Introduction

The term autophagy was introduced in 1963 at Rockefeller University by the Belgian scientist Christian de Duve (1917-2013), who examined and explained the mechanisms involved in cellular endocytosis, being awarded with Nobel Prize for Physiology and Medicine in 1974. Literally, autophagy means „self-eating”, while certain authors may refer to it as „auto-cannibalism”. The process of autophagy has recently gained increased attention, following intensive subsequent research by Japanese cell biologist Yoshinori sumi, ho also received the Nobel Prize for Medicine in 2016.

Autophagy is differentiated from proteasome-mediated degradation by its ability to remove whole intracellular organelles it can also remove aggregated protein complexes of large or non-selectively targeted substances. As a kind of cellular „self-feeding”, autophagy is the cellular process via which cells break down and recycle their internal components. Parts of the cytoplasm are trapped in cytosolic vesicles called autophagosomes, which have a double membrane. The autophagosome’s contents are transported to the lysosome, where they undergo degradation and recycling, re-entering the cytosol as amino acids. All eukaryotes engage in autophagy, which can be controlled in response to different nutritional shortages and becomes critical for survival in particular circumstances. Additionally, autophagy plays a significant role in preventing certain types of neurodegenerative diseases and cancer, as well as in eliminating invasive pathogens (Mizushima et al.,  2008).

To maintain intracellular homeostasis, protein synthesis and degradation must be balanced; specific environmental signals are translated by various regulatory mechanisms into adaptive responses for growth and development to maintain this balance. When it comes to deterioration, these reactions could be crucial for survival in a variety of stressful situations or for adjusting to particular nutrients (Longo and Anderson, 2022). Autophagy is also upregulated when cells prepare for structural reorganisation, like that which occurs during developmental transitions, or to eliminate detrimental cytoplasmic elements, like those formed after oxidative stress, infection, or protein aggregation buildup.

Nutritional status, hormonal variables, and other indicators like as temperature, oxygen concentrations, and cell density, all play a role in modulating autophagy. Adenosine monophosphate-activated protein kinase (AMPK), Protein Kinase B (Akt), Sirtuin 1 (SIRT1), and Mechanistic Target of Rapamycin Complex 1 (mTOR-1) are just a few of the signalling molecules that gracefully regulate the dynamic process of autophagy (Armstrong and Boggs, 2023). TOR is a key regulatory protein that, during the induction stage, controls the activity of a complex involving the Atg1 kinase. However, the specific target of Atg1 remains unknown.

Autophagy is typically thought to be non-specific; nevertheless, certain types of autophagy use proteins that bind and adjust, such Atg 11. It is unclear how Atg 11 ties the load to the autophagosome, a vesicle that sequesters. The process of forming the autophagosome is intricate, and the source of the donor membrane as well as the mechanism of vesicle creation are unknown. Well-characterized lysosomal / vacuolar proteases are necessary for the ultimate disintegration of the sequestered cargo; the functions of lipases, however, remain unclear, and it is not elucidated yet how the lysosome/vacuole membrane is kept intact throughout degradation (Longo and Anderson, 2022).

It may appear unlikely at first that cellular self-feeding might have any advantages. In its most basic form, autophagy reflects the adaptation of a single cell to starvation: in the absence of food in the environment, a cell must use some of its own reserves to sustain itself until conditions improve. For example, in organs such as the liver, autophagy is triggered every day or even every few hours to sustain metabolic processes that produce energy and amino acids via catabolism.

Even if there are in the recent period numerous studies investigating the molecular cascade that governs and executes autophagy, however, many uncertainties remain, despite the considerable progress in understanding the molecular basis of autophagy and identifying the proteins involved in its regulation.

The aim of this review is the revision of up-to-date information regarding the mechanism of autophagy, focusing on its role in protein metabolism and intracellular homeostasis.

Materials and Methods

Existing studies related to the mechanism of autophagy, its role in protein metabolism and intracellular homeostasis were gathered to discuss the currently available results. The literature research was carried out in the Scopus database, using initially the keyword “autophagy”. Then, the research was restricted to scientific papers focused on the molecular mechanism, using the keywords “autophagy” + molecular mechanisms intracellular homeostasis. The list of the publications was screened based on the title, authors, and year, and the rest of the studies was excluded. After identifying and screening, 8 research articles were selected.

Results and Discussions

As stated before, autophagy is a fundamental cellular process responsible for degrading and recycling intracellular components, playing a critical role in maintaining cellular homeostasis, responding to stress, and regulating various physiological functions such as metabolism, immunity, and aging.

