JOURNAL OF MEDICAL & CLINICAL CASE REPORTS

Figure 10 - Structure and composition of the cytoskeleton

This is not the place to describe the individual monomers, a topic that would require several chapters. We mention them only to underline the astonishing properties of proteins.

The cytoskeleton also participates in the formation of the Golgi apparatus with a continuous reciprocal feedback mechanism.

How these proteins manage to assemble the filamentous networks that transport the various organelles and chromosomes in cellular activity and during cell division, and above all, how they manage to follow the correct directions in their movements, remains a mystery.

Here, proteins have reached the maximum level of functional efficiency; not only do they reproduce in individual monomers, but they also know the correct direction to add them to complete the movement. Their ability to adapt and respond to functional demands is astonishing.

They are undoubtedly intelligent molecules.

Since they play a crucial role in the formation of the cytoskeleton, these proteins are also subject to the phenomenon of misfolding, which has direct, disastrous consequences on cellular structure and organization [22-23].

Since they are complex protein apparatuses that derive from the assembly of preformed monomeric proteins, their pathogenic effect has been defined as an aggresome [26-28].

Cell Membrane Proteins

We briefly mentioned the presence of proteins in the cell membrane.

These are proteins bound to the cell membrane that are either entirely contained within the thickness of the lipid bilayer or are attached to other membrane molecules and protrude from the cell surface.

These proteins represent a fundamental component of cells as they are the true effectors of cellular activity, from the transport of substances within the cell, to contact and recognition of other cells or foreign bodies, to receptor function, ion channels, etc.

In fact, the lipid bilayer of the cell membrane serves only to transport proteins to the same level and plays the role of a regulator of overall cellular homeostasis.

Protein Synthesis

To conclude the description of proteins, it's appropriate to mention the mechanism of protein synthesis. A detailed description of the entire process is beyond the scope of this article. We'll simply outline the simplified steps of the entire mechanism.

The mechanism, called "central dogma of Biology" by Watson and Crick, schematically goes through three fundamental stages [29-30]:

1. Transcription of the gene that codes for the protein from DNA to messenger RNA, mRNA.
2. Transport of amino acids onto mRNA by transfer RNA, Trna.
3. Formation of the peptide bond between amino acids.

The process takes place in ribosomes, complex macromolecules present in the cellular endoplasmic reticulum.

Fig. 11 shows a simplified diagram of the process,

Figure 11 – Schematic Mechanism of Protein Synthesis

Protein synthesis goes through several steps that include the transcription of the protein code from DNA to messenger RNA and the synthesis of the protein at the ribosome level. Fig. 10.

Once synthesized, the native protein undergoes a maturation process that serves to define the definitive sequence that the protein will assume in its target role.

This process is called post-transcriptional control of protein synthesis. During transcription, a nascent protein can be split into two different proteins, a process known as alternative RNA splicing, which can produce different forms of the same protein.

An interesting example of this process occurs in peptide hormones.

Hormones can be derived from lipids (steroid hormones) or from amino acids, as in peptide hormones [31].

Peptide hormones are naturally produced by various endocrine glands in our body. They consist of peptides of varying lengths and are water-soluble; therefore, they can easily travel through the bloodstream to their target cells, where they exert their biological effects.

Hormones are produced in immature forms as preprohormones, which will undergo a maturation process by splicing to become prohormones and then be processed into their final form as hormones [32-33].

This behavior, which recalls that of enzymes, another class of proteins, tells us that the behavior of proteins is very complex and is regulated by multiple biological events that have the task of maintaining the global homeostasis of the entire organism.

How do hormones know the exact sequence they need to interact selectively with their receptors? It's as if they knew in advance the conformation they need to assume to interact precisely with their receptors.

During the protein synthesis process, they know in advance the type and arrangement of amino acids they must contain and how they must be processed after transcription in order to function.

What feedback mechanism regulates the entire process?

These are currently unanswered questions; the only explanation we could venture is that the entire process is the result of the selection of thousands of years of evolution, and once achieved, it has been maintained in all multicellular organisms.

A similar process could have led to the stabilization of metabolic cycles, from glycolysis to the Krebs cycle, which we find identical, with very small variations, in all living things, from bacteria to humans.

Discussion

Proteins have the extraordinary ability to change their conformation in response to effective stimuli to assume a biologically useful transient conformation.

All proteins, from functional to structural (a meaningless distinction), have been selected by biological evolution precisely to fulfill the task of changing their steric conformation based on the needs required by biological activity.

Just to name a few examples:

Enzymes, channel proteins, receptor proteins, cell membrane-bound proteins, the antigen-antibody mechanism, etc.

It is as if they have the ability to assume the required three-dimensional shape when they receive a specific stimulus.

Another aspect that clarifies the function of proteins is the fact that they act by adaptive contact, a sort of formation of a molecular imprint, with the molecules with which they must interact, and only with those.

