biomolecules Review The Targeting of Native Proteins to the Endoplasmic Reticulum-Associated Degradation (ERAD) Pathway: An Expanding Repertoire of Regulated Substrates Deepa Kumari and Jeffrey L. Brodsky * Department of Biological Sciences, University of Pittsburgh, A320 Langley Hall, Fifth & Ruskin Ave, Pittsburgh, PA 15260, USA; [email protected] * Correspondence: [email protected] Abstract: All proteins are subject to quality control processes during or soon after their synthesis, and these cellular quality control pathways play critical roles in maintaining homeostasis in the cell and in organism health. Protein quality control is particularly vital for those polypeptides that enter the endoplasmic reticulum (ER). Approximately one-quarter to one-third of all proteins synthesized in eukaryotic cells access the ER because they are destined for transport to the extracellular space, because they represent integral membrane proteins, or because they reside within one of the many compartments of the secretory pathway. However, proteins that mature inefficiently are subject to ER-associated degradation (ERAD), a multi-step pathway involving the chaperone-mediated selection, ubiquitination, and extraction (or “retrotranslocation”) of protein substrates from the ER. Ultimately, these substrates are degraded by the cytosolic proteasome. Interestingly, there is an increasing number of native enzymes and metabolite and solute transporters that are also targeted Citation: Kumari, D.; Brodsky, J.L. for ERAD. While some of these proteins may transiently misfold, the ERAD pathway also provides The Targeting of Native Proteins to a route to rapidly and quantitatively downregulate the levels and thus the activities of a variety of the Endoplasmic Reticulum- Associated Degradation (ERAD) proteins that mature or reside in the ER. Pathway: An Expanding Repertoire of Regulated Substrates. Biomolecules Keywords: ERAD; molecular chaperones; ubiquitin; proteasome; protein quality control 2021, 11, 1185. https://doi.org/ 10.3390/biom11081185 Proteins play myriad roles in cells, and the acquisition of their final folded states and Academic Editors: Tommer Ravid structural integrity are imperative for function. However, the expression of misfolded or and Rasmus Hartmann-Petersen non-native protein structures can lead to a plethora of diseases [1–4]. To destroy these misfolded and potentially toxic proteins, eukaryotic cells survey structural integrity at Received: 27 July 2021 multiple checkpoints as a protein matures. The factors that survey these nascent proteins, Accepted: 8 August 2021 attempt to repair misfolded species, facilitate maturation, target misfolded substrates for Published: 11 August 2021 degradation, and trigger cellular responses to off-set proteotoxicity collectively maintain protein homeostasis, or “proteostasis”, in the cell. Publisher’s Note: MDPI stays neutral Between a quarter to one-third of all nascent proteins enter the endoplasmic retic- with regard to jurisdictional claims in ulum (ER) during or soon after synthesis. These diverse substrates then fold, become published maps and institutional affil- post-translationally modified, assemble into protein complexes, and/or insert into the lipid iations. bilayer in the membrane [5–7]. As a nascent protein undergoes these events, it is then subject to surveillance by several ER quality-control pathways. One of these pathways was named endoplasmic-reticulum-associated degradation (ERAD) [8,9], a pathway re- quiring a complex collection of factors that help eliminate aberrant proteins that pose a Copyright: © 2021 by the authors. threat to the ER and consequently cellular homeostasis [10–13]. Therefore, this protein Licensee MDPI, Basel, Switzerland. quality-control pathway clears the secretory pathway of potentially toxic proteins. During This article is an open access article ERAD, the aberrant protein is first recognized by chaperones, and after selection, it is then distributed under the terms and retrotranslocated into the cytosol and ubiquitinated by one of a collection of E3 ubiquitin conditions of the Creative Commons ligases. The ERAD substrate is finally transported to and degraded by the cytoplasmic Attribution (CC BY) license (https:// proteasome (Figure1). creativecommons.org/licenses/by/ 4.0/). Biomolecules 2021, 11, 1185. https://doi.org/10.3390/biom11081185 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, x 2 of 18 The ERAD substrate is finally transported to and degraded by the cytoplasmic pro- Biomolecules 2021, 11, 1185 2 of 16 teasome (Figure 1). Figure 1. The endoplasmic reticulum-associated degradation (ERAD) pathway consists of four steps. The degradation pathway for a generic integral membrane protein in the ER is shown, and misfolded