Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations

According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed) reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates). Cell function is considered as a transition between two states (two states model), the resting state and state of activity (this applies to the cell as a whole and to its individual structures). In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins), and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity.

"One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity."

To date, numerous mechanisms, signal pathways, and different factors have been found in the cell. Researchers are naturally eager to find commonalities in the mechanisms of cellular regulation. I would like to propose a substantial approach to problems of cell physiology - the structural ground that produces signals and underlies the diversity of cellular mechanisms.

The methodological basis for the proposed hypothesis results from studies by the scientific schools of Dmitrii Nasonov [1] and Gilbert Ling [2-6], which have gained new appreciation over the last 20-30 years owing to advances in protein physics [7] in the study of properties of globular proteins, their unfolding and folding, as well as the discovery of novel states of the protein molecule: the natively unfolded and the molten globule. The key statement for the rationale of the present paper is that the specificity of interactions of polypeptide chains with each other (at the intra- and inter-molecular levels) can be provided only by their secondary structures, primarily α-helices and β-sheets.

Nasonov's school discovered and studied a fundamental phenomenon -- the nonspecific reaction of the cell to external actions [1], while works by Ling [5] and his followers allow the mechanisms of this phenomenon to be understood.

The above-mentioned cell reaction has been called nonspecific because diverse physical and chemical factors produce the same complex of structural changes in the cell: an increase in the turbidity and macroscopic viscosity of the cytoplasm and in the adsorption of hydrophobic substances by cytoplasmic proteins. It is of primary importance that the same changes also occur in the cell during its transition into the active state: muscle contraction, action potential, enhancement of secretory activity (for details, see [8]). Hence, from the point of view of structural changes, there is no fundamental difference between the result of action on the cell of hydrostatic pressure and, for instance, muscle contraction. In both cases, proteins are aggregated.

Nasonov called the cause of these changes the stages of cell protein denaturation, as the changes of properties of isolated proteins during denaturation are very similar to the changes in the cytoplasm during the nonspecific reaction. As a result, the denaturational theory of cell excitation and damage was created [1]. The structural changes of protein denaturation were unclear in Nasonov's time. Nowadays, it is assumed that the denaturation is the destruction of the tertiary and secondary structure of a protein. Below I give two definitions, for the denaturation of natively folded (globular) proteins and for natively unfolded proteins.

A key notion in physiology is the resting state of the cell. This is implicit in the concept of the threshold character of the action of stimuli on the cell, which has played a historical role in the development of physiological science. It is the threshold that is the boundary between two states -- rest and activity. But in effect, all our knowledge about cells concerns active cells, not cells in the resting state. It is in the active cell that variable changes occur that can be recorded. Nothing happens in the resting cell, so there is nothing to be recorded in it. Nevertheless, it is obvious that the resting state is the initial cell state, the starting point for all changes occurring in the cell.

What characterizes the structural aspect of the cell in the state of rest? It is only in Ling's work [5] that I have found a clear answer to this question. The answer can be interpreted as follows: if all resting cell proteins were arranged in one line, it would turn out that most of the peptide bonds in this superpolypeptide would be accessible to solvent (water), while only a few would be included in secondary structures. When the cell is activated, the ratio between the unfolded and folded areas is changed sharply to the opposite: the proportion of peptide bonds accessible to solvent decreases markedly, whereas the proportion included in secondary structures rises significantly. These two extreme states of cell proteins, suggested by Ling, provide a basis for further consideration.

If Ling's approach is combined with Nasonov's theory, we obtain several interesting consequences. First of all, it is clear that proteins with maximally unfolded structures form the structural basis of resting cells because they are inactive, i.e., do not interact with other proteins or other macromolecules. The situation changes when an action on the cell exceeds the threshold: completely or partially unfolded key proteins begin to fold when new secondary protein structures are formed. Owing to these new secondary structures, the proteins become capable of reacting, i.e., intramolecular aggregation (folding of individual polypeptides into globules) and intermolecular aggregation (interaction of some proteins with others) begin. A distinguishing feature of these aggregational processes is their absolutely specific character, which is ensured by the amino acid composition, shape, and size of the secondary structures. The structures appearing have physiological meaning, so such aggregation is native and the secondary structures causing it are centers of native aggregation. Another source of secondary structures necessary for native aggregation is the molten globule.

The ability of cells to return to the initial state, the state of rest, means that native aggregation is completely reversible, and the structures appearing in the course of native aggregation are temporary and are disassembled as soon as they cease to be necessary. Native aggregation can involve both the whole cell and individual organelles, compartments, and structures, and activation of proteins is of a threshold rather than a spontaneous character.

The meaning of the proposed hypothesis of native aggregation is that the primary cause of any functional changes in cell is the appearance, as a result of native aggregation, of temporary structures, continually appearing and disintegrating during the life of the cell. Since native aggregation is initiated by external stimuli or regulatory processes and the structures appearing have a temporary character, these structures can be called signal structures.

