bims-apauto 2023-08-06 papers

Issue of 2023‒08‒06
five papers selected by
Su Hyun Lee, Harvard University

Methods Enzymol. 2023 ;pii: S0076-6879(23)00061-7. [Epub ahead of print]686 235-265

Characterization and chemical modulation of p62/SQSTM1/Sequestosome-1 as an autophagic N-recognin.

Su Jin Lee, Hye Yeon Kim, Min Ju Lee, Su Bin Kim, Yong Tae Kwon, Chang Hoon Ji.

In the Arg/N-degron pathway, single N-terminal (Nt) residues function as N-degrons recognized by UBR box-containing N-recognins that induce substrate ubiquitination and proteasomal degradation. Recent studies led to the discovery of the autophagic Arg/N-degron pathway, in which the autophagic receptor p62/SQSTM1/Sequestosome-1 acts as an N-recognin that binds the Nt-Arg and other destabilizing residues as N-degrons. Upon binding to Nt-Arg, p62 undergoes self-polymerization associated with its cargoes, accelerating the macroautophagic delivery of p62-cargo complexes to autophagosomes leading to degradation by lysosomal hydrolases. This autophagic mechanism is emerging as an important pathway that modulates the lysosomal degradation of various biomaterial ranging from protein aggregates and subcellular organelles to invading pathogens. Chemical mimics of the physiological N-degrons were developed to exert therapeutic efficacy in pathophysiological processes associated with neurodegeneration and other related diseases. Here, we describe the methods to monitor the activities of p62 in a dual role as an N-recognin and an autophagic receptor. The topic includes self-polymerization (for cargo condensation), its interaction with LC3 on autophagic membranes (for cargo targeting), and the degradation of p62-cargo complexes by lysosomal hydrolases. We also discuss the development and use of small molecule mimics of N-degrons that modulate p62-dependent macroautophagy in biological and pathophysiological processes.

Keywords: Autophagy flux assay; Autophagy-targeting ligand (ATL); Colocalization assay; In vitro oligomerization assay; Molecular modeling; N-degron pathway; N-terminal arginylation; Selective autophagy; Targeted protein degradation (TPD); p62/SQSTM1/Sequestosome-1

DOI: https://doi.org/10.1016/bs.mie.2023.02.005

Methods Enzymol. 2023 ;pii: S0076-6879(23)00194-5. [Epub ahead of print]686 165-203

Monitoring the interactions between N-degrons and N-recognins of the Arg/N-degron pathway.

Soon Chul Kwon, Jihoon Lee, Yong Tae Kwon, Ah Jung Heo.

As defined by the N-degron pathway, single N-terminal (Nt) amino acids can function as N-degrons that induce the degradation of proteins and other biological materials. Central to this pathway is the selective recognition of N-degrons by cognate N-recognins that direct the substrates to either the ubiquitin (Ub)-proteasome system (UPS) or autophagy-lysosome pathway (ALP). Eukaryotic cells have developed diverse pathways to utilize all 20 amino acids in the genetic code as pro-N-degrons or N-degrons which can be generated through endoproteolytic cleavage or post-translational modifications. Amongst these, the arginine (Arg) N-degron plays a key role in both cis- and trans-degradation of a large spectrum of cellular materials by the proteasome or lysosome. In mammals, Arg/N-degrons can be generated through endoproteolytic cleavage or post-translational conjugation of the amino acid L-Arg by ATE1-encoded R-transferases (EC 2.3.2.8), which requires Arg-tRNAArg as a cofactor. Arg/N-degrons of short-lived substrates are recognized by a family of N-recognins characterized by the UBR box for polyubiquitination and proteasomal degradation. Under stresses, however, the same degrons can be recognized for autophagic degradation by the ZZ domain of the N-recognin p62/SQSTSM-1/Sequestosome-1 or KCMF1. Biochemical tools were developed to monitor the interaction of Arg/N-degrons with its cognate N-recognins. These assays were employed to identify new N-recognins and to characterize their biochemical properties and physiological functions. The principles of these assays may be applied for other types of N-degron pathways. Below, we describe the methods that analyze the interaction of Arg/N-degrons and their chemical mimics to N-recognins.

Keywords: ATE1 R-transferases; Autophagy-lysosome system; N-terminal arginylation; Pulldown assay; UBR box; Ubiquitin-proteasome system; p62/SQSTSM-1/sequestosome-1

DOI: https://doi.org/10.1016/bs.mie.2023.05.009

Cell Signal. 2023 Jul 27. pii: S0898-6568(23)00244-9. [Epub ahead of print] 110830

N-degron-mediated degradation of the proteolytically activated form of PKC-theta kinase attenuates its pro-apoptotic function.

