The ubiquitin-proteasome system links NADPH metabolism to ferroptosis.

Ferroptosis is the type of cell death arising from uncontrolled and excessive lipid peroxidation. NADPH is essential for ferroptosis regulation because it supplies reducing equivalents for antioxidant defense systems and contributes to the generation of reactive oxygen species. Moreover, NADPH level serves as a biomarker for predicting the sensitivity of cells to ferroptosis. The ubiquitin-proteasome system governs the stability of many ferroptosis effectors. Recent research has revealed MARCHF6, the endoplasmic reticulum ubiquitin ligase, as an unprecedented NADPH sensor in the ubiquitin system and a critical regulator of ferroptosis involved in tumorigenesis and fetal development. This review summarizes the current understanding of NADPH metabolism and the ubiquitin-proteasome system in regulating ferroptosis and highlights the emerging importance of MARCHF6 as a vital connector between NADPH metabolism and ferroptosis.

Marchf6 E3 ubiquitin ligase critically regulates endoplasmic reticulum stress, ferroptosis, and metabolic homeostasis in POMC neurons.

The metabolic prohormone pro-opiomelanocortin (POMC) is generally translocated into the endoplasmic reticulum (ER) for entry into the secretory pathway. Patients with mutations within the signal peptide (SP) of POMC or its adjoining segment develop metabolic disorders. However, the existence, metabolic fate, and functional outcomes of cytosol-retained POMC remain unclear. Here, we show that SP-uncleaved POMC is produced in the cytosol of POMC neuronal cells, thus inducing ER stress and ferroptotic cell death. Mechanistically, the cytosol-retained POMC sequesters the chaperone Hspa5 and subsequently accelerates degradation of the glutathione peroxidase Gpx4, a core regulator of ferroptosis, via the chaperone-mediated autophagy. We also show that the Marchf6 E3 ubiquitin ligase mediates the degradation of cytosol-retained POMC, thereby preventing ER stress and ferroptosis. Furthermore, POMC-Cre-mediated Marchf6-deficient mice exhibit hyperphagia, reduced energy expenditure, and weight gain. These findings suggest that Marchf6 is a critical regulator of ER stress, ferroptosis, and metabolic homeostasis in POMC neurons.

Detection of Nα-terminally formylated native proteins by a pan-N-formyl methionine-specific antibody.

N-formyl methionine (fMet)-containing proteins are produced in bacteria, eukaryotic organelles mitochondria and plastids, and even in cytosol. However, Nα-terminally formylated proteins have been poorly characterized because of the lack of appropriate tools to detect fMet independently of downstream proximal sequences. Using a fMet-Gly-Ser-Gly-Cys peptide as an antigen, we generated a pan-fMet-specific rabbit polyclonal antibody called anti-fMet. The raised anti-fMet recognized universally and sequence context-independently Nt-formylated proteins in bacterial, yeast, and human cells as determined by a peptide spot array, dot blotting, and immunoblotting. We anticipate that the anti-fMet antibody will be broadly used to enable an understanding of the poorly explored functions and mechanisms of Nt-formylated proteins in various organisms.

N-Terminally arginylated ubiquitin is attached to histone H2A by RING1B E3 ligase in human cells.

Ubiquitin (Ub) is highly conserved in all eukaryotic organisms and begins at the N-terminus with Met and Gln. Our recent research demonstrates that N-terminally (Nt-) arginylated Ub can be produced in the yeast Saccharomyces cerevisiae. However, the existence of Nt-arginylated Ub in multicellular organisms remains unknown. Here we explore the mechanism for creating Nt-arginylated Ub using human embryonic kidney HEK293 cells that express various Nt-modified Ubs. We found that Gln-starting Q-Ub was converted into Glu-starting E-Ub by NTAQ1 Nt-deamidase and subsequently Nt-arginylated by ATE1 arginyltransferase in HEK293 cells. We also found that the resulting Arg-Glu-starting RE-Ub was mainly deposited on the Lys119 residue of histone H2A. Furthermore, RING1B E3 Ub ligase mediated the attachment of RE-Ub to H2A. These findings reveal a previously unknown type of histone ubiquitylation which greatly increases the combinatorial complexity of histone and ubiquitin codes.

The MARCHF6 E3 ubiquitin ligase acts as an NADPH sensor for the regulation of ferroptosis.