Autophagy classification

Autophagy can be classified based on several criteria, namely the molecular mechanisms involved, the specificity of selectivity, and the cellular organelles or structures that are degraded and recycled.

i) Pathways of cellular maintenance and degradation

Autophagy can be non-selective or selective depending on the specificity with which cellular components are selected and degraded. Non-selective autophagy involves the general degradation of cellular components, while selective autophagy targets specific cellular organelles or structures, such as mitochondria, protein aggregates, or pathogens.

Non-selective autophagy occurs when a cell degrades its cellular components in a general manner, without regard to their specificity or state. It is a more general process of recycling cellular components and helps maintain  cellular homeostasis by eliminating damaged organelles or denatured proteins. An example of non-selective autophagy would be when a cell needs additional energy or when it faces a stressful environment, such as famine or oxidative stress. In these situations, non-selective autophagy can be activated to degrade and recycle portions of the cytoplasm and cellular organelles, thereby releasing basic molecules and essential nutrients to maintain cell function. It is as the cell is cleaning out unwanted deposits to ensure proper functioning.

In contrast, selective autophagy is a process in which the cell selectively degrades certain cellular components. In addition to the non-selective recycling of nutrients, autophagy degrades specific cargoes such as lipids (lipophagy), pathogens (xenophagy), protein aggregates (aggrephagy / proteophagy), the endoplasmic reticulum (reticulophagy / ER-phagy), mitochondria (mitophagy), peroxisomes (pexophagy), the nucleus (nucleophagy), and even lysosomes themselves (lysophagy).

This process is regulated by certain proteins and specific receptors and helps eliminate cellular components that pose a risk to cellular integrity and function. Selective autophagy in mammals is mediated by a number of  receptors, such as toll interacting protein (TOLLIP), sequestosome1 (SQSTM1), neighbour NBR1 (breast cancer type 1), optineurin (OPTN), FUN14 domain containing 1 (FUNDC1), prohibitin 2 (PHB2), reticulophagy regulator 1 (RETREG1), calcium-binding and coiled-coil domain 2 (CALCOCO2), and nuclear FMR1 interacting protein 1 (Meijer and Codogno, 2006).

Thus, selective autophagy is a more specific and targeted process, while non-selective autophagy is more general and involves the general recycling of cellular components. Both types of autophagy are essential for maintaining proper cellular health and function.

ii) Molecular mechanism

Based on the molecular mechanisms involved in the formation process and degradation of autophagosomes, autophagy can be classified in microautophagy, macroautophagy, and chaperone-Mediated Autophagy (CMA), which differ in their mechanisms and functions (Figure 1). While CMA selectively destroys only soluble proteins, micro- and macroautophagy can engulf huge structures using both selective and non-selective methods processes (Mizushima et al., 2008).

Mechanism, microautophagy, involves septating or invaginating the lysosomal/vacuolar membrane in order to sequester cytoplasm. The most common kind of autophagy, called macroautophagy, is characterised by the production of double-membraned cytosolic vesicles that encapsulate parts of the cytoplasm (Figure 1). The sequestering vesicles known as autophagosomes during macroautophagy are not produced from the lysosomal/vacuolar membrane (Klionsky and Emr, 2000). An inner vesicle known as an autophagic body is delivered into the degradation compartment lumen by the union of the finished autophagosome with a vacuole or lysosome. The cargo of the vesicular membrane can eventually degrade and be recycled into amino acids and other compounds due to the membrane’s subsequent breakdown. In contrast to the other two processes, chaperone-mediated autophagy is a secondary reaction to starvation that requires the direct translocation of specific proteins across the lysosomal membrane. There isn’t a recognised yeast counterpart for this process yet (Mizushima et al., 2008).

Mechanisms that trigger autophagy

Nutritional Stress. Cellular starvation or nutritional deficiencies can trigger autophagy to provide essential nutrients to cells and maintain energy homeostasis. Nutritional stress can significantly impact the autophagy process in cells. When the body is subjected to poor or excessive nutrition, the autophagy system is activated to compensate. In conditions of starvation, autophagy can provide essential nutrients by breaking down cellular organs and proteins to maintain homeostasis and ensure cell survival. On the other hand, in conditions of excessive nutrition, excessive activation of autophagy can be linked to various metabolic and inflammatory conditions. Therefore, a proper balance of autophagy in response to nutritional stress is essential for maintaining cellular health and body function (Longo and Anderson, 2022).

