ACCUMULATION OF INTRACELLULAR PROTEIN ADVANCED GLYCATION END PRODUCTS ALTERS CELLULAR HOMEOSTASIS FOR PROTEIN CLEARANCE IN PANCREATIC DUCTAL EPITHELIAL CELLS

Abstract

Protein advanced glycation end products (AGEs) can be formed via non-enzymatic glycation and accumulated intracellularly to disrupt cellular homeostasis for protein clearance. Here, we investigated the formation particulars of intracellular protein AGEs and sought to elucidate the molecular events implicated in impacting cellular clearance systems. The formation and accumulation of intracellular protein AGEs increased protein aggregation and protease resistance, potentially overwhelming the ubiquitin-proteasome system (UPS). At high levels of protein AGEs, the abundance of many E3 ligases decreased, and the overall ubiquitination level was reduced, all of which indicated a decreased UPS activity. On the other hand, autophagy activity was stimulated, as evidenced by the upregulation of autophagy marker LC3II and important proteins in autophagosome and autolysosome formation, as well as downregulation of mTOR. Understanding the functional impacts of intracellular protein AGEs on the UPS and autophagy could pave the way for the future development of pharmaceutical agents targeting AGE-related diseases.

Graphical Abstract

Protein glycation and the formation of advanced glycation end products (AGEs) are widely studied for their implications in diseases and health¹. Protein AGEs are formed and accumulated in the body through increased intake of sugar-rich or dicarbonyls-rich foods, cigarette smoking, or due to certain comorbidities, such as diabetes^{2, 3}. The formation of protein AGEs generally occurs on lysine (K) and arginine (R) of the proteins⁴, and can take weeks to months in physiological conditions⁵. The formation and accumulation of protein AGEs can impair protein structure and function, alter cellular homeostasis, and predispose individuals to develop pathological conditions that lead to various diseases, including cancer^{1, 2}. The ubiquitin-proteasome system (UPS) and autophagy are two major cellular mechanisms to clear impaired proteins and recycle cellular components for maintaining cellular homeostasis⁶. As a consequence, the formation and accumulation of protein AGEs may trigger the UPS- and autophagy-mediated protein degradation^7-9.

Most of the cellular protein degradation occurs through the proteasome, which consists of proteolytic enzymes that rapidly cleave single, soluble-misfolded and regulatory proteins while the degradation of the bulk of cellular proteins is slower. This process is initiated by the sequential addition of ubiquitin chains to the proteins^{10, 11}. These proteins are ubiquitinated and transferred to the proteasome with the involvement of E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligase enzymes⁶. The proteolytic activities in proteasome occur in the β subunits, with three main proteolytic activities as follows: (I) β1 for caspase-like activity, (II) β2 for trypsin-like activity, and (III) β5 for chymotrypsin-like activity¹². With our attention to glycated proteins in this study, highly glycated proteins tend to aggregate and demonstrate resistance to proteases¹³ and can undergo autophagy mediated degradation¹¹. Macroautophagy (herein autophagy) has been widely studied among other two types of autophagy (microautophagy and chaperon mediate autophagy). During the autophagy process, the double membrane vesicle (autophagosome) engulfs the cargo molecules, and this cargo-trapped autophagosome is transported and fused with late-endosomes and lysosomes. The lysosomal hydrolases degrade these cargo molecules and provide building blocks (amino acids) for synthesizing new macromolecules (proteins)⁶. Currently, limited data is available in describing the molecular events implicated in regulating the UPS and autophagy upon the formation and accumulation of intracellularly formed protein AGEs, while most reported studies suggested that stimulation of receptor for advanced glycation end products (RAGE) might be a driver to enhance autophagy activities^{14, 15}.

In our previous study, we demonstrated that intracellularly formed protein AGEs in pancreatic ductal epithelial cells induced proteome impairment and cellular stress as indicated by increased lactose dehydrogenase activity, reactive oxygen species production, expression of RAGE and an elevated ER stress¹⁶. In this study, we further investigated the formation particulars of intracellular protein AGEs under mimic conditions of hyperglycemia or high exposure to reducing sugars or dicarbonyls using an accelerated cellular model. We sought to elucidate the molecular details implicated in the accumulation of intracellular protein AGEs and how it impacts UPS activity and autophagy in attempting to balance cellular homeostasis through clearance and recycling.

