try to keep track of how many different players are involved in the team of factory workers dedicated to one job: folding a single protein. And keep in mind that each team player is itself a protein, built with the same quality control. You can almost envision little factory workers, each skilled at their specific tasks, alert and knowing just what to do, but it’s all done with chemicals! Be patient in these extended quotes, because the awe is in the details.
- Redox regulation: A sensitive chemical balance is maintained between reducing and oxidizing (redox) conditions along the protein’s pathway through the folding factory. “The redox gradient between the ER and the cytosol seems to be important for intercompartmental signalling, particularly in the integrated response to oxidative stress, in which adaptive responses emanating from different compartments are coordinated. And redox reactions with opposite electron fluxes must take place in the ER to mediate formation, isomerization and reduction of disulphides. The wealth of redox assistants allows these fluxes to be separate, and channels electron transport through specific protein�protein interactions.”
- Location specificity: “Although most folding factors in the ER are ubiquitously expressed throughout the body, some are tissue-type specific or cell-type specific, and probably fulfil a particular synthetic task…. For example, efficient collagen production requires the expression of hsp47, whereas a tissue-specific protein-disulphide-isomerase-like protein, PDIp, is produced in the pancreas and probably permits the massive secretion of digestive enzymes.”
- Bridge builders: Junctions called disulphide bridges are common in protein folds, and these, although weak, are carefully maintained by a host of enzymes called oxidoreductases: “The impressive number of oxidoreductases in the ER suggests that catalysis and regulation of disulphide-bond formation is crucial for folding. Energywise, in most cases, the contribution of a disulphide bond is hardly more than that of a single hydrogen bond [i.e., quite weak], yet, without disulphide bonds, native conformations are not obtained. Disulphide bonds cannot force a folding protein into a given conformation: in the sampling of conformations during folding in the ER, native and non-native disulphide cross-links are transiently formed [i.e., correct and incorrect links form and break easily, and must be guided]. Continuous activity of oxidoreductases probably ensures that these covalent links remain flexible until folding is completed.”
- Correct fold recognition: Even though a string of amino acids could conceivably fold in large number of ways, like a Rubik’s cube, somehow the chaperones are able to tell a correct (native) fold from an incorrect one. “Besides providing a unique folding environment, the ER has a crucial quality-control role. How does it discriminate between native and non-native proteins? The answer to this question depends primarily on ER chaperones. When folding or assembly intermediates expose hydrophobic [i.e., water-avoiding] surfaces, unpaired cysteines or immature glycans, ER-resident chaperones or oxidoreductases interact with them, and as a consequence they are retained in the ER or retrieved from the Golgi complex [see 11/12/01 headline]. By forming multimolecular complexes, folding factors in the ER may provide matrices that couple retention to folding and assembly. Immature proteins may also form aggregates that are excluded from vesicles exiting from the ER” [i.e., such that they are not ejected before they are ready].
- Fail-safe inspection: A protein needs to pass multiple layers of monitoring: “All proteins are subjected to a ‘primary’ quality control that monitors their architectural design through ubiquitous folding sensors (Table 1). ‘Secondary’ quality-control mechanisms rely instead on cell-specific factors and facilitate export of individual proteins or classes of proteins…. the ER is the main test bench where molecules destined for the extracellular space are scrutinized for their potential toxicity.”
- Feedback regulation: The ER not only does quality control, but sends messages back to the nucleus to regulate the production of more chaperones: “The reasons for having a quality-control system in the ER are easy to understand where protein folding and function are concerned, especially in multicellular organisms where development relies on the fidelity of protein secretion. Quality control can also regulate the transport or the activity of certain proteins during differentiation or in response to stress or metabolic requirements.”