Ferroptosis is a unique form of cell death caused by excessive iron-dependent lipid peroxidation. The level of the anabolic reductant NADPH is a biomarker of ferroptosis sensitivity. However, specific regulators that detect cellular NADPH levels, thereby modulating downstream ferroptosis cascades, are largely unknown. We show here that the transmembrane endoplasmic reticulum MARCHF6 E3 ubiquitin ligase recognizes NADPH through its C-terminal regulatory region. This interaction upregulates the E3 ligase activity of MARCHF6, thus downregulating ferroptosis. We also found that MARCHF6 mediates the degradation of the key ferroptosis effectors ACSL4 and p53. Furthermore, inhibiting ferroptosis rescued the growth of MARCHF6-deficient tumours and peri-natal lethality of Marchf6-/- mice. Together, these findings identify MARCHF6 as a previously unknown NADPH sensor in the ubiquitin system and a crucial regulator of ferroptosis.

Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates.

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNAArg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNAArg, and a 3'-proximal segment of the tRNAArg moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe3+-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.

N-Terminal Modifications of Ubiquitin via Methionine Excision, Deamination, and Arginylation Expand the Ubiquitin Code.

Ubiquitin (Ub) is post-translationally modified by Ub itself or Ub-like proteins, phosphorylation, and acetylation, among others, which elicits a variety of Ub topologies and cellular functions. However, N-terminal (Nt) modifications of Ub remain unknown, except the linear head-to-tail ubiquitylation via Nt-Met. Here, using the yeast Saccharomyces cerevisiae and an Nt-arginylated Ub-specific antibody, we found that the detectable level of Ub undergoes Nt-Met excision, Nt-deamination, and Nt-arginylation. The resulting Nt-arginylated Ub and its conjugated proteins are upregulated in the stationary-growth phase or by oxidative stress. We further proved the existence of Nt-arginylated Ub in vivo and identified Nt-arginylated Ub-protein conjugates using stable isotope labeling by amino acids in cell culture (SILAC)-based tandem mass spectrometry. In silico structural modeling of Nt-arginylated Ub predicted that Nt-Arg flexibly protrudes from the surface of the Ub, thereby most likely providing a docking site for the factors that recognize it. Collectively, these results reveal unprecedented Nt-arginylated Ub and the pathway by which it is produced, which greatly expands the known complexity of the Ub code.

MTFMT deficiency correlates with reduced mitochondrial integrity and enhanced host susceptibility to intracellular infection.

Mitochondria behave as functional and structural hubs for innate defense against intracellular infection. While the mitochondrial membrane serves as a platform for the assembly of signaling complexes activated by intracellular infection, various danger molecules derived from impaired mitochondria activate innate signaling pathways. Using methionyl-tRNA formyl transferase (MTFMT)-deficient cells, which exhibit impaired mitochondrial activity, we examined the role of mitochondrial integrity in regulating innate defense against infection. Since MTFMT functions at the early steps of mitochondrial translation, its loss was expected to cause defects in mitochondrial activity. Under transient MTFMT gene silencing conditions, we observed shortened mitochondria along with reduced activity. MTFMT-silenced cells were more susceptible to intracellular infection, as examined by infection with RNA viruses and the intracellular bacterium Shigella flexneri. In support of this observation, MTFMT-silenced cells possessed lowered basal NF-κB activity, which remained low after S. flexneri infection. In addition, the mitochondrial accumulation of evolutionarily conserved signaling intermediate in Toll pathway (ECSIT), an adaptor protein for NF-κB activation, was significantly decreased in MTFMT-silenced cells, explaining the reduced NF-κB activity observed in these cells. Since impaired mitochondria likely release mitochondrial molecules, we evaluated the contribution of mitochondrial N-formyl peptides to the regulation of bacterial infection. Transient transfection of mitochondrial-derived N-formyl peptides favored S. flexneri infection, which was accompanied by enhanced bacterial survival, but did not affect host cell viability. However, transient transfection of mitochondrial-derived N-formyl peptides did not affect basal NF-κB activity. Altogether, these data suggest that the integrity of mitochondria is essential to their proper function in protecting against infection, as intact mitochondria not only block the release of danger molecules but also serve as signaling hubs for the downstream NF-κB pathway.

iNrich, Rapid and Robust Method to Enrich N-Terminal Proteome in a Highly Multiplexed Platform.

