Degradation Of Integral Membrane Proteins Research Articles

Integral Membrane Protein Degradation: A Novel Paradigm by PROTAC Technology

: Integral membrane proteins are attractive targets for therapeutic intervention due to their crucial roles in cellular functions and their involvement in various diseases. However, targeting these proteins for degradation has posed significant challenges due to their complex structures and diverse functions. The emergence of proteolysis-targeting chimeras (PROTAC) represents a groundbreaking paradigm shift in drug development, offering a novel approach to address the degradation of integral membrane proteins. This review explores the innovative application of PROTAC technology in inducing the degradation of integral membrane proteins. PROTACs are molecules that have two roles: they bring together specific target proteins and E3 ubiquitin ligases, which help with ubiquitination and the degradation of proteins by proteasomes. As PROTACs are made up of separate parts, they can be put together to make custom molecules that can target difficult targets, such as integral membrane proteins. This review covers recent developments and advancements in the design and optimisation of PROTACs tailored for integral membrane protein degradation. Furthermore, it also discusses the problems that come up when trying to target membrane proteins and how PROTACs solve these problems by using ligands that can pass cell membranes and specifically interact with target proteins. It delves into the potential therapeutic implications of degrading integral membrane proteins using PROTACs and elucidates the impact of this novel approach in treating diseases that involve aberrant membrane protein expression or function by highlighting specific examples and case studies.

Degradation of integral membrane proteins modified with the photosensitive degron module requires the cytosolic endoplasmic reticulum-associated degradation pathway.

Protein quality mechanisms are fundamental for proteostasis of eukaryotic cells. Endoplasmic reticulum–associated degradation (ERAD) is a well-studied pathway that ensures quality control of secretory and endoplasmic reticulum (ER)–resident proteins. Different branches of ERAD are involved in degradation of malfolded secretory proteins, depending on the localization of the misfolded part, the ER lumen (ERAD-L), the ER membrane (ERAD-M), and the cytosol (ERAD-C). Here we report that modification of several ER transmembrane proteins with the photosensitive degron (psd) module resulted in light-dependent degradation of the membrane proteins via the ERAD-C pathway. We found dependency on the ubiquitylation machinery including the ubiquitin-activating enzyme Uba1, the ubiquitin-­conjugating enzymes Ubc6 and Ubc7, and the ubiquitin–protein ligase Doa10. Moreover, we found involvement of the Cdc48 AAA-ATPase complex members Ufd1 and Npl4, as well as the proteasome, in degradation of Sec62-myc-psd. Thus, our work shows that ERAD-C substrates can be systematically generated via synthetic degron constructs, which facilitates future investigations of the ERAD-C pathway.

Accessibility from the Cytoplasm Is Critical for ssrA Tag-Mediated Degradation of Integral Membrane Proteins by ClpXP Protease.

The AAA+ protease ClpXP has long been established as the cellular rescue system that degrades ssrA-tagged proteins resulting from stalled ribosomes. Until recently, in all of these studies soluble proteins were used as model substrates, since the ClpXP complex and the related adapter SspB are all cytosolic proteins. In a previous study, we found that the introduction of an ssrA tag can facilitate complete degradation of a large and stable trimeric integral membrane protein AcrB, which is the first reported example of a membrane protein substrate. To investigate the mechanism of degradation of a membrane protein by a soluble protein complex, we experimented with the truncation of the C-terminal tail of AcrB. We found that the C-terminal tail is important for degradation, as systematic truncation of the tail diminished degradation. Thus, we hypothesize that membrane proteins need a cytosolic tail/domain for ClpXP-SspB to latch on to initiate degradation. To test this hypothesis, we introduced the ssrA tag at the C-terminal of several membrane proteins, including AqpZ, YiiP, YajR, as well as their truncation fragments, and examined their degradation. We found that the ssrA-facilitated degradation of membrane proteins by ClpXP-SspB depends on the presence of a CT tail or domain, which is critical for accessibility of the tag by ClpXP-SspB. When the ssrA tag is not well-exposed to the cytosol, FtsH can access and degrade the tagged protein, given that the substrate protein is metastable.

Investigating the Functional Role of the Transmembrane Segments of Yta10, a Subunit of the mAAA Protease, in Membrane Protein Degradation

The mAAA protease is present in the mitochondrial inner membrane and plays diverse roles in protein biogenesis and degradation. It is a hetero-oligomeric complex composed of Yta10 and Yta12. Yta10 carries two transmembrane segments (TMS) at its N-terminus and a large AAA+ domain and a proteolytic domain at its C-terminus. Previous studies have shown that the deletion of TMS does not compromise the proteolytic activity but, causes a defect in degradation of integral membrane proteins, hinting that the TMS of Yta10 is important for membrane protein degradation. The TMS of Yta10 has been either deleted or replaced by TMS of another mitochondrial inner membrane protein to elucidate their function. How Yta10 TMS mutants handle substrates have been tested using a set of Mgm1p of varying sequence composition in its 1st TMS. Mgm1p exists in two forms, l-Mgm1p and s-Mgm1p. The generation of s-Mgm1p is independent of the mAAA protease, but it becomes mAAA protease dependent when the first TMS of Mgm1p is replaced with a foreign TMS. A foreign TMS is dislocated from the membrane by the action of the mAAA protease and the downstream sequence is processed by Pcp1, generating s-Mgm1p. We speculated that if the TMS of Yta10 is required for the anchoring of the soluble domain to the membrane, only the deletion of TMS would impair the processing of Mgm1p variants to s-Mgm1p, not the substitution of TMS. In contrast, if the TMS required either for the recognition or dislocation of membrane proteins, both the deletion and substitution of TMS would impair the processing of Mgm1p variants to s-Mgm1p. This screening will elucidate the function of the TMS of Yta10 in membrane protein degradation.

Protein quality control at the inner nuclear membrane.

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

Cytosolic Degradation of T-cell Receptor α Chains by the Proteasome

The T-cell antigen receptor (TCR) is an hetero-oligomeric membrane complex composed of at least seven transmembrane polypeptide chains that has served as a model for the assembly and degradation of integral membrane proteins in the endoplasmic reticulum (ER). Unassembled TCRalpha chains fail to mature to the Golgi apparatus and are rapidly degraded by a non-lysosomal "ER degradation" pathway that has been proposed to be autonomous to the ER. In these studies we show that the degradation of core-glycosylated TCRalpha is blocked by N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) and lactacystin, implicating the proteasome in ER degradation. Either acute or chronic treatment of TCRalpha-transfected cells with proteasome inhibitors cause the core-glycosylated TCRalpha chains to progressively shift to an approximately 28-kDa form that lacks N-linked oligosaccharides and the N-terminal signal peptide. The susceptibility of this 28-kDa species to extravesicular protease indicates that it is not protected by the ER membrane and, hence, cytoplasmic. These data suggest a model in which TCRalpha chains that are translocated across the membrane, core-glycosylated, but fail to assemble are dislocated back to the cytoplasm for degradation by cytoplasmic proteasomes. Our data also suggest that covalent modification of TCRalpha with ubiquitin is not required for its degradation.