Signal structures can have different properties: (i) they can be centers of binding of ions, molecules (solutes), and proteins; (ii) they can have enzymatic activity; (iii) they can form channels and intercellular contacts; (iv) they can serve as matrices organizing the interactions of molecules in synthetic and transport processes; (iv) they can serve as receptors for signal molecules; (v) they can serve as the basis for constructing even more complex supramolecular structures. These structures "flash" in the cell space like signal lights, perform their role, and disappear, to appear in another place and at another time. The meaning of the existence of the structural "flashes" is that during transition into the active state the cell needs new resources, functions, mechanisms, regulators, and signals. As soon as the cell changes to the resting state, the need for these structures disappears, and they are disassembled. Extreme examples of native aggregation are muscle contraction, condensation of chromosomes, the appearance of the division spindle, and interactions of ligands with receptors.

Thus, the present paper will consider the meaning and significance of native aggregation as the universal structural basis of the active cell. The basis of pathological states is the inability of the cell to return to the resting state and errors in the formation of signal structures. The presentation of native aggregation is based on three pillars: (i) reversible protein aggregation is a structural basis of cell activity (Nasonov's School); (ii) the operation of the living cell or its individual structures can be regarded as a repetitive sequence of transitions between two states (active and resting), a key role in which belongs to natively unfolded proteins (Ling's approach); (iii) the specificity of interactions of separate parts of a single polypeptide chain with each other (folding) or the interaction of separate polypeptide chains among themselves (self-assembly, aggregation) can be provided only by protein secondary structures.

The goal of this paper is the enunciation of principles, rather than a review of facts corresponding to these principles.

The best-studied nonspecific response of cells to external actions might possibly be the response to fixatives. For a long time in the history of science, cells were considered optically empty structures by researchers. The appearance of methods of fixation and staining wrought a revolution in cytology, as these approaches opened to the researchers' sight numerous cell structures whose existence had not even been suspected. After a period of euphoria, doubts were cast: were these structures real or were they the results of fixation, denaturation of the cell's native substance?

The danger of serious errors when artifacts of fixation might be considered real structures became a subject of general attention after 1899 (see [9], Ch. 1 for details), when coagulation of homogenous protein solutions was shown to lead to the appearance of structures quite similar to those observed in fixed cell preparations (see [10], Fig. twenty-four). The shape of such artificial structures depended on the chemical nature of the fixative, its concentration, the protein concentration in solution, the temperature, and other conditions. This brought about an obvious crisis in the study of cell morphology.

However, other things were also obvious. In the optically empty part of the cell, visible structures could appear not only during fixation, but also during the transition of the cell to the active state. Comparative observations on fixed preparations and living cells showed that where the structure appeared in vivo, it was also observed in a fixed preparation. The obvious resemblance between native structures and the structures obtained as a result of fixation gave grounds for considering that several cell structures are formed not only at fixation, but also during activation of some particular cell fraction, when new structures absent in the resting cell are formed by self-assembly (see [9], Ch. 1 for details).

This discussion has led to the rather important conclusion that despite the dangers of producing artifacts, another thing is beyond doubt: in the process of aggregation, the denatured cell proteins interact with each other not chaotically, but regularly, in accordance with a certain plan (this is what I call native aggregation). The laws of this interaction lead to the formation of temporary structures necessary for the cell to function under new conditions. During fixation and dehydration, this process initially occurs "as it should" (the self-assembly of real cellular structures takes place), but it goes too far when the process of making the preparation is completed, when aggregation becomes irreversible and the structure appearing as a result of aggregation becomes a "corpse". If the interaction of proteins during aggregation had been chaotic, we would still know little about cell structure.

The course of native aggregation seems to be determined by the non-homogeneity of the content of the resting cell; it has structure that is invisible under the light microscope, but reveals itself at the onset of native aggregation. The role of structure guiding native aggregation may be played, for instance, by Porter's "microtrabecular lattice" [11], which can be envisaged «...as that which is in the background of all the visible membranous organelles and all the visible elements of the cytoskeleton; e.g., that which has been invisible up until now and which we wish to "see" microscopically» [12]. Such a lattice might act as the center of "crystallization" or the center of "attachment" of aggregating proteins. However, this is merely an example that I cite for clarity. The centers of crystallization can also comprise the most sensitive proteins that are the first to respond by conformational alterations to changes in the medium and become aggregation-competent. In any event, as a result of native aggregation, the hidden structures become visible under the microscope.

Fulton [13], a convinced Porter devotee, moved even further: she put forward a point of view that "the cytoplasm is so compact that it is only occasionally more open than a crystal". Sufficient data have probably accumulated in the literature to establish that the content of a cell is to be considered a structured system that guides native aggregation into the required course. As one example, one can indicate the data of Balό-Banga et al. [14]: the birefringence of lymphocyte nuclei was enhanced after fixation with ethanol, i.e., correct fixation leads to the appearance of new, more ordered structures. However, especially interesting are the cases when native aggregation, as I call it, takes place in the process of normal cell functioning. Thus, in the same work by Balό-Banga et al. [14], activation of lymphocytes by specific antigens or haptens was shown to lead to a significant enhancement of nuclear birefringence. The same phenomenon was also observed in the case of activation of peripheral blood lymphocytes with allergens in drug-allergic patients [15].

