PARP14 mediated SQSTM1/p62 cysteine ADP-ribosylation is counteracted by the SARS-CoV-2 macrodomain

PARP14 mediated SQSTM1/p62 cysteine ADP-ribosylation is counteracted by the SARS-CoV-2 macrodomain | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results PARP14 mediated SQSTM1/p62 cysteine ADP-ribosylation is counteracted by the SARS-CoV-2 macrodomain View ORCID Profile David Kubon , View ORCID Profile Deena M. Leslie Pedrioli , View ORCID Profile Michael O. Hottiger doi: https://doi.org/10.1101/2024.06.29.601315 David Kubon 1 Department of Molecular Mechanisms of Disease (DMMD); University of Zurich ; 8057 Zurich; Switzerland 2 Life Science Zurich Graduate School; Molecular Life Science Ph.D. Program; University of Zurich , 8057 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for David Kubon Deena M. Leslie Pedrioli 1 Department of Molecular Mechanisms of Disease (DMMD); University of Zurich ; 8057 Zurich; Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Deena M. Leslie Pedrioli Michael O. Hottiger 1 Department of Molecular Mechanisms of Disease (DMMD); University of Zurich ; 8057 Zurich; Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael O. Hottiger For correspondence: michael.hottiger{at}dmmd.uzh.ch Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Several ADP-ribosyltransferases are upregulated during viral infections and are crucial for the cellular immune response. While interferon-induced PARP14 ADP-ribosylates various substrates, viruses such as SARS-CoV-2 counteract this by reversing ADP-ribosylation. The exact mechanism of PARP14’s antiviral activity and the targets of viral macrodomains remain unknown. Here, we observe that PARP14 mono-ADP-ribosylates the selective autophagy adaptor SQSTM1/p62 at cysteine residues 113, 289/90, and 331 following interferon treatment. This correlates with the ADP-ribosylation of cytoplasmic p62 foci that colocalize with ubiquitin and PARP14 but not with LC3, thereby distinguishing them from classical autophagosomes. Moreover, the SARS-CoV-2 macrodomain effectively prevented this p62 modification, suggesting an antiviral function for this ADP-ribosylated target. Furthermore, our results indicate that TRIM21 prevents the autophagic degradation of ADP-ribosylated p62, suggesting that the identified p62 foci may have autophagy-independent roles. This study contributes to our understanding of the molecular dynamics involved in host-virus interactions and highlights the potential role of ADP-ribosylation in the regulation of innate immunity. INTRODUCTION ADP-ribosyltransferases (ARTs) play an important role in the regulation of cellular immune responses 1 – 3 . The diphtheria toxin-like ART subfamily (ARTD) comprises 17 members that catalyze the transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD + ) to protein and nucleic acid substrates, resulting in substrate ADP-ribosylation 4 . Remarkably, mono-ADP-ribosylating (MARylating) ARTs such as PARP9, 10, 12, 13, and 14 are highly upregulated during innate immune responses as a result of the activation of pattern recognition receptors (PRRs) following viral infection 4 , 5 . These MARylating ARTs are thought to play an essential role in the antiviral response by ADP-ribosylating both host and viral factors 6 . Viruses have evolved various strategies to evade the innate immune response, such as evading detection by PRRs, disrupting IFN-dependent signaling, or hijacking autophagy 7 , 8 , 9 . Certain virus families, such as Togaviridae, Hepeviridae, and the Coronaviridae (including SARS-CoV-2) encode macrodomains within their non-structural proteins that are tightly linked to ADP-ribose metabolism. These macrodomains can bind to and, in some cases, reverse ADP-ribosylation 10 – 12 . The SARS-CoV-2 non-structural protein 3 (nsP3) is a membrane-bound multi-domain protein that contains three macrodomains 13 , 14 . Macrodomain1 (Mac1) functions as a MAR hydrolase, a role conserved within the Coronaviridae family 15 , 16 . Studies have demonstrated the critical role of Mac1 for efficient viral replication and pathogenesis, both in vitro and in vivo 17 , 18 . However, the specific ADP-ribosylated host factors targeted by Mac1 remain to be defined. PARP14 is a mono-ADP-ribosyl transferase that plays a role in the regulation of immunity, transcription, DNA replication, and stress responses 19 – 24 . Recent studies have shown that PARP14 is a dual-function enzyme, exhibiting ADP-ribosyltransferase and -hydrolase activities 25 , 26 . In particular, the macrodomain 1 of PARP14 functions as an active ADP-ribosylhydrolase 25 , 26 . The role of PARP14 in innate immunity has been increasingly recognized, with its identification as one of 38 genes comprising the interferon-stimulated gene (ISG) signature across tumors 27 . It has also been explicitly implicated in the innate immune response by attenuating SARS-CoV-2 replication 28 . Furthermore, PARP14 is one of the few ARTs under positive selection in humans, suggesting an ongoing host-virus conflict 29 . Despite this growing evidence for its role in regulating host immunity, its ADP-ribosylation activity, substrate specificity, and the reversal mechanisms of PARP14-dependent ADP-ribosylation remain poorly understood. A recent study reported an increase in punctate cytosolic ADP-ribosylation foci in human A549 cells upon stimulation with interferons and the dsRNA mimetic poly(I:C) 30 . These foci did not colocalize with known cytoplasmic structures. The formation of these structures was associated with the PARP9/DTX3L