When Degradation Elicits the Alarm: N-Terminal Degradation of NLRP1B Unleashes Its Inflammasome Activity.

Despite being among the first discovered mammalian innate immune sensor, NLRP1B (NLR pyrin domain-containing1B) activation and its molecular basis have remained elusive. Two recent studies have unveiled N-terminal degradation as a common mechanism for pathogen-mediated NLRP1B inflammasome activation in mammals.

Modifications in the T arm of tRNA globally determine tRNA maturation, function, and cellular fitness.

Almost all elongator tRNAs (Transfer RNAs) harbor 5-methyluridine 54 and pseudouridine 55 in the T arm, generated by the enzymes TrmA and TruB, respectively, in Escherichia coli. TrmA and TruB both act as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wild type. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion of trmA and truB in E. coli causes a global decrease in aminoacylation and alters other tRNA modifications such as acp3U47. While overall protein synthesis is not affected in ΔtrmA and ΔtruB strains, the translation of a subset of codons is significantly impaired. As a consequence, we observe translationally reduced expression of many specific proteins, that are either encoded with a high frequency of these codons or that are large proteins. The resulting proteome changes are not related to a specific growth phenotype, but overall cellular fitness is impaired upon deleting trmA and truB in accordance with a general protein synthesis impact. In conclusion, we demonstrate that universal modifications of the tRNA T arm are critical for global tRNA function by enhancing tRNA maturation, tRNA aminoacylation, and translation, thereby improving cellular fitness irrespective of the growth conditions which explains the conservation of trmA and truB.

ER-associated Protein Degradation at Atomic Resolution.

The endoplasmic reticulum-associated degradation (ERAD) pathway eliminates misfolded proteins. The Hrd1 complex represents the main gate mediating retrotranslocation of ER luminal misfolded (ERAD-L) substrates to the cytosol. A recent cryo-electron microscopy (cryo-EM) study by Wu et al. unveils the structural features of active Hrd1, providing mechanistic insights into the movement of proteins directed for degradation across ER membranes.

Formylation of Eukaryotic Cytoplasmic Proteins: Linking Stress to Degradation.

Unlike prokaryotes, N-terminal formylation has been confined to a handful of mitochondrial proteins in eukaryotes. A recent study unveils a new role for eukaryotic cytoplasmic N-terminal formylation linking diverse cellular stresses to N-terminal-dependent protein degradation. These findings suggest broad cellular implications in higher eukaryotes for N-terminal methionine formylation.

Degron-mediated proteolysis of CrhR-like DEAD-box RNA helicases in cyanobacteria.

Conditional proteolytic degradation is an irreversible and highly regulated process that fulfills crucial regulatory functions in all organisms. As proteolytic targets tend to be critical metabolic or regulatory proteins, substrates are targeted for degradation only under appropriate conditions through the recognition of an amino acid sequence referred to as a "degron". DEAD-box RNA helicases mediate all aspects of RNA metabolism, contributing to cellular fitness. However, the mechanism by which abiotic-stress modulation of protein stability regulates bacterial helicase abundance has not been extensively characterized. Here, we provide in vivo evidence that proteolytic degradation of the cyanobacterial DEAD-box RNA helicase CrhR is conditional, being initiated by a temperature upshift from 20 to 30 °C in the model cyanobacterium, Synechocystis sp. PCC 6803. We show degradation requires a unique, highly conserved, inherently bipartite degron located in the C-terminal extension found only in CrhR-related RNA helicases in the phylum Cyanobacteria. However, although necessary, the degron is not sufficient for proteolysis, as disruption of RNA helicase activity and/or translation inhibits degradation. These results suggest a positive feedback mechanism involving a role for CrhR in expression of a crucial factor required for degradation. Furthermore, AlphaFold structural prediction indicated the C-terminal extension is a homodimerization domain with homology to other bacterial RNA helicases, and mass photometry data confirmed that CrhR exists as a dimer in solution at 22 °C. These structural data suggest a model wherein the CrhR degron is occluded at the dimerization interface but could be exposed if dimerization was disrupted by nonpermissive conditions.

Does Too Much MAGIC Lead to Mitophagy?