- Waste control: When a protein cannot be folded after repeated attempts, more assistants are on hand to ensure proper dismantling and recycling: “Mutations or unbalanced subunit synthesis make folding or assembly � and hence exit from the ER � impossible. To maintain homeostasis [dynamic equilibrium], terminally misfolded molecules are ‘retrotranslocated’ or ‘dislocated’ across the ER membrane to be degraded by cytosolic proteasomes” [organelles equipped to break up badly-folded proteins and recycle their parts].
- Time limits: Somehow, the cell knows when a protein has had enough time to shape up or ship out: “A fascinating problem is how molecules that have not been given the time to fold (and therefore are unfolded) are discriminated from those that have failed to fold after many attempts (misfolded), and must therefore be disposed of. One way of timing glycoprotein quality control involves the sequential processing of N-glycans and in particular mannose trimming in the ER. It remains to be seen how substrates are eventually targeted to the retrotranslocation channels, how these are opened, and to what extent proteins must be unfolded to negotiate dislocation.”
- Workforce regulation: Like a company’s human resources department that responds to managers’ calls for more workers, the cell keeps track of how many chaperones are available, and sends out “help wanted” ads to the nucleus. “To maintain the efficiency of quality-control mechanisms in diverse physiological conditions, living cells have evolved regulatory circuits that monitor the levels of available chaperones. This is true for both the cytosol and the ER, and compartment-specific responses clearly exist that selectively restore optimal levels of the desired folding factors.”
- Emergency squads: The authors provide two examples of rapid-response traffic control teams: “The accumulation of aberrant proteins in the cytosol triggers the heat-shock response, resulting in de novo synthesis of hsp70 and other cytosolic chaperones. But if aberrant proteins accumulate in the ER, cells activate a different response, the unfolded protein response (UPR), which leads to the coordinated synthesis of ER-resident chaperones and enzymes.”
- Failure consequences: The authors give an example of what can go wrong when the system gets swamped, starved, or sent defective parts: “Physiologically, ER stress (a condition in which the folding machinery in the ER cannot cope with its protein load) can be caused by synthesis of mutated or orphan proteins, absence of cofactors (an example being scurvy, in which collagen cannot fold because of the lack of vitamin C), or a drastic increase in otherwise normal cargo proteins.”
- Unified response to varied inputs: A variety of signals can lead to the same Unfolded Protein Response (UPR) pathway: “How do the diverse unfolded or misfolded proteins that accumulate in the ER provoke the same pathway? The unifying concept is that BiP and other primary quality-control factors maintain the stress sensors in the ER in the inactive state, so that chaperone insufficiency triggers UPR whatever the nature of the cargo.”
- Meltdown regulation: What happens when the damage is so great, that further operation of the factory would be dangerous? Three independent controls make sure an orderly shutdown occurs (apoptosis, or cell death: see 04/09/02 headline). “The mammalian ER sensors, Ire1, PERK and ATF6, guarantee a tripartite response with synergic strategies. By phosphorylating eIF2alpha, PERK transiently attenuates translation, limiting protein load. ATF6 drives the transcriptional upregulation of many ER-resident proteins and folding assistants. Ire1 activates XBP-1, which in turn induces transcription of factors that facilitate ER-associated degradation (ERAD). The two-step activation of XBP-1 (transcriptionally induced by ATF6 and post-transcriptionally regulated by Ire1) guarantees the proper timing of the UPR [unfolded protein response] attempts to fold proteins precede the decision to degrade them. If the response fails to clear the ER, apoptosis is induced through several pathways.” The authors explain that “The UPR is multi-faceted and regulates proteins involved in quality control, ERAD and many aspects of the secretory pathway.”
- Balancing act: The quality control mechanisms walk a tightrope, with serious consequences for falling off: “Quality control must be a balance between retaining and degrading potentially harmful products and not preventing export of biologically active proteins. CFTR mutants in cystic fibrosis illustrate an overzealous quality control, where biologically active mutants cannot leave the ER. In this case, relaxing the quality control could cure the patient. But disease can also originate from defective degradation. If the rate of synthesis of a protein exceeds the combined rates of folding and degradation, a fraction of it will accumulate intracellularly.” Misfolded proteins have to make it across the ER membrane in time, and get degraded by the proteasome in time, or else aggregations (aggresomes) can build up inside or outside the ER. These are implicated in a number of “ER storage diseases.”