This means that their conformation is not definitive, and that they are able to modify it.

Therefore, when studying proteins, it is possible to do so through the crystallization process, which causes them to assume a rigid structure, so that they can be studied with X-ray crystallography.

This dynamic behavior can explain the properties of some unusual molecules, such as prions and amyloid structures, which, as we described above, both arise from misfolding compared to what they should have.

This property is probably the key to understanding the behavior of intrinsically disordered proteins that play an important role in the pathogenesis of some diseases.

We can consider these molecules as being made up of a stable polypeptide component in its quaternary structure, which acts as a handle and vehicle that transports them to their final position, while their variable component does not yet have a defined structure.

Then one could think of this variable component as seeking the correct steric coupling with other molecules or metabolites with which it must interact in order to collapse and produce the final functional result.

We can imagine this beady portion, similar to a rubber hose, oscillating and probing the environment in search of other structures with which to mate.

This could be their biological role, and it helps us understand how thermodynamically unstable they are and how susceptible to misfolding and incorrect couplings that produce the most diverse pathologies.

Viewing prions and amyloid structures as intrinsically defective degenerate proteins with the potential to pair with and deform other similar proteins would provide us with clues for attempting to halt the degenerative process, and perhaps even the diseases they cause.

Let us ask ourselves then: where does the ability of misfolded proteins to modify their structure and promote the deformation of other similar proteins in the modified form come from?

Misfolded proteins that we know best is the transition of the alpha-helical portions into beta- sheet forms (Fig. 12). When they assume this shape, they are no longer recognized by the chaperone system and the immune system and are free to act undisturbed, allowing them to assume conformations capable of sterically coping with similar molecules and producing their conformational distortion. Essentially, they function as enzymes.

Figure 12 - The conformational transition from alpha helix to beta sheet exposes hydrophobic amino acid residues and promotes protein aggregation

Factors that Alter Protein Structures

In addition to the events described above that intervene in the conformational transformation of proteins from native to their misfolded forms, there are other causes capable of determining their deformation. In particular, some substances have clinical and pathological relevance in the pathogenesis linked to their ability to induce protein misfolding.

Some of these substances have assumed the value of pathogenic markers in clinical diagnosis.

Here we highlight just three of these substances: homocysteine, glucose, and cholesterol. These substances, when present in excess in the blood, are considered important risk factors.

Homocysteine (Hcy) and N- Homocysteinylated Proteins

Homocysteine is a sulfur-containing amino acid, chemically it is a γ– thio –α– aminobutyric acid (C ₄ H ₉ NO ₂ S), which is formed, as an intermediate product, in the enzymatic transformation of methionine (C ₅ H ₁₁ NO ₂ S) into cysteine (C ₃ H ₇ NO ₂ S), through the removal of methyl groups: the prefix homo- (derived from the Greek ὁμο -) indicates similarity between two compounds [34].

The conversion of homocysteine to methionine (a process called remethylation) or its conversion to cysteine (transsulfuration) represents the main metabolic pathways capable of maintaining its intracellular levels within a narrow range; its release into the bloodstream allows us to measure its plasma concentrations, which represent an index of the tissue homocysteine status Fig.13.

Figure 13 – Homocysteine Metabolism

Homocysteine levels in the blood must be kept under strict control in order to prevent the numerous pathogenic effects reported in the literature and schematically indicated in Fig.14.

Figure 14 – Blood Values of Homocysteine and Pathologies Resulting from Hyperhomocysteinemia

Homocysteine, present in high concentrations in the blood, facilitates the binding of the substance to lysine residues present in proteins, or is selected in place of methionine by methioninyl RNA synthetase during protein synthesis.

This process results in the formation of homocysteine-thiolactone in the human body.

It is a chemically reactive thioester metabolite that modifies protein lysine residues in a process called N-homocysteinylation. This modification causes protein damage/aggregation, a hallmark of many diseases. N-homocysteinylated proteins lose their normal function, are cytotoxic, promote immune, inflammatory, thrombotic, and atherogenic processes, and have been linked to cardiovascular disease, Alzheimer's disease, diabetes, kidney disease, neural tube defects, and male infertility.

For this reason, hyperhomocysteinemia is considered a primary risk factor for cardiovascular and other pathogenic events.

The primary pathogenic effects induced by hyperhomocysteinemia are found on the endothelium [35].

The endothelium is the functional unit of the cardiovascular system and is made up of epithelial cells that line the inside of all blood vessels.

The normal endothelium functions as a semipermeable cell layer and acts as a sensor and transmitter of signals in the circulating environment. Endothelial cells are important for maintaining the homeostatic balance of vessels by producing factors that regulate intracellular signaling, vascular tone, coagulation, neutrophil recruitment, lipid transport, hormone trafficking, and the proliferative cellular response, among others.