Neurodegeneration-associated hallmarks include an abundance of protein aggregates and amelioration of mitochondrial function. Despite the knowledge of molecular counteracting mechanisms, the molecular dialogue between protein aggregate accumulation and aberrant mitochondrial import is poorly understood. Recent work unraveled a novel role for the mitochondrial import machinery in regulating cytosolic proteostasis.

The N-terminal domain of the Schaaf-Yang syndrome protein MAGEL2 likely has a role in RNA metabolism.

MAGEL2 encodes the L2 member of the melanoma-associated antigen gene (MAGE) protein family, truncating mutations of which can cause Schaaf-Yang syndrome, an autism spectrum disorder. MAGEL2 is also inactivated in Prader-Willi syndrome, which overlaps clinically and mechanistically with Schaaf-Yang syndrome. Studies to date have only investigated the C-terminal portion of the MAGEL2 protein, containing the MAGE homology domain that interacts with RING-E3 ubiquitin ligases and deubiquitinases to form protein complexes that modify protein ubiquitination. In contrast, the N-terminal portion of the MAGEL2 protein has never been studied. Here, we find that MAGEL2 has a low-complexity intrinsically disordered N-terminus rich in Pro-Xn-Gly motifs that is predicted to mediate liquid-liquid phase separation to form biomolecular condensates. We used proximity-dependent biotin identification (BioID) and liquid chromatography-tandem mass spectrometry to identify MAGEL2-proximal proteins, then clustered these proteins into functional networks. We determined that coding mutations analogous to disruptive mutations in other MAGE proteins alter these networks in biologically relevant ways. Proteins identified as proximal to the N-terminal portion of MAGEL2 are primarily involved in mRNA metabolic processes and include three mRNA N 6-methyladenosine (m6A)-binding YTHDF proteins and two RNA interference-mediating TNRC6 proteins. We found that YTHDF2 coimmunoprecipitates with MAGEL2, and coexpression of MAGEL2 reduces the nuclear accumulation of YTHDF2 after heat shock. We suggest that the N-terminal region of MAGEL2 may have a role in RNA metabolism and in particular the regulation of mRNAs modified by m6A methylation. These results provide mechanistic insight into pathogenic MAGEL2 mutations associated with Schaaf-Yang syndrome and related disorders.

An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21.

DDX21 is a newly discovered RNA G-quadruplex (rG4) binding protein with no known biological rG4 targets. In this study we used label-free proteomic MS/MS to identify 26 proteins that are expressed at significantly different levels in cells expressing an rG4-binding deficient DDX21 (M4). MS data are available via ProteomeXchange with identifier PXD013501. From this list we validate MAGED2 as a protein that is regulated by DDX21 through rG4 in its 5'-UTR. MAGED2 protein levels, but not mRNA levels, are reduced by half in cells expressing DDX21 M4. MAGED2 has a repressive effect on TRAIL-R2 expression that is relieved under these conditions, resulting in elevated TRAIL-R2 mRNA and protein in MCF-7 cells, rendering them sensitive to TRAIL-mediated apoptosis. Our work identifies the role of DDX21 in regulation at the translational level through biologically relevant rG4 and shows that MAGED2 protein levels are regulated, at least in part, by the potential to form rG4 in their 5'-UTRs.

Does N-Terminal Protein Acetylation Lead to Protein Degradation?

The N-end rule denotes the relationship between the identity of the amino-terminal residue of a protein and its in vivo half-life. Since its discovery in 1986, the N-end rule has generally been described by a defined set of rules for determining whether an amino-terminal residue is stabilizing or not. However, recent studies are revealing that this N-end rule (or N-degron concept) is less straightforward than previously appreciated. For instance, it is unveiled that N-terminal acetylation of N-terminal residues may create a degradation signal (Ac-degron) that promotes the degradation of target proteins. A recent high-throughput dissection of degrons in yeast proteins amino termini intriguingly suggested that the hydrophobicity of amino-terminal residues-but not the N-terminal acetylation status-may be the indispensable feature of amino-terminal degrons. Herein, these recent advances in N-terminal acetylation and the complexity of N-terminal degradation signals in the context of the N-degron pathway are analyzed.

Label-free proteomic analysis reveals large dynamic changes to the cellular proteome upon expression of the miRNA-23a-27a-24-2 microRNA cluster.