regions in the lumen, membrane, Figureand cytosol 1. The are endoplasmic represented withinreticulum-associated the polypeptide degradat chain asion condensed (ERAD) regions.pathway Recognition: consists of four Integral steps. membrane The degradation proteins pathwayenter the for ER a concomitantgeneric integral with membrane protein translationprotein in the on ER ER-associated is shown, and ribosomes misfoldedand regions with inthe the assistancelumen, membrane, of the Sec61 and cytosoltranslocon. are represented A misfold region within in the a po nascentlypeptide polypeptide chain as condensed is recognized regions. by cytosolic Recognition: or lumenal Integral chaperones. membrane Ubiquitination: proteins enter theThe ER ubiquitination concomitant machinery—and with protein translation more specifically on ER-associated an E3 ubiquitin ribosomes ligases, and often with alongthe assistance with an E2of ubiquitinthe Sec61 conjugatingtranslocon. Aenzyme misfold (not region shown)—is in a nascent next recruitedpolypeptide to the is recognized misfolded protein,by cytosolic which or is lumenal then conjugated chaperones with. Ubiquitination: a polyubiquitin The chain. ubiqui- The tinationchain most machinery—and commonly contains more specifically Lys-48 isopeptide an E3 ubiquitin linkages, andligases, a minimum often along of fourwith ubiquitins an E2 ubiquitin is required conjugating for proteasome- enzyme (notdependent shown)—is degradation next recruited [14]. Retrotranslocation: to the misfolded protein, The ubiquitinated which is then protein conjugated is retrotranslocated with a polyubiquitin through anchain. ER-integrated The chain most commonly contains Lys-48 isopeptide linkages, and a minimum of four ubiquitins is required for proteasome-de- retrotranslocon. Retrotranslocation requires ATP-dependent extraction mediated by the Cdc48 (in yeast) or p97 (in mammals) pendent degradation [14]. Retrotranslocation: The ubiquitinated protein is retrotranslocated through an ER-integrated complex. The Cdc48/p97 complex also consists of two associated factors, Npl4 and Ufd1, which aid in ubiquitinated retrotranslocon. Retrotranslocation requires ATP-dependent extraction mediated by the Cdc48 (in yeast) or p97 (in mam- mals)substrate complex. capture. The Degradation: Cdc48/p97 complex During and/oralso consists after retrotranslocation,of two associated factors, the misfolded Npl4 and ubiquitination Ufd1, which substrate aid in ubiquitinated is degraded substrateby the 26S capture. proteasome Degradation: into short During peptide and/or fragments. after retrotranslocation, the misfolded ubiquitination substrate is degraded by the 26S proteasome into short peptide fragments. Although ERAD was first discovered and was initially thought to primarily target misfoldedAlthough proteins ERAD [8 ,was15–19 first], the discovered ERAD pathway and was also initially regulates thought steady-state to primarily levels target and misfoldedthus, the activities proteins of[8,15–19], several apparently the ERAD nativelypathway folded also regulates proteins (seesteady-state Table1 for levels select and ex- thus,amples) the (also activities see [20 of– 22several]). In retrospect, apparently this nati isvely not surprising folded proteins since there (see areTable>7000 1 for proteins select examples)that interact (also with see the [20–22]). ER at someIn retrospect, point during this is biogenesis not surprising in human since there cells [are23], >7000 and these pro- proteins support myriad physiological processes, many of which are regulated. In fact, teins that interact with the ER at some point during biogenesis in human cells [23], and one regulated wild-type substrate, HMG CoA-reductase (see below), appears to become these proteins support myriad physiological processes, many of which are regulated. In unfolded in response to a metabolic signal/lipid, which destabilizes and targets the enzyme fact, one regulated wild-type substrate, HMG CoA-reductase (see below), appears to be- for ERAD [24]. It is likely that a similar scenario is evident for other regulated, functioning come unfolded in response to a metabolic signal/lipid, which destabilizes and targets the enzymes. In effect, the rapid degradation of these proteins represents the best and most enzyme for ERAD [24]. It is likely that a similar scenario is evident for other regulated, complete way to downregulate function. Overall, ERAD maintains quality control—i.e., the functioning enzymes. In effect, the rapid degradation of these proteins represents the best degradation of misfolded and improperly modified proteins—as well as quantity control and most complete way to downregulate function. Overall, ERAD maintains quality con- in the secretory pathway. trol—i.e.,