The field of terminal proteomics is limited in that it is optimized for large-scale analysis via multistep processes involving liquid chromatography. Here, we present an integrated N-terminal peptide enrichment method (iNrich) that can handle as little as 25 µg of cell lysate via a single-stage encapsulated solid-phase extraction column. iNrich enables simple, rapid, and reproducible sample processing, treatment of a wide range of protein amounts (25 µg ∼ 1 mg), multiplexed parallel sample preparation, and in-stage sample prefractionation using a mixed-anion-exchange filter. We identified ∼5000 N-terminal peptides (Nt-peptides) from only 100 µg of human cell lysate including Nt-formyl peptides. Multiplexed sample preparation facilitated quantitative and robust enrichment of N-terminome with dozens of samples simultaneously. We further developed the method to incorporate isobaric tags such as a tandem mass tag (TMT) and used it to discover novel peptides during ER stress analysis. The iNrich facilitated high-throughput N-terminomics and degradomics at a low cost using commercially available reagents and apparatus, without requiring arduous procedures.

Cross-Species Functional Conservation and Possible Origin of the N-Terminal Specificity Domain of Mitochondrial Presequences.

Plants have two endosymbiotic organelles, chloroplast and mitochondrion. Although they have their own genomes, proteome assembly in these organelles depends on the import of proteins encoded by the nuclear genome. Previously, we elucidated the general design principles of chloroplast and mitochondrial targeting signals, transit peptide, and presequence, respectively, which are highly diverse in primary structure. Both targeting signals are composed of N-terminal specificity domain and C-terminal translocation domain. Especially, the N-terminal specificity domain of mitochondrial presequences contains multiple arginine residues and hydrophobic sequence motif. In this study we investigated whether the design principles of plant mitochondrial presequences can be applied to those in other eukaryotic species. We provide evidence that both presequences and import mechanisms are remarkably conserved throughout the species. In addition, we present evidence that the N-terminal specificity domain of presequence might have evolved from the bacterial TAT (twin-arginine translocation) signal sequence.

N-terminal methionine excision of proteins creates tertiary destabilizing N-degrons of the Arg/N-end rule pathway.

All organisms begin protein synthesis with methionine (Met). The resulting initiator Met of nascent proteins is irreversibly processed by Met aminopeptidases (MetAPs). N-terminal (Nt) Met excision (NME) is an evolutionarily conserved and essential process operating on up to two-thirds of proteins. However, the universal function of NME remains largely unknown. MetAPs have a well-known processing preference for Nt-Met with Ala, Ser, Gly, Thr, Cys, Pro, or Val at position 2, but using CHX-chase assays to assess protein degradation in yeast cells, as well as protein-binding and RT-qPCR assays, we demonstrate here that NME also occurs on nascent proteins bearing Met-Asn or Met-Gln at their N termini. We found that the NME at these termini exposes the tertiary destabilizing Nt residues (Asn or Gln) of the Arg/N-end rule pathway, which degrades proteins according to the composition of their Nt residues. We also identified a yeast DNA repair protein, MQ-Rad16, bearing a Met-Gln N terminus, as well as a human tropomyosin-receptor kinase-fused gene (TFG) protein, MN-TFG, bearing a Met-Asn N terminus as physiological, MetAP-processed Arg/N-end rule substrates. Furthermore, we show that the loss of the components of the Arg/N-end rule pathway substantially suppresses the growth defects of naa20Δ yeast cells lacking the catalytic subunit of NatB Nt acetylase at 37 °C. Collectively, the results of our study reveal that NME is a key upstream step for the creation of the Arg/N-end rule substrates bearing tertiary destabilizing residues in vivo.

N-terminal acetylation and the N-end rule pathway control degradation of the lipid droplet protein PLIN2.

Perilipin 2 (PLIN2) is a major lipid droplet (LD)-associated protein that regulates intracellular lipid homeostasis and LD formation. Under lipid-deprived conditions, the LD-unbound (free) form of PLIN2 is eliminated in the cytosol by an as yet unknown ubiquitin (Ub)-proteasome pathway that is associated with the N-terminal or near N-terminal residues of the protein. Here, using HeLa, HEK293T, and HepG2 human cell lines, cycloheximide chase, in vivo ubiquitylation, split-Ub yeast two-hybrid, and chemical cross-linking-based reciprocal co-immunoprecipitation assays, we found that TEB4 (MARCH6), an E3 Ub ligase and recognition component of the Ac/N-end rule pathway, directly targets the N-terminal acetyl moiety of Nα-terminally acetylated PLIN2 for its polyubiquitylation and degradation by the 26S proteasome. We also show that the TEB4-mediated Ac/N-end rule pathway reduces intracellular LD accumulation by degrading PLIN2. Collectively, these findings identify PLIN2 as a substrate of the Ac/N-end rule pathway and indicate a previously unappreciated role of the Ac/N-end rule pathway in LD metabolism.