In deciphering the regulatory networks of gene expression controlled by the small non-coding RNAs known as microRNAs (miRNAs), a major challenge has been with the identification of the true mRNA targets by these RNAs within the context of the enormous numbers of predicted targets for each of these small RNAs. To facilitate the system-wide identification of miRNA targets, a variety of system wide methods, such as proteomics, have been implemented. Here we describe the utilization of quantitative label-free proteomics and bioinformatics to identify the most significant changes to the proteome upon expression of the miR-23a-27a-24-2 miRNA cluster. In light of recent work leading to the hypothesis that only the most pronounced regulatory events by miRNAs may be physiologically relevant, our data reveal that label-free analysis circumvents the limitations of proteomic labeling techniques that limit the maximum differences that can be quantified. The result of our analysis identifies a series of novel candidate targets that are reduced in abundance by more than an order of magnitude upon the expression of the miR-23a-27a-24-2 cluster.

The necdin interactome: evaluating the effects of amino acid substitutions and cell stress using proximity-dependent biotinylation (BioID) and mass spectrometry.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by the loss of function of a set of imprinted genes on chromosome 15q11-15q13. One of these genes, NDN, encodes necdin, a protein that is important for neuronal differentiation and survival. Loss of Ndn in mice causes defects in the formation and function of the nervous system. Necdin is a member of the melanoma-associated antigen gene (MAGE) protein family. The functions of MAGE proteins depend highly on their interactions with other proteins, and in particular MAGE proteins interact with E3 ubiquitin ligases and deubiquitinases to form MAGE-RING E3 ligase-deubiquitinase complexes. Here, we used proximity-dependent biotin identification (BioID) and mass spectrometry (MS) to determine the network of protein-protein interactions (interactome) of the necdin protein. This process yielded novel as well as known necdin-proximate proteins that cluster into a protein network. Next, we used BioID-MS to define the interactomes of necdin proteins carrying coding variants. Variant necdin proteins had interactomes that were distinct from wildtype necdin. BioID-MS is not only a useful tool to identify protein-protein interactions, but also to analyze the effects of variants of unknown significance on the interactomes of proteins involved in genetic disease.

A molecular toolbox for studying protein degradation in mammalian cells.

Protein degradation is a crucial regulatory process in maintaining cellular proteostasis. The selective degradation of intracellular proteins controls diverse cellular and biochemical processes in all kingdoms of life. Targeted protein degradation is implicated in controlling the levels of regulatory proteins as well as eliminating misfolded and any otherwise abnormal proteins. Deregulation of protein degradation is concomitant with the progression of various neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. Thus, methods of measuring metabolic half-lives of proteins greatly influence our understanding of the diverse functions of proteins in mammalian cells including neuronal cells. Historically, protein degradation rates have been studied via exploiting methods that estimate overall protein degradation or focus on few individual proteins. Notably, with the recent technical advances and developments in proteomic and imaging techniques, it is now possible to measure degradation rates of a large repertoire of defined proteins and analyze the degradation profile in a detailed spatio-temporal manner, with the aim of determining proteome-wide protein stabilities upon different physiological conditions. Herein, we discuss some of the classical and novel methods for determining protein degradation rates highlighting the crucial role of some state of art approaches in deciphering the global impact of dynamic nature of targeted degradation of cellular proteins. This article is part of the Special Issue "Proteomics".

The sole LSm complex in Cyanidioschyzon merolae associates with pre-mRNA splicing and mRNA degradation factors.

Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1-7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2-8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga Cyanidioschyzon merolae and found that it has only seven LSm proteins. The identities of CmLSm2-CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the C. merolae LSm complex and experimentally validate our bioinformatic predictions of a reduced spliceosome in this organism. Copurification of Pat1 and its associated mRNA degradation proteins with the LSm proteins, along with evidence of a cytoplasmic fraction of CmLSm complexes, argues that this complex is involved in both splicing and cytoplasmic mRNA degradation. Intriguingly, the Pat1 complex also copurifies with all four snRNAs, suggesting the possibility of a spliceosome-associated pre-mRNA degradation complex in the nucleus.

Isoniazid induces a monocytic-like phenotype in HL-60 cells.