Oxidative stress. An increase in the level of reactive oxygen species (ROS) in cells can activate autophagy. This helps remove cellular components damaged or affected by oxidative stress.

DNA damage. Autophagy can be triggered in response to injuries or damage at the DNA level, thus contributing to the maintenance of genomic integrity.

Infections and inflammation. The presence of infections or inflammation in cells can activate autophagy to eliminate pathogens and limit excessive inflammatory responses.

Endoplasmic stress. Dysfunctions at the level of the rough endoplasmic reticulum (a cell component responsible for protein synthesis) can trigger autophagy to manage cellular stress.

Mitochondrial stress. Damage at the mitochondrial level can activate autophagy to remove damaged mitochondria, a process known as mitophagy.

Regulatory Mechanisms

Various processes can regulate autophagy. Microautophagy is a mechanism in which lysosomes actively consume cytosolic components by invaginating their membrane. Similar processes in fungi selectively degrade organelles, but the function of microautophagy in higher eukaryotes is unknown.

Macroautophagy is another type of autophagy that engulfs cellular cargo within a double-membrane vesicle known as an autophagosome. The autophagosome then unites with lysosomes, where the contents are destroyed and recycled.

Sequestration can be selective, focusing on payloads like organelles or invasive microorganisms, or non-specific, involving the engulfment of bulk cytoplasm (Shabkhizan et al., 2023). The phagophore expands to form the autophagosome, yet it is unclear where the membrane originated. Hydrolases are produced when the autophagosome fuses with a lysosome or an endosome (not shown). Membrane permeases facilitate the release of the resultant macromolecules back into the cytosol when the inner membrane of the autophagosome is lysed by the autolysosome and breaks down its contents. Chaperone-mediated autophagy (CMA) is a process in which the integral membrane receptor LAMP-2A (Lysosome-associated membrane protein type 2A) and the cytosolic and lysosomal chaperone protein hsc70 work together to directly translocate unfolded substrate proteins across the lysosomal membrane (Mizushima et al., 2008).

Severe degradation is crucial to autophagic activity, but it brings some danger because unchecked cytoplasmic degradation can be fatal. However, basal autophagy levels are necessary to preserve regular cellular homeostasis. Because of this, it’s critical that autophagy be strictly controlled (Figure 2), triggered when needed, but otherwise kept at a low level. Many facets of the control of autophagy have been discussed in recent reviews, despite the lack of a comprehensive picture.

Figure 2. A shortened framework for autophagy regulation.

Autophagy in survival and death cells

In situations, such as microbial infection, efficiency of nutrients and growth factors, and illnesses marked by the buildup of protein aggregates, have been shown to highlight autophagy’s pro-survival role at both the cellular and organism levels. Although this pro-survival role is usually thought of as adaptive, it might not be the case in cancer. A prevalent characteristic of the tumour microenvironment is metabolic stress, which is also induced in cells by most of chemotherapy drugs. Therefore, whether autophagy-based survival should be inhibited under certain circumstances, to encourage tumour cell death, is a topic that is being thoroughly researched (DiLoreto and Murphy, 2015).

Complex interactions occur between the autophagy process, which is mostly involved in cell survival, and the apoptosis pathway, which always results in cell death. The two routes have common components, are regulated by similar circumstances, and each has the ability to control and alter the other’s activity. Autophagy is induced by many signals that were firstly investigated in relation to apoptosis activation, while autophagy is inhibited by signals that block apoptosis. Pro-apoptotic factors, including BH3-only proteins, break this inhibitory connection and trigger autophagy. Anti-apoptotic proteins, like those in the Bcl-2 family, inhibit Beclin proteins. The finding that Atg5 can be cleaved by calpain to produce a pro-apoptotic component that participates in the intrinsic mechanism of mitochondrial death provides another connection between autophagy and apoptosis. These are just a few examples, as our understanding of the intricate chemical relationship between autophagy and apoptosis is still developing. Nonetheless, it appears likely that several features tissue homeostasis, and disease pathogenesis are governed by the methodical control of „self-digestion” through autophagy and „self-killing” through apoptosis (Mizushima et al., 2008).

Adaptive and innate immunity

Like cellular organelles, the removal of intracellular organisms presents a steric barrier for cellular breakdown mechanisms, one that autophagy is ideally suited to meet. The process known as xenophagy involves the selective transport of microbes to lysosomes using similar autophagic machinery that is utilised to capture certain cellular components. Autophagy has a likely broader function in antimicrobial defence in mammals (and possibly other metazoan animals) than only eliminating infections directly. Autophagy plays a part in both preserving lymphocyte homeostasis and supplying the innate and adaptive immune systems’ antigenic material from microbes.

