Ubiquitin receptors and protein quality control

Abstract

Protein quality control (PQC) is essential to intracellular proteostasis and is carried out by sophisticated collaboration between molecular chaperones and targeted protein degradation. The latter is performed by proteasome-mediated degradation, chaperone-mediated autophagy (CMA), and selective macroautophagy, and collectively serves as the final line of defense of PQC. Ubiquitination and subsequently ubiquitin (Ub) receptor proteins (e.g., p62 and ubiquilins) are important common factors for targeting misfolded proteins to multiple quality control destinies, including the proteasome, lysosomes, and perhaps aggresomes, as well as for triggering mitophagy to remove defective mitochondria. PQC inadequacy, particularly proteasome functional insufficiency, has been shown to participate in cardiac pathogenesis. Tremendous advances have been made in unveiling the changes of PQC in cardiac diseases. However, the investigation into the molecular pathways regulating PQC in cardiac (patho)physiology, including the function of most ubiquitin receptor proteins in the heart, has only recently been initiated. A better understanding of molecular mechanisms governing PQC in cardiac physiology and pathology will undoubtedly provide new insights into cardiac pathogenesis and promote the search for novel therapeutic strategies to more effectively battle heart disease.This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".

Figure 1. An illustration of the ubiquitin-proteasome system-mediated proteolysis

A substrate protein molecule is first covalently tagged with a chain of ubiquitin (Ub) protein molecules, a process known as ubiquitination which is performed by a cascade of enzymatic reactions catalyzed sequentially by E1 (Ub activating enzyme), E2 (Ub conjugating enzyme), and E3 (Ub ligase). The conjugated Ub can be removed from the substrate via a process known as deubiquitination which counters ubiquitination and is performed by deubiquitinating enzymes (DUBs). Ubiquitinated substrates may be directly recognized and bound by Rpn10/S5a of the 19S proteasome, but often require extraproteasomal Ub receptor proteins (i.e., UBA-UBL proteins) to be delivered to the 26S proteasome (26S) and degraded by the latter.

Figure 2. An illustration of intracellular protein quality control (PQC)

Chaperones assist in protein folding and help to maintain protein integrity. The ubiquitin-proteasome system (UPS) is responsible for the degradation of most proteins, normal and abnormal, in the cell. The highly regulated process involves attachment of a ubiquitin (Ub) chain by E1 (Ub activating enzymes), E2 (Ub conjugating enzymes), and E3 (Ub ligase) to the targeted protein, a process known as ubiquitination. The Ub-tagged protein is then transferred to the proteasome for degradation. Recognition and target degradation of misfolded proteins can be carried out by the UPS as well, and additionally by chaperone-mediated autophagy (CMA). CMA unfolds and translocates individual misfolded proteins to the lysosome for degradation through the formation of a chaperone-substrate complex involving chaperone heat shock cognate 70 (Hsc70). Misfolded proteins that have escaped the surveillance of the UPS and CMA form aggregates and aggresomes. Proteins in the aggregated forms can only be degraded by macroautophagy, in which aggregates or aggresomes are segregated by formation of a double-membrane autophagosome. The formed vesicle then fuses with a lysosome and the degradation of autophagosome contents ensues. In addition to targeting proteins for degradation, ubiquitination can direct proteins along other pathways through site specific and varying chain length ubiquitination.

Figure 3. A schematic illustration of the exon composition and domain structure of Ubiquilin-1 transcript variants (TV) and Ubiquilin1 protein expression in the heart of a mouse model of desmin-related cardiomyopathy

A, The full-length form of ubiquilin-1 (TV1) contains 11 exons. A ubiquitin-like (UBL) domain at the N-terminus of ubiquilin-1 protein is encoded primarily by exon 2 (shaded in red) and binds to the proteasome. The C-terminal ubiquitin-associated (UBA) domain is coded by exon 11 (shaded in blue) and binds poly-ubiquitinated proteins. In variable central region, there are 4 STI1 motifs (indicated with purple bars) located respectively in exons 4, 5, 7, and 8. TV2 lacks exon 8 (shaded in green), causing deletion of one of the 4 STI motifs. TV3 lacks exons 2, 3 and 4, and thus the majority of the UBL domain. TV4 consists of the first 3 exons. A frame shift leading to a 32-amino acid insertion after the exon 3/5 junction creates a unique short C-terminus (dark blue) and therefore TV4 lacks the UBA domain. B, Western blot analyses of myocardial Ubqln1 in mice. Total myocardial proteins extracted from three pairs of D7-des transgenic (TG+) and non-TG (−) mice were fractionated using 12%SDS-PAGE under the standard denatured and reduced conditions, transferred onto PVDF membrane, and immuno-probed for Ubqln1 (using the upper part of the membrane, >40 kDa) and for GAPDH (using the lower part of the membrane, <40 kDa). The band marked as “Ublqn1 TV1” shows the same mobility as a full length murine Ubqln1 transgenic protein (data not shown). Bands a and b show a molecular weight substantially higher than the TV1, whereas bands c and d display a molecular weight clearly lower than TV1.

Figure 4. Models of p62 in cardiac protein quality control

(A) Upregulation of p62 in response to proteotoxic stress sequesters NRF2 from its interaction with Keap1, leading to the stablization and activation of NRF2, which in turn induces p62 expression. (B) Proteasome functional insufficiency leads to the accumulation of protein aggregates and compensatory activation of selective autophagy in a p62-dependent manner. Casein kinase 2 (CK2)-mediated p62 phosphorylation regulates selective degradation of protein aggregates by autophagy. Autophagy activation protects cardiomyocytes from defective UPS-induced proteotoxic stress. (C) Defective autophagy accumulates p62, which binds ubiquitinated proteins and promotes their agregation, hindering proteasomal proteolysis. Both defective autophagy and impaired UPS function are detrimental to cardiomyocyte function and survival. (Adopted from Figure 1 of Su et al. [96], with permission)