Protein degradation and aging

Continuous turnover of intracellular proteins is essential for the maintenance of cellular homeostasis and for the regulation of multiple cellular functions. The first reports showing a decrease in total rates of protein degradation with age are dated more than 50 years ago, when the major players in protein degradation where still to be discovered. The current advances in the molecular characterization of the two main intracellular proteolytic systems, the lysosomal and the ubiquitin proteasome system, offer now the possibility of a systematic search for the defect(s) that lead to the declined activity of these systems in old organisms. We discuss here, in light of the current findings, how malfunctioning of these two proteolytic systems can contribute to different aspects of the phenotype of aging and to the pathogenesis of some age-related diseases.     

Glycan degradation promotes macroautophagy

Macroautophagy promotes cellular homeostasis by delivering cytoplasmic constituents to lysosomes for degradation [Mizushima, Nat. Cell Biol. 20, 521–527 (2018)]. However, while most studies have focused on the mechanisms of protein degradation during this process, we report here that macroautophagy also depends on glycan degradation via the glycosidase, α-l-fucosidase 1 (FUCA1), which removes fucose from glycans. We show that cells lacking FUCA1 accumulate lysosomal glycans, which is associated with impaired autophagic flux. Moreover, in a mouse model of fucosidosis—a disease characterized by inactivating mutations in FUCA1 [Stepien et al., Genes (Basel) 11, E1383 (2020)]—glycan and autophagosome/autolysosome accumulation accompanies tissue destruction. Mechanistically, using lectin capture and mass spectrometry, we identified several lysosomal enzymes with altered fucosylation in FUCA1-null cells. Moreover, we show that the activity of some of these enzymes in the absence of FUCA1 can no longer be induced upon autophagy stimulation, causing retardation of autophagic flux, which involves impaired autophagosome–lysosome fusion. These findings therefore show that dysregulated glycan degradation leads to defective autophagy, which is likely a contributing factor in the etiology of fucosidosis.

