What are cell processes

The chaperones of proteins

The French term "chaperone" refers to chaperones who take care that unmarried girls do not mischief. In molecular biology, this refers to proteins that help other proteins to mature, prevent unwanted contacts and correct errors. Molecular chaperones organize the world of proteins, they are to a certain extent obstetricians, inspectors and doctors and thus fundamental for all cell processes as well as higher-level processes such as aging and evolution. They are also important for the development of diseases such as cancer, infections and heart attacks. Bernd Bukau from the Center for Molecular Biology describes the tasks of the chaperones and explains how they fulfill their protective function.

Proteins are made in high-molecular protein factories called ribosomes. The biosynthetic performance of the ribosomes inside the cells consists in the chemical linkage - the formation of peptide bonds - of twenty different amino acid building blocks to form a linear polypeptide chain, which is typically composed of several hundred amino acids. The ribosomes carry out the synthesis step by step, starting at one end of the polypeptide chain and linking amino acid by amino acid until the other end of the chain is reached.

The order of the building blocks of a polypeptide chain is the primary sequence. It is determined by the genetic information of the coding gene and is different for each protein species. This variety of amino acid chains determines the diversity of proteins and enables, for example, insulin to regulate blood sugar and hemoglobin to transport oxygen. In order for a protein to develop its biological activity, the amino acid chain must first be "folded" into a three-dimensional structure. Folding represents the maturation of the protein and is a fascinatingly complex process that has occupied generations of researchers. It is based on interactions between the amino acids of a polypeptide chain, which form within seconds to minutes. Here, the hydrophobic (water-repellent) amino acids, which do not mix well with the aqueous environment of the cell, are hidden inside the protein structure.

In folded proteins there are mainly two secondary structures, alpha helices and beta sheets, which are combined with one another in a variety of ways and connected with loops, thereby forming three-dimensional structural units, the so-called folding domains, with typically 100 to 200 amino acids. Many proteins consist of several folding domains that interact with one another and thereby form a superordinate tertiary structure. The complexity of the proteins is even higher, since the formation of a functional entity often requires the ordered assembly of several folded proteins of the same or different species in a quaternary structure.

The growing polypeptide chain is continuously transported from the place of its formation inside the ribosome through a narrow tunnel to the surface of this molecular machine. This is where the protein begins to fold. This is a complex process that is highly error-prone and can lead to the formation of "misfolded" proteins that are nonfunctional and sometimes even toxic. Protein misfolding can also affect existing, native proteins and is therefore a much more general problem for cells. Proteins can misfold when cells are exposed to stress, such as heat treatment. Or when proteins are destabilized by mutations, for example in diseases such as cancer or cystic fibrosis (an inherited metabolic disease; the genetic defect leads to a membrane protein that is important for normal mucus production being missing or impaired in its function). Finally, long-term changes in the proteins can also take place, for example in the Aß protein of Alzheimer's plaques or in prions.

Misfolded proteins often differ from correctly folded, "native" proteins in that hydrophobic amino acids reach the surface from the inside of the protein and enter into undesired hydrophobic interactions with other proteins (mostly of the same species). This process of "protein aggregation" is facilitated by the fact that proteins are packed so closely together in the cell (approx. 200 to 300 grams per liter of cell volume) that there is a "macromolecular crowding" - a constriction that it brings with it that proteins are constantly colliding with each other.

Protein aggregation thus reflects protein folding problems and manifests itself in a variety of ways: protein aggregates when you boil an egg; Biotechnologically produced proteins form "inclusion bodies" in their host cells; Many neurodegenerative diseases are associated with the formation of protein aggregates, the amyloid fibrils; Misfolding and aggregation of proteins contribute significantly to the aging of cells and death under stress.

To counter these problems, cells have an arsenal of molecular chaperones, which make up around ten percent of the total proteins in the cell. Chaperones bind and protect the newly synthesized proteins until they have obtained their native three-dimensional structure. They prevent unwanted protein aggregations by shielding exposed hydrophobic areas and are able to actively assist the native folding of proteins. They are even able to dissolve proteins that have already been aggregated and fold them back into their native state. In the last few years my working group has been able to explain some of the working methods of molecular chaperones.

The first chaperone that comes into contact with newly synthesized proteins in bacteria and helps with folding during their biosynthesis is the ribosome-associated protein "trigger factor". He sits directly at the tunnel exit of the ribosome and receives the polypeptide chains. The trigger factor was discovered in our laboratory as a chaperone for newly synthesized proteins and its importance for folding was determined. How it actually helps the proteins to fold was initially unknown. In 2004, in collaboration with Nenad Ban (ETH Zurich) and Elke Deuerling (Center for Molecular Biology at the University of Heidelberg), the atomic structure of the trigger factor in the complex with the ribosome was elucidated: The trigger factor bulges over the tunnel exit of the ribosome and forms a hydrophobic one Cave into which the amino acid chain growing out of the ribosome is taken up. We suspect that the first folding steps of a protein take place in this cavity. The folding of the protein could be delayed by hydrophobic interactions until the amino acid chain, due to the ongoing synthesis, has reached a length that allows a stable folding domain to be formed. The biochemical properties of the interior of the cave enable individual domains of the "newborn" protein to be folded in a protected environment. The protein may not be released from the cavity until it has been folded, although the molecular details of this folding process are still largely unclear. It is also still unclear how the first folding steps on the ribosome take place in evolutionarily more developed cells. Although higher cells do not have a trigger factor, they do have several other ribosome-associated chaperones, which, however, have not yet been assigned any specific functions in the folding of proteins.