Mohamed A Eldeeb, Wenbin Zhou, Mansoore Esmaili, Alaa M Elgohary, Hai Wei, Richard P Fahlman.

Cellular signalling cues lead to the initiation of apoptotic pathways and often result in the activation of caspases which in turn cause the generation of proteolytically generated protein fragments with new or altered functions. Mounting number of studies reveal that the activity of these proteolytically activated protein fragments can be counteracted via their selective degradation by the N-degron degradation pathways. Here, we investigate the proteolytically generated fragment of the PKC theta kinase, where we study the first report on the stability of this pro-apoptotic protein fragment. We have determined that the pro-apoptotic cleaved fragment of PKC-theta is unstable in cells because its N-terminal lysine targets it for proteasomal degradation via the N-end rule pathway and this degradation is inhibited by mutating the destabilizing N-termini, knockdown of the UBR1 and UBR2 E3 ligases. Tellingly, we demonstrate that the metabolic stabilization of the cleaved fragment of PKC-theta or inhibition of the N-end rule augments the apoptosis-inducing effect of staurosporine in Jurkat cells. Notably, we have demonstrated that the cleaved fragment of PKC theta, per se, can induce apoptotic cell death in Jurkat T-cell leukemia. Our results expand the functional scope of mammalian N-degron pathway, and support the notion that targeting N-degron degradation machinery may have promising therapeutic implications in cancer cells.

Keywords: Cell death; Cellular signalling; Proteasome; Protein degradation; Protein quality control; Proteolysis; Ubiquitin

DOI: https://doi.org/10.1016/j.cellsig.2023.110830

Methods Enzymol. 2023 ;pii: S0076-6879(23)00058-7. [Epub ahead of print]686 99-123

In vitro production of N-degron fused proteins and its application.

Bong Heon Kim, Do Hoon Kwon, Leehyeon Kim, Woo Seok Yang, Hyun Kyu Song.

The N-degron pathway, first discovered several decades ago by Varshavsky's laboratory, controls the half-life of target proteins depending on their N-terminal residues. In vivo cell biology studies have established the physiological role of the N-degron pathway. However, in vitro studies such as biochemical assays and structural biology studies are relatively limited. The N-degron substrates cannot be obtained via simple protein expression. The N-degron residues are exposed via the proteolytic process from the translated nascent polypeptide chains. Thus, methods for the fusion expression with several cleavable tags and subsequent treatment with specific proteases to design the exposed N-degron signals have been introduced. Recently, we developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B (LC3B), a key marker protein of autophagy, to obtain a high yield of the purified target proteins with variable N-terminal residues for various biochemical studies including enzymatic and binding assays, and crystallization of N-degron complex. This chapter describes the protocols that include the vector map designed for producing LC3B fused target proteins, methods for expression and purification of an example protein, p62/SQSMT1, using different N-terminal residues, and methods to obtain the purified ATG4B protease, which is used for processing LC3B tag and exposing the required N-terminal residues of the target protein.

Keywords: ATG4B; Crystallization; LC3B-fusion; N-degron; N-end rule; N-recognin; ZZ-domain; p62/SQSTM1

DOI: https://doi.org/10.1016/bs.mie.2023.02.002

Methods Enzymol. 2023 ;pii: S0076-6879(23)00081-2. [Epub ahead of print]686 205-220

In vitro autoubiquitination activity of E3 ubiquitin ligases of the N-degron pathway.

Alexander Sandmann, Nico Dissmeyer.

As a part of the ubiquitin-proteasome system, E3 ubiquitin ligases play an important role in the regulation of the proteome in eukaryotic cells. These enzymes are extensively studied because of their crucial function, however it can be challenging to observe E3 ubiquitin ligases in action. Here, we outline a method for determining whether a known or potential E3 ubiquitin ligase exhibits autoubiquitination activity in vitro using PROTEOLYSIS1 (PRT1, AT3G24800), the first identified N-degron pathway E3 ubiquitin ligase from plants as an example. The approach provided here makes it possible to analyze mutations that could reduce or eliminate activity, to test for interaction with E2 ubiquitin conjugating enzymes, as well as to check for in vitro substrate ubiquitination.

Keywords: E3 ubiquitin ligase; N-degron pathway; Protein degradation; Ubiquitin

DOI: https://doi.org/10.1016/bs.mie.2023.02.014