Glyceraldehyde (GA) is an important intermediate in sugar metabolism and a key source of toxic AGEs, including: glyceraldehyde-derived AGE (GLAP), argpyrimidine (ArgP), and methylglyoxal-derived hydroimidazolone 1 (MG-H1)¹⁷. Our mass spectrometric analysis showed a dose-dependent increase in the accumulation of intracellular protein AGEs in GA-treated cells. The formation of AGEs was substantially more noticeable on K compared to R residues, especially at the higher GA-doses (Figure 1A). This observation was consistent with the analysis of protein AGEs in human plasma samples (Figure 1B). Glycation motif analysis (GMA) suggested that physiochemical properties of the neighboring amino acid of a glycation site could influence the prevalence of glycation occurrence. As shown in Figure 1C, the neighboring amino acids of the glycated residues were prominently acidic (e.g. glutamic acid) and hydrophobic (e.g. alanine and leucine). These factors may assist to distinguish potential glycation sites that are more vulnerable to be influenced by exogenous sources or disease conditions from those that are less likely to be glycated.

Figure 1.

Formation of intracellular protein AGEs and their direct impacts on protein aggregation and protease resistance. (A) GA-mediated formation of intracellular protein AGEs. (B) Mass spectrometric analysis of plasma proteins derived from healthy individuals. (C) Bioinformatic prediction of neighboring amino acids of glycated sites, revealing neighboring regions that are mostly acidic. (D) Aggregation prediction analysis, revealing the overlap of neighboring regions of glycation sites with the in-silico aggregation hits. Red: glycation site-neighboring region and in-silico aggregation hits, green: in-silico aggregation hits, and purple: glycation sites. (E) Protease resistance is induced by the formation of intracellular protein AGEs.

Protein aggregation and protease resistance induced by the formation and accumulation of protein AGEs are implicated in the etiologies of many diseases^{1, 2, 13, 18}. We found that most of the neighboring regions of the glycation sites overlapped with in silico aggregation hits, as shown in several exemplified proteins in Figure 1D. Our protease-resistant analysis (Figure 1E) revealed that, in comparison to the untreated cells (lane 2), formation of protein AGEs caused the proteins to be difficult to separate by the SDS-Gel, which appeared to be smeared and blurry (lane 4). As a consequence, the glycated proteins were resistant to trypsin digestion, resulting in more “peptide” bands with high molecular masses remaining in the SDS-Gel (lane 5) compared to the untreated control (lane 3). Similar results were observed in glycated human serum and albumin protein (Figure S1).

Additionally, because of protein aggregation and potential changes to the native structure, the accessibility of proteasomal enzymes could be challenging. In our study, the proteasome activity decreased as the glycation increased, and a significantly lower proteasome activity was observed at 4mM GA treatment (Figure 2A). We further evaluated ubiquitination levels of proteins after the GA treatment. The overall ubiquitination was slightly increased at 1mM treatment (low glycation level), but significantly reduced at 4mM treatment (high glycation level) compared to the control (Figure 2B). Furthermore, our previous proteomic study¹⁶ and the current study have empirically demonstrated that AGEs modification is prominent in K. As such, protein AGEs conjugated in K might negatively influence the intrinsic ubiquitination, which also occurs on K of target proteins^{6, 19} and is critical in regulation of protein degradation in UPS. As we observed a decrease in both ubiquitination and proteasome activity at high glycation levels, we further carried out proteomic analyses to evaluate the abundant changes in E3 ligases. Notably, many of the E3 ligases were downregulated in cells treated with higher doses of GA, especially starting at >2 mM dose (Figure 2C). As an example of LC-MS/MS analysis, the elution profile of peptide ALLDASETTSTR derived from an E3 ligase, TRIM25, was presented in Figure 2D. Furthermore, western blot was performed on two E3 ligases, TRIM25 and STUB1(CHIP), for validating the mass spectrometric data, and their expressions were downregulated as the treatment doses increased (Figure 2E, 2F, and 2G).

Figure 2.

Decrease in UPS activity and downregulation of multiple E3 ligases induced by the formation of intracellular protein AGEs through GA treatment. (A) Proteasome activity of cells. (B) Western blot (WB) of ubiquitination. (C) Proteomic data of E3 ligases. (D) Exemplification of MS analysis of E3 ligases - the elution profile of peptide ALLDASETTSTR from TRIM25 E3 ligase. (E) WB analysis performed on TRIM25 and STUB1in cells treated with GA. (F) Quantitative expression of TRIM25. (G) Quantitative expression of STUB1. Data represent mean ± SE (n=3) with statistical significance *P<0.05, **P<0.01, ***P<0.001 and ****P< 0.0001.

The UPS-mediated degradation is mostly occurred for short-lived and soluble-misfolded proteins, while autophagy is activated for the degradation of long-lived insoluble protein aggregates⁶. A study conducted on investigating the intracellular protein clearance mechanisms of GA-derived AGEs suggests that UPS and p62 may play a vital role in the degradation of protein AGEs at the early stage of AGEs formation, while high molecular-weight protein aggregates cannot be degraded by the proteasome pathway²⁰. When the UPS was overwhelmed by the accumulation of protein AGEs, autophagy and other detoxification activities could be triggered to balance cellular homeostasis through clearing and recycling impaired proteins^{7, 8}. We observed that the autophagy marker LC3II was gradually induced in cells treated with GA with increasing levels of protein glycation (Figure 3A and 3B). The downregulation of UPS was most prominent with 4mM GA treatment where the autophagy was also induced significantly, likely due to the diminished function of UPS at a high level of protein glycation. Ultimately, these events led to a shift in the machinery of protein clearance from UPS to autophagy in response to the overwhelmed UPS caused by the accumulation of protein AGEs.