For many newly synthesized proteins, the folds on the ribosome are not sufficient to achieve the native spatial structure. Members of the heat shock protein families 70 (Hsp70) and 60 (Hsp60), which together with the trigger factor, form a cooperating chaperone network, provide further assistance.

The name of the Hsp70 and Hsp60 proteins was based on the finding that they are not only important for the folding of newly synthesized proteins, but also function as central components of the cellular repair system for misfolded proteins and are therefore essential for the survival of cells under stress conditions. Part of the survival strategy of cells exposed to stress is the synthesis of Hsp70 and Hsp60 in what is known as a "heat shock response".

Hsp70 and Hsp60 chaperones require the energy of ATP, the cellular "energy currency", in order to bind in a controlled manner to their substrates - the folding and misfolded proteins - and then to release them again for further folding. Both chaperones bind to superficial hydrophobic areas of the proteins, but in extremely different ways.

Our investigations showed that Hsp70 chaperones have a small binding pocket for only four to five amino acids of a polypeptide chain, the only characteristic of which is the enrichment of hydrophobic amino acids and the lack of negatively charged amino acids. As soon as such a hydrophobic section (sequence) is exposed on the surface of a protein, Hsp70 can bind to it, thus protecting the protein from aggregation and preparing further steps for folding. This mode of recognition of a short hydrophobic sequence on the surface of a protein therefore represents a molecular definition of the cell for misfolding. For binding, Hsp70 must convert (hydrolyze) ATP (adenosine triphosphate) to ADP (adenosine diphosphate), whereby the open binding pocket is closed and the substrate in the Is locked inside. This critical step in the binding cycle requires a cofactor, the "DnaJ co-chaperone". It couples the substrate binding with the hydrolysis of ATP and thus directs Hsp70 to its substrates. The binding cycle is complete when another co-chaperone, GrpE, causes Hsp70 to release bound ADP. This creates space for a new ATP molecule that opens the binding pocket and frees the substrate from its grip by the chaperone.

Hsp60 chaperones (also called GroEL) form complex structures made up of 14 subunits that come together to form a ring with a central opening, a kind of chamber. Studies by various working groups have shown that the protein substrates bind inside the chamber and are held there by several hydrophobic binding sites. As soon as a protein substrate is bound, the GroES co-chaperone binds over the bound substrate and closes the chamber like a lid. The substrate is then detached from its binding sites within the chamber in an ATP-regulated manner and can now fold in the protected chamber - in infinite dilution, so to speak, far from any aggregation problems. Driven by the ATP hydrolysis cycle from GroEL, the GroES lid opens after about 15 seconds and releases the protein substrate into the aqueous solution of the cell. If the protein substrate fails to fold into its native structure during this period, this chaperone binding cycle can be repeated.

The main strategy of the cellular quality management of already existing proteins is to prevent protein aggregations from occurring due to the temporary binding of the misfolded proteins to Hsp70 chaperones - similar to what is described for newly synthesized proteins. The interaction with Hsp70 allows a subsequent refolding into the native structure or a transfer of the substrate to Hsp60 (GroEL), which can also bring about the refolding. Although this chaperone duo works very efficiently, it is overwhelmed, especially after strong stress, for example after a massive increase in cell temperature to 45 ° C or the biotechnological production of large amounts of protein in bacteria. As a consequence, protein aggregates occur.

Until a few years ago it was assumed that the aggregation of proteins could no longer be reversed. Only recently it was shown that a chaperone system exists in yeast cells, which consists of two components and is able to dissolve protein aggregates again. Our own research led to the identification of a bacterial chaperone system that performs the same function. It consists of the ClpB chaperone, which forms a ring with a central pore with its six subunits, and of Hsp70 with its DnaJ co-chaperone. The cooperation of both chaperones is essential for the dissolution of the protein aggregate: The aggregated proteins must first interact with the Hsp70 chaperone, which may loosen the aggregate structure.

ClpB itself is a fascinating molecular machine: It uses the energy of ATP to bind individual polypeptide chains of the aggregated proteins and transport them through the central pore. The polypeptide chains initially bind at the entrance to the pore, namely at one of the two ends of the ClpB ring. The polypeptide chain is then transported through the pore, whereby the substrate is likely to be completely unfolded. We do not yet understand the mechanics of this process, but we assume that the hydrolysis of ATP generates the force that is required to push the substrate through the pore and thus pull it out of the aggregate. Once the polypeptide chain has reached the exit of the ClpB ring, the refolding process can begin with the help of Hsp70. This bi-chaperone system is incredibly potent: it can be used to dissolve almost all proteins that have aggregated in the cell after a heat shock. If this system is missing because the genes that carry the instructions for the construction of these proteins have mutated, the cell dies after heat treatment.

The entirety of the molecular chaperones, of which only a few important ones have been mentioned here, constitute an efficient quality control system for proteins. This system is also used by cells to control dozens of signaling pathways, including the signaling pathways that cells use in response to environmental changes and growth factors are induced. For this purpose, chaperones bind to key proteins of the signaling pathways - kinases and transcription factors - and prevent their aggregation. This function in signal transduction makes chaperones particularly important for the regulation of cellular activity. Rapidly growing tumor cells have a greater need for chaperones, which is why they are more sensitive to active substances that are directed against chaperones. There is currently a keen interest in the pharmaceutical industry in developing substances which are specific for chaperones and which have been shown to be effective against tumors in some test cases. So there are plenty of reasons to explore the world of chaperones further and understand the variety of their modes of action.

Author:
Prof. Dr. Bernd Bukau
Heidelberg University, Center for Molecular Biology
Im Neuenheimer Feld 282, 69120 Heidelberg
Telephone (0 62 21) 54 67 95
E-mail: [email protected]