Isoniazid (INH) is one of the oldest drugs for the treatment of tuberculosis (TB) and is of continual clinical and research interest. The aim of the current study is to investigate the ability of INH to induce monocyte differentiation and the underlying signaling pathway involved in this phenomenon using HL-60 cells. In this study, HL-60 cells were treated with different non-cytotoxic concentrations of INH or vitamin D (a well-known inducer of monocytic differentiation) to determine key functional changes in the phenotype of these cells using several biochemical and cytobiological experiments. HL-60 cells are derived from human promyelocytic leukemia and bear some resemblance to promyelocytes, which differentiate into various cell types. INH-induced differentiation was confirmed to occur in a concentration-dependent manner through several functional markers such as nonspecific esterase activity, NADPH oxidase activity and expression of surface markers CD14 and CD16 (characteristic of monocytes). INH-induced monocytic-like differentiation in HL-60 cells and demonstrated that at least 25% of cells were differentiated within the range of the pharmacological concentrations of INH. To determine the effects of INH on HL-60 cells, we applied quantitative proteomics that revealed 32 proteins were altered significantly in pathways that could involve differentiation signals. Lastly, INH activated the ERK-1/MAPK signaling pathway based on detection of phosphorylated ERK-1. These in vitro findings in HL-60 cells warrant further study using promyelocytes or hematopoietic stem cells to evaluate the physiological capability of INH to induce monocytic differentiation that may aid in host defense against TB.

Emerging branches of the N-end rule pathways are revealing the sequence complexities of N-termini dependent protein degradation.

The N-end rule links the identity of the N-terminal amino acid of a protein to its in vivo half-life, as some N-terminal residues confer metabolic instability to a protein via their recognition by the cellular machinery that targets them for degradation. Since its discovery, the N-end rule has generally been defined as set of rules of whether an N-terminal residue is stabilizing or not. However, recent studies are revealing that the N-terminal code of amino acids conferring protein instability is more complex than previously appreciated, as recent investigations are revealing that the identity of adjoining downstream residues can also influence the metabolic stability of N-end rule substrate. This is exemplified by the recent discovery of a new branch of N-end rule pathways that target proteins bearing N-terminal proline. In addition, recent investigations are demonstrating that the molecular machinery in N-termini dependent protein degradation may also target proteins for lysosomal degradation, in addition to proteasome-dependent degradation. Herein, we describe some of the recent advances in N-end rule pathways and discuss some of the implications regarding the emerging additional sequence requirements.

Regulating Apoptosis by Degradation: The N-End Rule-Mediated Regulation of Apoptotic Proteolytic Fragments in Mammalian Cells.

A pivotal hallmark of some cancer cells is the evasion of apoptotic cell death. Importantly, the initiation of apoptosis often results in the activation of caspases, which, in turn, culminates in the generation of proteolytically-activated protein fragments with potentially new or altered roles. Recent investigations have revealed that the activity of a significant number of the protease-generated, activated, pro-apoptotic protein fragments can be curbed via their selective degradation by the N-end rule degradation pathways. Of note, previous work revealed that several proteolytically-generated, pro-apoptotic fragments are unstable in cells, as their destabilizing N-termini target them for proteasomal degradation via the N-end rule degradation pathways. Remarkably, previous studies also showed that the proteolytically-generated anti-apoptotic Lyn kinase protein fragment is targeted for degradation by the UBR1/UBR2 E3 ubiquitin ligases of the N-end rule pathway in chronic myeloid leukemia cells. Crucially, the degradation of cleaved fragment of Lyn by the N-end rule counters imatinib resistance in these cells, implicating a possible linkage between the N-end rule degradation pathway and imatinib resistance. Herein, we highlight recent studies on the role of the N-end rule proteolytic pathways in regulating apoptosis in mammalian cells, and also discuss some possible future directions with respect to apoptotic proteolysis signaling.

Proteomic characterization of EL4 lymphoma-derived tumors upon chemotherapy treatment reveals potential roles for lysosomes and caspase-6 during tumor cell death in vivo.

The murine mouse lymphoblastic lymphoma cell line (EL4) tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research, little is known regarding the molecular details of in vivo tumor cell death. Here, we report the first in-depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label-free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the downregulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected downregulation of caspase-3 and an upregulation of caspase-6 in addition to a global upregulation of lysosomal proteins in the bulk of the tumor.