Targets of xenophagy include newly synthesised virions when they leave the nucleus through the cytoplasm, parasites and bacteria living inside vacuoles, phagosomes, or the cytosol harbouring pathogens, and external bacteria that infiltrate intracellularly. The xenophagy’s cell biology is not as well understood as that of conventional autophagy. Although the molecular components needed for both processes are comparable, it is still unclear if the biogenesis of the membranes that engulf microorganisms differs or is similar from that of classical autophagosomes. The autophagic process’s ability to absorb microorganisms larger than its own organelles is demonstrated by the fact that LC3-positive compartments containing pathogens can be significantly larger than classical autophagosomes formed entirely of cellular constituents. This ability shows the distinct autophagosome formation mechanism (Figure 1). Little
is known about the process by which bacteria (or the membrane compartments hosting microbes) are chosen for autophagy, even though xenophagy seems to be essentially a selective type of autophagy.

Beyond its direct involvement in the removal of pathogens, autophagy has the ability to mediate the trafficking steps required for both innate and adaptive immunity as well as to have cytoprotective effects in infected cells. Autophagy is required in some RNA viral infections for the viral nucleic acids to reach the endosomal TLR7 receptor and activate type I interferon signalling. Certain endogenously synthesised viral antigens are also presented on the class II Major Histocompatibility Complex (MHC) by means of autophagic machinery. In addition to its role in both adaptive and innate immunity, the autophagic process is also stimulated by several innate and adaptive immune mediators that are involved in the management of intracellular pathogens (Mizushima et al., 2008).

Aging and longevity

Even if there are no mutations that lead to pathogenic cells, such as mutant proteins prone to aggregation, the buildup of damaged proteins and organelles is a common characteristic of all ageing cells (Figure 3). Differentiated cells that do not divide, such as neurons and cardiomyocytes, are particularly vulnerable to these deposits of altered components because their typical aging-related functional decline usually occurs earlier than in other types of cells.

Figure 3. Autophagy and aging.

Both macroautophagy and CMA (chaperone-mediated autophagy) activities decrease with age. The dramatic changes observed in the phenotypes of recently created genetically modified mice, where autophagy function is impaired, suggest that a gradual decline in autophagic activity as organisms age could play a significant role in the functional decline observed in aging organisms (Longo and Anderson, 2022).

In contrast, caloric restriction, the only known intervention for slowing aging, appears to increase autophagy induction, presumably due to reduced insulin levels, which hinder autophagy (Figure 3). Current initiatives to avoid or reverse the loss in age-related macroautophagy focus on replicating the positive effect of caloric restriction using anti-lipolytic medications (which simulate the sense of hunger caused by caloric restriction) (DiLoreto and Murphy, 2015).

In nearly all tissues of aging species, ranging from nematode worms to mammals, defective lysosomal function has been observed. Additionally, the activities of macroautophagy and chaperone-mediated autophagy (CMA) decrease with age. Age-related functional loss in macroautophagy is partly explained by problems with its regulation and execution. Older organisms lose the ability to regulate macroautophagy through elevated blood glucagon levels (i.e., between meals). This may be because the insulin receptor’s basal activity is constantly boosted responding to the ageing cells’ increased levels of reactive oxygen species concentration.

Furthermore, the build-up of lipofuscin, an undigested substance, within autolysosomes and other secondary lysosomes hinders their capacity to combine with autophagosomes and break down their cargo. The CMA receptor becomes unstable as we age because of modifications to the lysosomal membrane. The lower CMA activity in older organisms is caused by an age-dependent drop in this receptor’s levels. Some expected effects of the aging-related decrease in autophagy include a weak stress response, an ineffective way of eliminating damaged components, and a possible way of causing disease (Mizushima et al., 2008).

Regulatory process of the autophagy

Sirtuins, a special class of enzymes, are essential for many vital cellular functions, including survival, metabolism, division, aging, programmed cell death (apoptosis), and DNA repair. These enzymes are activated by nicotinamide adenine dinucleotide (NAD+), a coenzyme involved in redox reactions at the cellular level. There are seven types of sirtuins in mammals (SIRT 1-7), grouped into four distinct classes, SIRT1-3, which are involved in deacetylation, belong to Class I. SIRT1 shuttles between the nucleus and the cytoplasm, SIRT2 resides in the cytoplasm, while SIRT3 is found in both the nucleus and the mitochondria.