The pathogenic effect of homocysteine-thiolactone is expressed through the alteration of the proteins that make up the endothelial scaffolding, which produces the breaking of the bonds between the endothelial cells and consequent alteration of the homeostatic functions Fig.15.

Figure 15- Schematic of the Pathogenic Activity of Homocysteine

Another class of compounds that are implicated in protein misfolding and conformational changes of other metabolic components are carbohydrates, which are therefore also considered pathogenic causes of many diseases [36-38].

The products thus modified are called advanced glycation end-products (AGEs ) or glycated substances, and are the result of exposure to high concentrations of sugar. The mechanism is called glycation.

Their presence is a pathognomonic index and marker of many degenerative diseases, such as diabetes, atherosclerosis, renal failure, Alzheimer's, etc.

Advanced glycation products are the result of a spontaneous mechanism that occurs between reducing sugars (a reducing sugar is any sugar that, in solution, has an aldehyde or ketone group that acts as a reducing agent) and the autoxidation derivatives with amino groups of proteins, DNA, or lipids, which occur under conditions of hyperglycemia. The formation of AGEs produces negative effects on the structures of macromolecules and determines pathological events.

Their tendency to accumulate takes on a very important pathogenic significance in molecular interactions and can determine macromolecular misfolding similar to that described for proteins.

As an example, we can point out glycated hemoglobin, which is a significant marker of the glycemic status of a diabetic patient.

The glycosylation of macromolecules takes on different meanings based on the group of carbohydrates involved in the process.

Glycans attached to proteins by enzymatic glycosylation can be classified into two types:

• N-glycans, which are those attached to the amide side chain of Asn residues, and
• O-glycans, including those attached to the hydroxyl group of the side chains of Ser or Thr residues.

Cholesterol

Excess cholesterol in the blood is considered a major cardiovascular risk factor, and all primary prevention guidelines recommend keeping it under close monitoring and maintaining its blood concentration below normal values.

Little attention is paid to the fundamental role this substance plays in stabilizing cell membranes, and it represents the most common component within the lipid bilayer of the cell wall.

It contributes to the stability, fluidity, and resistance of the cell membrane, as well as having an important function in modulating the activity of most wall proteins [39-41].

And it is thanks to the association of cholesterol with membrane proteins that the latter function properly. This indissoluble synergistic association between cholesterol and proteins is present in all living organisms and has evolved to perform the functions of the proteins mentioned above.

Until now, only the negative aspects of cholesterol as a potential risk factor have been studied, but the positive role it plays has been overlooked.

Several studies on the cholesterol-protein association have allowed us to examine the association in detail, both from a structural-morphological and functional point of view, and have allowed us to recognize and assign a fundamental role to cholesterol as an essential element for the correct functioning of proteins.

Conclusion

From what we have previously explained, some considerations emerge on the evolution, structural, and functional role of proteins, which makes these substances the fundamental biological component of all vital functions.

Proteins are the result of molecular evolution lasting millions of years, which has produced complex polymeric molecules with the unique ability to change their shape to adapt to the needs imposed by environmental conditions.

Some of them, once they have obtained the optimal size and shape, are able to reproduce and act as a template for forming other proteins of the same shape and size.

Examples of these are prions and amyloid proteins.

These remind us of the behavior of crystals in the formation of crystalline structures.

For this reason, they have played a fundamental role in biological evolution.

This ability to reproduce is a necessary but not sufficient condition for the formation of the extraordinary life forms known to us. Another molecular mechanism was needed to orchestrate the random reproduction of proteins, resulting in autonomous structures designed for reproduction and the necessary adaptations imposed by environmental conditions.

The necessary evolutionary step was the formation of RNA, capable of assembling functionally effective protein forms to carry out useful activities.

The symbiotic relationship between the two RNA-Protein molecular entities has proven to be effective and has been preserved for millions of years.

This system lasted until the revolution with DNA, a molecule capable of collecting and storing environmental information and translating it into phenotypes aimed at reproduction for survival.

To draw an analogy with a system that is more familiar and in continuous use to us, we could compare proteins to writing and language.

Here too, the building blocks are the letters of the alphabet, which consist of 21 characters, at least in Indo-European languages, arranged in succession. Each sentence is like a protein, which must follow logical rules to make sense. An incorrect arrangement of the letters leads to a lack of meaning, just as happens with proteins.

Just as happens with proteins, with the letters of the alphabet, or with the sounds of individual phonemes, it is possible to compose an infinite number of messages, which are exemplified in this article.

Even the gaps between one word and another find analogy in protein synthesis, during which the genetic codes of proteins are separated by stretches of DNA that are not transcribed, and are called introns.

But the analogy doesn't end there. Both in words and with proteins, the messages and forms they express in nature leave us amazed by their beauty and the multiplicity of forms and contents they can convey, making us feel alive and part of a wonderful, complex, and exciting experience, which, in this case, we cannot express in words.