The authors put this all into perspective: “Over the past few years, much has been learned about how proteins are handled by the ER folding and quality-control machineries, and some of this knowledge has begun to be translated to industry and to the clinic. Yet, many questions remain….” They hope that further elucidation of these complex, coordinated systems will allow drug designers to target faulty elements that cause degenerative diseases, or induce apoptosis in tumors to make them self-destruct. Clearly, though, in spite of the complexity already described, much remains to be learned about cell quality control.
1Robert Sitia and Ineke Braakman, “Quality control in the endoplasmic reticulum protein factory,” Nature 426, 891 – 894 (18 December 2003); doi:10.1038/nature02262.
If you endured the heavy reading in the above paragraphs, you were undoubtedly rewarded with a sense of how incomprehensibly amazing and mind-boggling a living cell is. Think of it: all this activity is going on right now in every cell in your body. The authors describe it in typical scientific jargon, but sprinkled here and there are hints of their own wonder and fascination at how all this quality control works. Who could help but to be awe-struck at the choreography and efficiency of so many coordinated parts? No human enterprise comes close.
Only twice do the authors mention evolution just in passing, and in both cases, they merely assume it rather than explain it. The first reference almost argues against it: “A certain degree of freedom from quality control is essential for the evolution of proteins. However, it comes at a price for multicellular organisms. Indeed, many proteins that cause systemic amyloidosis [clumping, runaway misfolding] … can adopt more than one conformation and can undergo uncontrolled aggregation outside of the cells.” This means that mutations have a hard time getting past the guards, and even when they do, the consequences could be catastrophic. How then, could evolution ever get a beneficial mutation past the controls, without mucking up the whole works or triggering the cell-death alarm? Don’t look for an explanation from these authors. If there was ever a need for more examples of irreducible complexity, this is surely a contender.
This afternoon, after reading this scientific paper, I was sitting in a fast food restaurant eating a hamburger and looking out the window at a sunset. I paused for a moment to think about how many tiny miracles of sophisticated, coordinated interaction were going on inside my body to make possible the enjoyment of tasting food and seeing a beautiful sky. If more people knew just a fraction of this information, and really thought about it with any common sense at all, they would be impelled to acknowledge that an all-wise Creator with superior intelligence must exist. The beauty and balance of all these systems should move us to fear Him and want to know Him. A big part of the problem in society is that so few individuals have any vague idea of what is required to allow them to do something as routine as eating a hamburger, talking with a friend, or looking at the colors in the sky.
Scientific papers are difficult for most people to read, requiring understanding of specialized vocabulary and abstract concepts. O, that more talented people could translate this information into everyday terms, and visualize it for more to see, that it might rekindle their sense of wonder. Do you have such talent? Has this article struck a chord with you? Would you think and pray about how you might make this kind of worship-inducing information accessible to children and adults alike? Some possibilities come to mind: radio, print, audio-visual media, film, tracts, church bulletins, classroom teaching, educational games and role playing, computer animations… did you get any ideas while reading? If this kind of amazing information excites you, how could you make it accessible to those in your sphere of influence? If you have an inspiration, write in and share it.
Our perception of our lives and the world around us is often far too simplistic. Science should be, like James Joule said, a worshipful quest. He felt the study of nature and nature’s laws was “essentially a holy undertaking,” and of “great importance and absolute necessity in the education of youth.” He said, “After the knowledge of, and obedience to, the will of God, the next aim must be to know something of His attributes of wisdom, power and goodness as evidenced by His handiwork…. It is evident that an acquaintance with natural laws means no less than an acquaintance with the mind of God therein expressed.” Let’s hear a hearty Amen. Take a moment to thank God for quality control that keeps your machinery humming.