Formyl-methionine as an N-degron of a eukaryotic N-end rule pathway.

In bacteria, nascent proteins bear the pretranslationally generated N-terminal (Nt) formyl-methionine (fMet) residue. Nt-fMet of bacterial proteins is a degradation signal, termed fMet/N-degron. By contrast, proteins synthesized by cytosolic ribosomes of eukaryotes were presumed to bear unformylated Nt-Met. Here we found that the yeast formyltransferase Fmt1, although imported into mitochondria, could also produce Nt-formylated proteins in the cytosol. Nt-formylated proteins were strongly up-regulated in stationary phase or upon starvation for specific amino acids. This up-regulation strictly required the Gcn2 kinase, which phosphorylates Fmt1 and mediates its retention in the cytosol. We also found that the Nt-fMet residues of Nt-formylated proteins act as fMet/N-degrons and identified the Psh1 ubiquitin ligase as the recognition component of the eukaryotic fMet/N-end rule pathway, which destroys Nt-formylated proteins.

Control of protein degradation by N-terminal acetylation and the N-end rule pathway.

Nα-terminal acetylation (Nt-acetylation) occurs very frequently and is found in most proteins in eukaryotes. Despite the pervasiveness and universality of Nt-acetylation, its general functions in terms of physiological outcomes remain largely elusive. However, several recent studies have revealed that Nt-acetylation has a significant impact on protein stability, activity, folding patterns, cellular localization, etc. In addition, Nt-acetylation marks specific proteins for degradation by a branch of the N-end rule pathway, a subset of the ubiquitin-mediated proteolytic system. The N-end rule associates a protein's in vivo half-life with its N-terminal residue or modifications on its N-terminus. This review provides a current understanding of intracellular proteolysis control by Nt-acetylation and the N-end rule pathway.

Degradation of Serotonin N-Acetyltransferase, a Circadian Regulator, by the N-end Rule Pathway.

Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-end rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two "complementary" forms of rat AANAT are targeted for degradation by two "complementary" branches of the N-end rule pathway. Specifically, the N(α)-terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt-acetylated N-terminal Met residue by the Ac/N-end rule pathway, whereas the non-Nt-acetylated AANAT is targeted by the Arg/N-end rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine-to-arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer-lived than its rat counterpart and appears to be refractory to degradation by the N-end rule pathway. Together, these and related results indicate both a major involvement of the N-end rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N-terminal sequences.

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways.

N-Terminal Acetylation-Targeted N-End Rule Proteolytic System: The Ac/N-End Rule Pathway.

Although Nα-terminal acetylation (Nt-acetylation) is a pervasive protein modification in eukaryotes, its general functions in a majority of proteins are poorly understood. In 2010, it was discovered that Nt-acetylation creates a specific protein degradation signal that is targeted by a new class of the N-end rule proteolytic system, called the Ac/N-end rule pathway. Here, we review recent advances in our understanding of the mechanism and biological functions of the Ac/N-end rule pathway, and its crosstalk with the Arg/N-end rule pathway (the classical N-end rule pathway).

Protein Degradation of RNA Polymerase II-Association Factor 1(PAF1) Is Controlled by CNOT4 and 26S Proteasome.

The PAF complex (PAFc) participates in various steps of the transcriptional process, from initiation to termination, by interacting with and recruiting various proteins to the proper locus for each step. PAFc is an evolutionarily conserved, multi-protein complex comprising PAF1, CDC73, CTR9, LEO1, yRTF1 and, in humans, hSKI8. These components of PAFc work together, and their protein levels are closely interrelated. In the present study, we investigated the mechanism of PAF1 protein degradation. We found that PAF1 protein levels are negatively regulated by the expression of CNOT4, an ortholog of yNOT4 and a member of the CCR4-NOT complex. CNOT4 specifically controls PAF1 but not other components of PAFc at the protein level by regulating the polyubiquitination of PAF1 and its subsequent degradation by the 26S proteasome. The degradation of PAF1 was found to require nuclear localization, as no PAF1 degradation by CNOT4 and the 26S proteasome was observed with NLS (nucleus localization signal)-deficient PAF1 mutants. However, chromatin binding by PAF1 was not necessary for 26S proteasome- or CNOT4-mediated degradation. Our results suggest that CNOT4 controls the degradation of chromatin-unbound PAF1 via the 26S proteasome.