Maintaining the fidelity of cellular constituents is critical for cell viability and organismal health. Autophagy (literally “self-eating”) is a group of catabolic processes that deliver cytoplasmic constituents to lysosomes for degradation (1). The best-studied form of autophagy is macroautophagy, which is characterized by specialized organelles called autophagosomes that facilitate the delivery of cargoes destined for degradation (1).Macroautophagy is initiated by the formation of a membranous structure termed an isolation membrane, which forms from a variety of sources within the cell (2–5). Two ubiquitin-like conjugation systems then grow this membrane via the action of evolutionarily conserved autophagy-related (ATG) proteins. The growing double membrane finally fuses to form the ball-like autophagosome, which encapsulates cargoes including damaged/misfolded proteins and organelles. Autophagosomes can then fuse with endosomes to form amphisomes, but ultimately fusion occurs with lysosomes to form autolysosomes, within which the cargo of the autophagosome is degraded by lysosomal hydrolases. Finally, the degraded constituent parts—including amino acids, lipids, sugars, and minerals—are then recycled into the cytoplasm, where they are used in biosynthetic pathways or, in some cases, further catabolized to generate ATP (6). Since macroautophagy is a major mechanism for the degradation of long-lived proteins and the only mechanism to degrade organelles described so far, perturbation of autophagy can lead to a variety of diseases, including neurodegenerative diseases, inflammatory diseases, diabetes, and cancer (7).Glycosylation is essential for the correct functioning of cellular machinery. The importance of this process is exemplified by the fact that ∼50% of all proteins are glycosylated and over 50 diseases involve deregulated glycosylation (8, 9). Glycosylation of proteins is complex but occurs via two major pathways: N-linked glycosylation, in which glycans are attached to asparagine residues, and O-linked glycosylation, in which glycans are attached to either serine or threonine. In both types, glycans are attached to proteins by glycosyltransferases and, conversely, glycans are removed by glycosidases.The lysosomal enzyme α-l-fucosidase 1 (FUCA1, EC 3.2.1.51) is critical for the degradation of N-linked glycans. FUCA1 cleaves fucose from both the terminal branches of glycans, as well as the moiety known as “core fucose” on N-glycans, which is linked to the N-acetylglucosamine that is attached to asparagine in the peptide backbone (Fig. 1A) (10–13). Cleavage of core fucose is an essential event for further degradation of the glycan, and therefore FUCA1 is critically important for the breakdown of the glycan component of N-linked glycoproteins (12, 14).Open in a separate windowFig. 1.Generation of a mouse model of fucosidosis. (A) Schematic showing that FUCA1 cleaves both terminal fucose and core fucose linked to an N-acetylglucosamine (purple diamond: N-acetylneuraminic acid; yellow circle: galactose; blue square: N-acetylglucosamine; green circle: mannose; red triangle: fucose). (B) Schematic representation of the Fuca1-targeted allele and the mutated allele after Cre recombination. (C) Relative Fuca1 mRNA expression from different organs was determined by qRT-PCR (three mice per genotype). Results are represented as mean ± SEM (two-way ANOVA with Bonferroni correction. Fuca1^+/+ vs. Fuca1^+/− or Fuca1^−/− **P < 0.01. Fuca1^+/− vs. Fuca1^{−/− $$}P < 0.01). (D) FUCA1 activity was measured from lysates of the indicated organs (five mice per genotype, mean ± SEM) and expressed as arbitrary units (a.u.) per microgram of protein (Kruskal–Wallis test; *P < 0.05, **P < 0.01). (E and F) Organ sections of both Fuca1^+/+ (Upper) and Fuca1^−/− (Lower) mice were stained with H&E (E) and AAL (F). Red arrows indicate cytoplasmic vacuolation, purple arrows indicate Purkinje cell loss, black arrows indicate accumulation of glycan species in the specified tissues. Pictures were taken using a Zeiss AX10 microscope with a 40× objective. The images shown are representative of changes observed in six mice (aged between 90 and 220 d). (Scale bars, 20 µm.) Insets are magnified (2×) crops from the same images to show specific staining.The importance of FUCA1 is exemplified by mutations in the gene, which lead to the congenital lysosomal storage disorder fucosidosis (15). Individuals carrying two mutated alleles of FUCA1 often have no fucosidase activity and have lysosomal accumulation of glycans in multiple tissues. As a result, fucosidosis patients develop multiple pathologies, including neurodegeneration, growth retardation, impaired immunity, and visceromegaly. The majority of patients with fucosidosis die before 30 y of age (16, 17).Due to the large proportion of proteins that are glycosylated, we postulated that macroautophagy must be dependent not only on enzymes that degrade polypeptides, but also on those that degrade glycans. To test this, we generated mice that are deficient in FUCA1 and examined the impact on macroautophagy. These experiments clearly revealed that fucosidase activity promotes both autophagosome–lysosome fusion as well as the turnover stage of macroautophagy, and that modulation of autophagy has a significant impact on the accumulation of glycans associated with fucosidosis.     

Disorders of glycoprotein degradation

Summary The intracellular degradation of glycoproteins occurs predominantly in the lysosomes through the concerted action of proteases and glycosidases. Genetic defects in any of the enzymes cleaving the oligosaccharide side chains lead to specific diseases because of an excessive lysosomal accumulation of partially degraded material, mostly oligosaccharides.This paper presents an overview of the biochemistry and the clinical spectrum of this group of diseases including sialidosis, galactosialidosis,-and-mannosidosis, fucosidosis, aspartylglucosaminuria, and-N-acetylgalactosaminidase deficiency (Schindler disease). In addition, the sialic acid storage disorder (Salla disease) which is caused by a defect in the lysosomal transport of this acidic monosaccharide is included because of functional and clinical correlations.     

