A. Validity of in Vitro Experiments
Most of this volume discusses recent observations and interpretations of the structure and functions of the chaperonins, but a summary of key points may be useful here to orient the reader new to this field. A distinction should be drawn between those experiments performed in vitro with either defined components or isolated organelles and crude cell-free extracts, and those performed in vivo, usually by genetic means. The former experiments greatly outnumber the latter and have the advantage that they allow sophisticated techniques to be used to analyze, for example, the kinetics of protein refolding, the order of complex interactions, and the structures of partially folded forms of polypeptides. However, in vitro experiments suffer from the disadvantage that the conditions under which they are performed are different in important respects from those operating within the intact cell (see Section V,B). Since the chaperonins evolved to assist protein folding inside the living cell, and not in the test tube, this is not a trivial distinction.
It is my view that biochemistry is a branch of biology and not a branch of chemistry, since its declared aim is to understand how organisms work. On this basis I suggest that the ultimate aim of research into protein folding should be to understand how this process operates in vivo. This view appears not to be shared by all workers in this field, but I suspect that had protein chemists in the past considered more closely the intracellular environment in which proteins have evolved to fold, the chaperonins could have been discovered much sooner. One encouraging trend that has become evident recently, and one that I predict will increase in importance, is that more attention is being paid to trying to mimic in vitro some of the conditions that may influence how the chaperonins function in vivo, conditions such as the presence of other molecular chaperones acting on folding polypeptides (see Chapter 8) and the degree of macromolecular crowding (see Section V).
The majority of published evidence about chaperonin function concerns the GroEL and GroES chaperonins of E. coli and the chaperonins of yeast mitochondria; much less information is available about the functions of the TCP-1 chaperonins (see Chapter 5 for the latter). This evidence supports the provisional conclusion that these proteins serve at least three functions: (1) They prevent the aggregation of partially folded polypeptides that have either just been released by the ribosome or just emerged from a membrane after transport from the cytosol (see Chapters 4,7-9). (2) They bind partially folded polypeptides that may be kinetically trapped in a conformation unable to proceed spontaneously to the functional conformation; this binding and/or subsequent release causes the alteration of some noncovalent interactions that allow correct folding to proceed (see Chapter 7). (3) They protect correctly folded proteins from aggregation after denaturation by stresses such as high temperatures and trap denatured proteins so that they can be degraded; these activities account for the observation that in many, but not all, cell types the GroE and TCP-1 chaperonins are induced by stress (see Chapter 8).
These functions are not mutually exclusive, but complementary, and all involve the binding of the cpn60 component to exposed hydrophobic surfaces of the protein substrate. The protein binding sites of GroEL lie within the top of each ring (see Chapter 9). This location serves to encapsulate the protein substrate to some degree and prevents GroEL oligomers from binding to one another. The binding is reversed by ATP hydrolysis, and this reversal releases the substrate protein in a manner whereby it is more likely to avoid aggregation and fold correctly. The precise manner by which these ends are achieved is the subject of intense current debate (see Chapters 7-9). Two aspects of these issues that relate to chaperonin function in vivo are discussed in Section V.
The majority of the evidence for the above conclusions is derived from in vitro experiments, but experiments in which cpn60 function is disrupted in vivo by genetic means support the view that cpn60 prevents the aggregation of a significant number of newly imported proteins in yeast mitochondria (Cheng et al., 1989; Hallberg et al., 1993) and newly synthesized proteins in E. coli (Gragerov etal., 1992; Horwicheia/., 1993).
The biological significance of the chaperonins does not end with their roles in protein assembly. The GroE chaperonins are some of the most potent stimulators of the immune system yet discovered, and indeed are the dominant immunogens in all human bacterial infections studied. There is thus considerable medical interest in the possible role of the chaperonins in human disease, especially in protection against infection, cancer, and autoimmune disease (see Chapter 10).
There is some indication that the chaperonins may also play extracellular roles in animals. It is claimed that the early pregnancy factor, a protein detected by bioassay in the maternal serum of mammals within 24 h of fertilization, is identical to mitochondrial cpnlO (Cavanagh and Morton, 1994). However, there has been no report of the detection of cpnlO in serum by chemical methods, perhaps because the concentration of cpnlO may be very low. One of the cpn60 proteins from mycobacteria stimulates cytokine secretion by human monocytes, whereas cpnlO from Mycobacterium tuberculosis induces apoptosis in human T lymphocytes. These and related observations have been used to formulate the idea that the chaperonins are multiplex antigens; i.e., they act simultaneously on a range of cell types in stress situations so that immune messages such as cytokines cause immune effector cells to adapt to cope with the stress (see Chapter 10). There are several reports that cpn60 occurs at sites outside the mitochondria in some animal cells, including at the cell surface (Kaur et ah, 1993). These studies use indirect immunological methods as the main means of detection, and more detailed characterization is required before the significance of these observations becomes clearer. Perhaps the peptide binding abilities of cpn60 enable it to play an extracellular role in binding peptides for transport or detoxification.
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