Ubiquitin Proteasome Pathway–Mediated Degradation of Proteins: Effects Due to Site-Specific Substrate Deamidation

With the world's population aging and incidences of age-related diseases taking heavy tolls on personal life quality and health care budgets, there is increasing urgency to understand the mechanisms of aging. The accumulation of aggregates of cellular proteins modified by oxidation, nitration, glutathiolation, glycation, truncation, and deamidation have been etiologically associated with risk for age-related diseases, including Parkinson's, Alzheimer's and other neurodegenerative diseases, age-related macular degeneration, compromised immune function, and cataract.1–19 Levels of these altered proteins reflect their rates of formation and their rates of degradation. Thus, the ability of cells to efficiently clear these modified proteins from the intracellular environment is essential for cellular homeostasis. Deamidation of proteins occurs with aging, and deamidated proteins accumulate in cataracts.19 Surprisingly, little is known about how deamidated proteins are removed from cells. A major pathway by which modified proteins are cleared from cells is through proteasomal degradation involving two related proteolytic complexes, the 20S and 26S proteasomes.20–26 The 26S proteasome is formed on the addition to the 20S proteasome of two “cap” components containing subunits for adenosine triphosphate (ATP) binding and hydrolysis and polyubiquitin chain recognition.27–29 This ubiquitin-dependent degradation pathway is referred to as the ubiquitin proteasome pathway (UPP). In the UPP, ubiquitin, a highly conserved 8500-Da protein present in all eukaryotic organisms, is usually covalently linked to an e-amino group of a specific lysine on the target protein. This process is catalyzed by a series of enzymes referred to as E1, E2, and E3. Polyubiquitination results in the high molecular weight (HMW) protein-ubiquitin conjugates that are usually recognized for degradation by the 26S proteasome.30,31 Low molecular weight (LMW) conjugates, with cellular functions other than as degradation substrates, have also been described.32 The lens of the eye is one of only two clear organs in the body, and clarity is required for the lens to collect and focus light on the retina. For clarity, proteins must remain soluble. Cataract can be considered a paradigmatic protein conformation or amyloid disease because aggregation and precipitation of proteins from the normally clear soluble lens milieu result in opacification and cataract. This makes it easy to observe in vivo. The three major classes of vertebrate lens proteins are α-, β-, and γ-crystallins, and they are required to maintain refractive capacity or provide chaperone function.33–39 These proteins are also expressed, albeit at lower levels, in other tissues.40–44 The β- and γ-crystallins are part of the same superfamily. Importantly, in the lens α-, β-, and γ-crystallins are all long-lived proteins, and they accumulate many age-related protein modifications that compromise their function and that are related to insolubilization.6,45–49 Deamidation of βB2 and βB1 crystallins is associated with age- and cataract-related protein insolubilization.19 Motivated to understand why these modified proteins accumulate, we examined the susceptibility of wild-type (WT) and specific deamidated mutants of βB2 and βB1 crystallins to ubiquitination and UPP-mediated degradation in several physiologically relevant proteolytic systems, including human lens epithelial cell (HLEC), bovine lens fiber (LF) cell lysate, and rabbit reticulocyte lysate (RRL). We demonstrate that WTβB1 and the deamidated βB1 crystallins are inherently excellent substrates for ubiquitination and UPP-mediated degradation using RRL. In comparison, WTβB2 crystallin is a poor substrate for ubiquitination and UPP-mediated degradation, but site-specific deamidation dramatically enhances its ubiquitination and degradation by the UPP. Related γD crystallins are refractory to the UPP. Importantly, lens epithelial cell and fiber lysates catalyze ubiquitination effectively but fail to complete proteolysis of the deamidated proteins as efficiently as RRL, even though the lens is known to contain active proteasome.50 This finding may explain their accumulation on aging in many tissues and, specifically, their cataractogenic potential.