Crystal-induced inflammatio and cartilage degradation

In the past year, there have been advances in our understanding of the induction of cartilage damage by calcium-containing crystals. Mechanisms of deposition and the biologic effects of crystals have been further characterized, as has the interaction between crystals and leukocytes. Studies of the clinical diagnosis of crystal deposition diseases suggest that accuracy with microscopy needs to be enhanced. Normal val ues for serum NTPPPHase have been established and optimal diagnostic imaging strategies for calcium pyrophosphate deposition disease have been suggested. There are still no available drugs to inhibit deposition or effect reabsorption of calcium-containing crystals.     

Versican degradation and vascular disease

Versican is an abundant proteoglycan in the blood vessel wall that is increased after vascular injury and accumulates in advanced atherosclerotic plaques. Versican is a large molecule with domains that mediate binding to cytokines, enzymes, lipoproteins, other extracellular matrix molecules, and signaling receptors. There is evidence that versican exists in the normal, as well as the diseased, vessel wall as discrete fragments, which represent these functional domains. We review the literature on versican degradation in vascular tissue and the function of versican domains, all of which suggest that proteolytic modification of versican may have physiologic as well as pathologic implications for the vascular system.     

Lignin degradation in wood-feeding insects

The aromatic polymer lignin protects plants from most forms of microbial attack. Despite the fact that a significant fraction of all lignocellulose degraded passes through arthropod guts, the fate of lignin in these systems is not known. Using tetramethylammonium hydroxide thermochemolysis, we show lignin degradation by two insect species, the Asian longhorned beetle (Anoplophora glabripennis) and the Pacific dampwood termite (Zootermopsis angusticollis). In both the beetle and termite, significant levels of propyl side-chain oxidation (depolymerization) and demethylation of ring methoxyl groups is detected; for the termite, ring hydroxylation is also observed. In addition, culture-independent fungal gut community analysis of A. glabripennis identified a single species of fungus in the Fusarium solani/Nectria haematococca species complex. This is a soft-rot fungus that may be contributing to wood degradation. These results transform our understanding of lignin degradation by wood-feeding insects.     

Investigation of polymer electrolyte membrane chemical degradation and degradation mitigation using in situ fluorescence spectroscopy

A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO(2) nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO(2) to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO(2) into the catalyzed membrane provided an eightfold reduction in ROS generation rate.     

Synthesis and degradation of methylated proteins of mouse organs: Correlation with protein synthesis and degradation

L-(Methyl-¹⁴C)-methionine was administered i.p. to mice, and the incorporation of radioactive methionine into proteins and methyllysine and methylarginine residues formed by the transfer of the methyl-¹⁴C group of methionine were measured.Tissue protein was actively methylated in organs having a high activity of protein synthesis, and the in vivo methylating activity in organs was not correlated with the protein methylating activity of the organs determined in vitro. Puromycin inhibited both protein synthesis and protein methylation in mouse organs to a similar degree. Neither the formation of S-adenosyl-(methyl-¹⁴C)-methionine nor protein methylase was inhibited by puromycin. The data suggests that proteins are methylated immediately after protein synthesis, that is, newly synthesized proteins are the substrates of protein methylation.Radioactive methionine and the [C¹⁴] methyl groups of methyllysine and methylarginine residues of tissue proteins are degraded in parallel over a period of 3 wk, suggesting that protein methylation is an irreversible type of protein modification.     

Cartilage destruction by matrix degradation products

Abstract

The progressive destruction of articular cartilage is one of the hallmarks of osteoarthritis and rheumatoid arthritis. Cartilage degradation is attributed to different classes of catabolic factors, including proinflammatory cytokines, aggrecanases, matrix metalloproteinases, and nitric oxide. Recently, matrix degradation products generated by excessive proteolysis in arthritis have been found to mediate cartilage destruction. These proteolytic fragments activate chondrocytes and synovial fibroblasts via specific cell surface receptors that can stimulate catabolic intracellular signaling pathways, leading to the induction of such catalysts. This review describes the catabolic activities of matrix degradation products, especially fibronectin fragments, and discusses the pathologic implication in cartilage destruction in osteoarthritis and rheumatoid arthritis. Increased levels of these degradation products, found in diseased joints, may stimulate cartilage breakdown by mechanisms of the kind demonstrated in the review.