If the factor affecting the cell becomes more intense, its activating effect will be replaced by a damaging one. Thus, the studies of Inners and Bendet [16] on thermal DNA denaturation in bacteriophage T2 and spermatozoa [17] showed that during irreversible denaturation of structures their capacity for birefringence is lost. Such data indicate that under certain conditions, the actions of heat, organic solvents, etc. on cells produce not native aggregation, but destruction, disorganization of intracellular structure; in other words, destruction of structure can follow native aggregation. Unfortunately, there is a marked tendency in the literature towards rough alterations in the structure of the cytoplasm and organelles, because they are easier to study.

Thus, the retrospective considered shows that when adequate methods of study are used, native (programmed) protein aggregation leading to self-assembly of various cell structures is the usual phenomenon of cell life. An example of this is the universal reaction of the living cell [8].

Why does native aggregation not occur in cells in the resting state but begins only on activation (for instance, muscle contraction, action potential) or damage? To answer this question, let us return to Nasonov's denaturation theory [1]. According to this theory, excitation of the cell takes place only when its proteins are subjected to the initial stages of denaturation.

Mirsky seems to have been the first to pay attention to the similarity between changes in active cytoplasm and the denaturation of isolated proteins [18]. Mirsky came to the conclusion that denaturational protein changes appear when an egg cell is fertilized [19] and during photoreception [20]. This is what he says about it in the latter of the above-cited works: "...There is evidence indicating that light denatures a conjugated protein, visual purple, and that denaturation reverses in the dark." However, his studies in this direction were not systematic.

Nasonov and his followers studied the effects of quite different factors (chemical substances, pH, hydrostatic pressure, mechanical action) on cells of different types. As a result, a regularity was revealed: regardless of the character of the action and the type of cell, the response reaction represented a monotypic (nonspecific) complex of synchronous changes. These changes were of two-phase character: macroscopic viscosity first decreased, then rose; binding of vital dyes by cell structures (under conditions of diffuse equilibrium) first decreased, then increased; in the first phase of the reaction the cytoplasm became clear, in the second phase it became turbid. Other parameters (see [8] for review) were also studied. The first phase of this reaction is not related to the subject of the present paper, as it is a variation of the resting state. Of interest to us is the second phase, whose structural basis is protein aggregation (Fig. 1). It is this phase that is the phase of activation of cell functions [1].

This second phase was called the phase of excitation and damage by Nasonov's school. Substantial changes in the cell in this phase are remarkably reminiscent of denaturation of isolated proteins; therefore, Nasonov called his theory explaining the cell response reaction the denaturational theory of excitation and damage. According to this theory, the initial stages of denaturational changes, when they still are reversible, underlie cell excitation (activation of secretory function, muscle contraction, action potential, etc.). More profound protein changes lead to disturbances of normal cell functioning, but may still be reversible. Then, with further development of damage, denaturational changes become irreversible and the cell dies.

The peculiarity of the cellular reaction discovered and studied by Nasonov's school was its nonspecific character: whatever the action on the cell was, its proteins were aggregated (as in fixation); any cellular activity was also accompanied by protein aggregation (this is especially well seen in the case of muscle contraction). The behavior of isolated proteins during denaturation was the same: any denaturing agent caused their aggregation (except for denaturation under non-physiological conditions, e.g. denaturation by concentrated solutions of urea).

In this universalism of the cellular response, a puzzle was hidden, but in an era concerned with specific interactions, nonspecific phenomena drew no attention. Nevertheless, it is obvious that the nonspecific cellular reaction discovered by Nasonov's school is a fundamental natural phenomenon - like cell division or carcinogenesis. Attention to it is justified because the phenomena of nature, unlike theories, cannot be erroneous.

The nonspecific character of the cellular reaction considered is a superficial impression. Death is also a nonspecific phenomenon, but the processes leading to it are characterized by diversity and can be extremely specific. In exactly the same way, aggregation of proteins can be based on specific interactions. If we deny the existence of specific mechanisms in cell protein aggregation, we will not be able to understand why cell stress initiates such processes as proliferation, differentiation, senescence, apoptosis, necrosis, or mitotic cell death [21]. On the other hand, it is obvious that with all the specificity of interactions leading to protein aggregation, the cellular reaction looks nonspecific because any aggregation, whether specific or nonspecific, ends in the formation of protein complexes. Therefore, it is more correct to focus not on the nonspecificity, but on the universality of the complex of structural and functional cellular changes studied by Nasonov's school. That is why I have proposed to name this typical cellular response a universal reaction of the living cell or protoreaction, because there are grounds to consider it the most ancient type of cellular reaction to external actions [8].