Phosphorylation Impacts N-end Rule Degradation of the Proteolytically Activated Form of BMX Kinase.

Cellular signaling leading to the initiation of apoptosis typically results in the activation of caspases, which in turn leads to the proteolytic generation of protein fragments with new or altered cellular functions. Increasing numbers of reports are demonstrating that the activity of many of these proteolytically activated protein fragments can be attenuated by their selective degradation by the N-end rule pathway. Here we report the first evidence that selective degradation of a caspase product by the N-end rule pathway can be modulated by phosphorylation. We demonstrate that the pro-apoptotic fragment of the bone marrow kinase on chromosome X (BMX) generated by caspase cleavage in the prostate cancer-derived PC3 cell line is metabolically unstable in cells because its N-terminal tryptophan targets it for proteasomal degradation via the N-end rule pathway. In addition, we have demonstrated that phosphorylation of tyrosine 566 relatively inhibits degradation of the C-terminal BMX catalytic fragment, and this phosphorylation is crucial for its pro-apoptotic function. Overall, our results demonstrate that cleaved BMX is a novel N-end rule substrate, and its degradation exhibits a novel interplay between substrate phosphorylation and N-end rule degradation, revealing an increasing complex regulatory network of apoptotic proteolytic signaling cascades.

Functional analyses of phosphorylation events in human Argonaute 2.

Argonaute 2 (Ago2) protein is a central effector of RNA interference (RNAi) pathways and regulates mammalian genes on a global level. The mechanisms of Ago2-mediated silencing are well understood, but less is known about its regulation. Recent reports indicate that phosphorylation significantly affects Ago2 activity. Here, we investigated the effect of mutating all known phospho-residues within Ago2 on its localization and activity. Ago2 associates with two different cytoplasmic RNA granules known as processing bodies (P-bodies) and stress granules, but the nature of this phenomenon is controversial. We report that replacing serine with a phospho-mimetic aspartic acid at position 798 completely abrogates association of Ago2 with P-bodies and stress granules. The effect of this mutation on its activity in gene silencing was modest, which was surprising because association of Ago2 with cytoplasmic RNA granules is thought to be a consequence of its role in RNAi. As such, our data indicate that targeting of Ago2 to P-bodies and stress granules is separable from its role in RNAi and likely requires dynamic phosphorylation of serine 798.

Cyanobacterial RNA Helicase CrhR Localizes to the Thylakoid Membrane Region and Cosediments with Degradosome and Polysome Complexes in Synechocystis sp. Strain PCC 6803.

UNLABELLED: The cyanobacterium Synechocystis sp. strain PCC 6803 encodes a single DEAD box RNA helicase, CrhR, whose expression is tightly autoregulated in response to cold stress. Subcellular localization and proteomic analysis results indicate that CrhR localizes to both the cytoplasmic and thylakoid membrane regions and cosediments with polysome and RNA degradosome components. Evidence is presented that either functional RNA helicase activity or a C-terminal localization signal was required for polysome but not thylakoid membrane localization. Polysome fractionation and runoff translation analysis results indicate that CrhR associates with actively translating polysomes. The data implicate a role for CrhR in translation or RNA degradation in the thylakoid region related to thylakoid biogenesis or stability, a role that is enhanced at low temperature. Furthermore, CrhR cosedimentation with polysome and RNA degradosome complexes links alteration of RNA secondary structure with a potential translation-RNA degradation complex in Synechocystis IMPORTANCE: The interaction between mRNA translation and degradation is a major determinant controlling gene expression. Regulation of RNA function by alteration of secondary structure by RNA helicases performs crucial roles, not only in both of these processes but also in all aspects of RNA metabolism. Here, we provide evidence that the cyanobacterial RNA helicase CrhR localizes to both the cytoplasmic and thylakoid membrane regions and cosediments with actively translating polysomes and RNA degradosome components. These findings link RNA helicase alteration of RNA secondary structure with translation and RNA degradation in prokaryotic systems and contribute to the data supporting the idea of the existence of a macromolecular machine catalyzing these reactions in prokaryotic systems, an association hitherto recognized only in archaea and eukarya.