Figure 3.

Upregulation of autophagy activity and glyoxalase enzymes. (A) WB analysis showing upregulation of autophagy marker LC3-II induced by formation of intracellular protein AGEs. (B) Quantitative expression of autophagy marker -LC3-II. (C and D) Expression of autophagy-related proteins at proteomic (C) and RNA levels (D). (E and F) Expressions of AGEs detoxifying enzymes at proteomic (E) and RNA levels (F). (G) Expression of sialytransferases at RNA level, ST3GAL: ST3 beta-galactoside alpha-2,3-sialyltransferase, ST6GAL: ST6 beta-galactoside alpha-2,6-sialyltransferase, ST6GALNAC: ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase, ST8SIA: ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase. Data represent mean ± SE (n=3) with statistical significance *P<0.05, **P<0.01 and ***P<0.001.

Moreover, our data revealed the downregulation of mTOR with GA treatment as an indication of induction of autophagy (Figure 3C and 3D). mTOR is a master regulator of cellular metabolism with an inhibitory effect on autophagy⁶. Alternatively, molecules (at protein and/or mRNA levels) that are important in autophagosome and autolysosome formation, such as ATG12, LAMP1, VAMP8, and STX7^{21, 22} were upregulated with GA treatments (Figure 3C and 3D). Functional annotations of proteomic and RNA data revealed that genes responsible for oxidative phosphorylation were upregulated by GA treatment (Figure S2D and S3D). The common genes under this category are mainly ATP synthase. This is a notable indication that autophagy is favored as it consumes a lot of energy/ATP for the initiation, and progression of autophagosomes and maintains the lysosomal pH²³. Besides HPDE, autophagy marker LC3II was also upregulated in pancreatic cancer cells PANC1 and MIA-PaCa2 treated with GA to induce glycation (Figure S4). Notably, while it is important to consider that proteins in growth media may undergo glycation and potentially influence AGE-RAGE signalling to promote autophagy^{14, 15}, our data suggests that the systemic effect of protease resistance due to intracellular protein glycation and the resulting diminished UPS activity could be a major contributor to the shift in the machinery of protein clearance from UPS to autophagy.

In addition, the glyoxalase system also acts as a defence mechanism to reduce the AGEs accumulation. Glyoxalase 1, which detoxifies the methylglyoxal precursor of AGEs²⁴, was upregulated at both proteomic and RNA levels (Figure 3E and 3F). Deglycase enzyme DJ-1(PARK7), which deglycates the early glycation²⁵, was upregulated at the RNA level (Figure 3F). This detoxification system forms an additional defence line to inhibit certain protein AGEs via specific enzymatic-driven reactions. Interestingly, the accumulation of protein AGEs also resulted in the downregulation of many sialyltransferases at the transcriptional level (Figure 3G), supported by the GO annotation that transferase activity was highly enriched among the downregulated genes after GA treatment (Figure S3C). These observations may imply that glycation and formation of protein AGEs can negatively affect enzymatic-driven glycosylation²⁶ and thereby disturb protein stability due to improper formation of glycan structures.

Our data elucidated protein AGE formation and their direct impacts on cellular clearance systems, including the UPS and autophagy. The intracellular protein AGEs increase protein aggregation and protease resistance, potentially overwhelming the UPS. Due to the proteome-wide systemic impact of protease resistance and reduced UPS activity, we speculate that a feedback mechanism may be triggered to downregulate the abundance of many E3 ligases. This occurs as many impaired, aggregated proteins are not directed to the proteasome for degradation (Figure 4).

Figure 4.

A model to show how intracellular glycation and protein AGEs can potentially impair the cellular homeostasis for protein clearance. Under normal physiological conditions, intracellular proteins are comparatively less glycated, most of which are ubiquitinated and degraded by proteasome with normal UPS activity. However, at higher levels of protein AGEs, protein aggregation and protease resistance can compromise UPS activity with downregulated E3 ligases and ubiquitination level, leading to the shift in the machinery of protein clearance from UPS to autophagy.

Understanding the impacts of protein AGEs on UPS and autophagy could lead to the development of pharmaceutical agents targeting AGE-related diseases. This knowledge may offer new insights into the etiologies of these diseases, including cancer, and inform novel strategies to reduce the formation and accumulation of toxic protein AGEs for disease prevention and early intervention.