SIRT4, which predominantly functions as an ADP-ribosyl transferase in the mitochondria, belongs to Class II. SIRT5 and SIRT6, on the other hand, belong to Class III and provide a variety of roles. SIRT5 is located in the mitochondria and serves as a deacetylase, demalonylase, and desuccinylase. SIRT6 is located in the nucleus and regulates nuclear ADP-ribosylation and deacetylation. Finally, Class IV includes SIRT7, a deacetylase that plays a role in the nucleus’ RNA polymerase I transcription machinery (Kim et al., 2022).

Among these, sirtuin 1 (SIRT1) is the largest and the most extensively studied, playing a crucial role in various cellular processes. SIRT1 is composed of 11 exons and 33,715 base pairs, located on chromosome 10q21.3 (Kim et al., 2022).

SIRT1 regulates autophagy. SIRT1 influences this complex process at multiple levels, interacting not only with proteins directly involved in autophagy (known as ATGs) but also with various regulators that coordinate different phases of autophagy: initiation, expansion, maturation, fusion with lysosomes, and final degradation.

SIRT1 controls stability when autophagy starts (Figure 4). Through inactivating the brain-enriched Ras homolog (Rheb), which triggers mechanistic Target of Rapamycin Complex 1 (mTORC1), Tuberous Sclerosis Complex 2 (TSC2) will inhibit the mTORC1 signalling pathway. When Rheb binds to GTP, it activates mTORC1, and GAP (the TSC2 GTP activating protein) promotes GTP hydrolysis. TSC2’s enhanced stability will decrease mTOR signalling and start the autophagy process when it improves GTP hydrolysis to inactivate Rheb. The deacetylase activity of SIRT1 prevents the ubiquitin-mediated destruction of the TSC2 complex, maintaining its stability in the absence of nutrition.

Figure 4. SIRT1 influences on the initiation of autophagy.

When faced with a lack of food, the deacetylase enzyme activity of SIRT1 intensifies, contributing to greater stability of the TSC2 protein. This facilitates the breakdown of GTP by Rheb, a crucial step in stopping the signaling of the mTOR pathway, which is essential for controlling cell growth and metabolism. On the other hand, in situations of oxygen deficit (hypoxia), SIRT1 acts on FOXO3 proteins, through deacetylation, making them bind to the control sections (promoters) of the BNIP-3 gene. This leads to an increase in the production of BNIP-3, an important factor in activating autophagic processes, through which cells recycle their damaged or unnecessary components to survive in unfavourable conditions (Longo and Anderson, 2022).

Additionally, recent research has shown that, under hypoxic conditions, SIRT1 moderates the initiation of autophagy, which is mediated by BNIP-3 (Kim et al., 2022). SIRT1 deacetylates the nuclear protein FOXO3, thus facilitating the binding of FOXO3 to the BNIP-3 promoter and inducing BNIP-3 expression. BNIP-3 expression stimulates autophagy by promoting the separation of Beclin1 from the Bcl-2-Beclin1 complex. Activated Beclin1 triggers the PI3K complex necessary for the initiation of autophagy. SIRT1 and autophagy have a reciprocal relationship that shows that SIRT1 controls autophagic activity and that autophagy can affect SIRT1 levels by degrading SIRT1 in lysosomes (Kim et al., 2022).

Depending on the cellular state, autophagy can adjust SIRT1 levels. Dysfunction of the autophagic process disrupts normal metabolism, which can lead to the emergence of endocrine disorders. Thus, current studies indicates that disturbances in the SIRT1-regulated autophagy process can contribute to the occurrence of obesity, type 2 diabetes, and diabetes-associated cardiomyopathy (Meijer and Codogno, 2006).

Conclusions

In conclusion, autophagy plays a critical role in maintaining cellular homeostasis and is intricately regulated by conserved signalling pathways such as mTOR, AMPK, Akt, and SIRT1. Dysfunctions in autophagy are closely associated with various diseases, including neurodegenerative disorders, cancer, and aging, underscoring its significance in health and disease. Ongoing research in autophagy has the potential to uncover novel therapeutic targets and improve treatment strategies for these conditions.

Understanding and manipulating autophagic processes could provide significant advancements in promoting health, preventing disease, and extending lifespan. Therefore, continuing studies on autophagy is essential for progress in understanding cellular biology and for developing more precise and effective therapeutic interventions.