complex, though the inactive catalytic role of PARP9 leaves the exact enzyme responsible for the modification unidentified 30 – 32 . Notably, overexpression of the SARS-CoV-2 macrodomain reversed the ADP-ribosylation foci, suggesting a potential antiviral function of these structures. Two recent publications reported that PARP14 is the major driver of IFN-induced ADP-ribosylation and that the PARP9/DTX3L complex regulates its activity via protein-protein binding and posttranslational mechanisms 33 , 34 . Autophagy is an evolutionarily conserved process with a widely recognized impact on both innate and adaptive immunity 35 , 36 . Selective autophagy is facilitated by autophagy receptors that specifically bind ubiquitinated substrates and sequester them into autophagosomes. Lysosomes fuse with autophagosomes and provide the environment and hydrolases for the degradation of the cargo 37 – 39 . Sequestosome 1 (SQSTM1)/p62 (hereafter p62) is an autophagy receptor involved in the autophagic clearance of ubiquitinated proteins, which are sequestered and degraded together with p62 40 , 41 . p62 contains several domains, including a Phox1 and Bem1p (PB1) domain, a microtubule-associated protein 1 light chain 3 (LC3)-interacting region (LIR), and a ubiquitin-associated (UBA) domain, all of which work together to facilitate selective autophagy 41 , 42 . p62 facilitates the collection and elimination of ubiquitinated proteins through the formation of p62-liquid droplets 43 . These droplets exhibit liquid-like properties formed by liquid-liquid phase separation induced by polyubiquitin chains and are essential to facilitate p62-mediated selective autophagy 42 – 46 . Its liquid-liquid phase separation behavior is controlled by various p62-binding partners and posttranslational modifications, thereby altering p62-mediated selective autophagy 47 – 50 . Here, we report that p62 is ADP-ribosylated on cysteine residues by PARP14 following interferon treatment, forming a significant proportion of ADP-ribosylated foci in the cytoplasm. ADP-ribosylated p62 is targeted by the SARS-CoV-2 macrodomain, which efficiently prevents this modification. These foci colocalize with ubiquitin and PARP14 but do not represent classical autophagosomes due to the absence of LC3. We show that autophagic turnover of ADP-ribosylated p62 is prevented by tripartite motif-containing protein 21 (TRIM21), suggesting that the modified p62 may have degradation-independent functions. RESULTS SQSTM1/p62 is mono-ADP-ribosylated at specific cysteine residues To gain further insights into how mono-ARTs regulate the innate immune response during viral infection in an ADP-ribosylation-dependent manner, we used A549 cells, a widely used model for SARS-CoV-2 infection 51 . This epithelial non-small cell lung cancer (NSCLC) cell line exhibits low basal expression of interferon-stimulated genes (ISGs) and low levels of ADP-ribosylation but shows robust induction of ISGs and ADP-ribosylation upon treatment with interferons and the dsRNA mimetic poly(I:C) 51 . First, we aimed to identify the targets that are ADP-ribosylated in a viral context. A549 cells were thus treated for 24 hours with IFNβ, a type I interferon that is remarkably suppressed during SARS-CoV2 infection to evade innate immunity 52 , 53 . Using an ADP-ribose-specific mass spectrometry (MS)-based proteomics approach 54 , we observed a dramatic change in ADP-ribosylated proteins (i.e., the ADP-ribosylome) in A549 cells comparing basal (untreated) to IFNβ-treated conditions. The modification profile shifted from predominantly detecting PARP1 and histones towards modifications on PARP12, DTX3L, and p62 after IFNβ treatment ( Fig. 1A ). While some physiological ADPr sites on PARP12 have been previously mapped 55 , both DTX3L and p62 emerged as novel proteins modified by ADPr. We focused on p62, which is modified exclusively at cysteine residues C113 and either C289 or C290 ( Fig. 1B ) in A549 cells due to p62’s well-described functions in viral clearance 56 , 57 . In addition, a modification at C331 was identified in NCI-H1975 58 , which is located in the LC3 interaction region (LIR). The remaining modifications are in intrinsically disordered regions near the ZZ-type zinc finger (ZZ domain) and the nuclear localization/export sequence (NLS/NES). These p62-specific cysteine residues were also identified in the NSCLC cell line NCI-H1975 58 under unstimulated conditions, supporting that these cells are in a viral mimicry primed state 59 . Download figure Open in new tab Figure 1: SQSTM1/p62 is ADP-ribosylated at cysteine residues in a PARP14 dependent manner (A) Pie chart showing unique ADP-ribosylated proteins identified by LC-MS/MS in A549 cells treated for 24h with IFNβ (1000U/mL), in comparison to untreated control cells. (B) p62 domain structure with annotated cysteine ADP-ribosylation sites identified by LC-MS/MS. Figure was created with a licensed version of BioRender.com (License number for Fig. 1B : LV26ZULJXG) (C) Immunoblot following p62 immunoprecipitation (IP) from A549 cells treated with IFNβ (1000U/mL) for 24h and detection of ADP-ribosylation using eAf1521. (D) p62 immunoprecipitation from A549 cells treated with IFNβ (1000U/mL) and the PARP14 inhibitor RBN012759 (20nM) for 24h. (E) Immunoblots of A549 cells following the knockdown of p62, PARP14, PARP9, and DTX3L for 48h. IFNβ and PARP14i treatment according to Fig. 1D . (F) Immunoblots of A549 cells with stable overexpression of a doxycycline-inducible FLAG-tagged SARS-COV-2 nsP3 macrodomain1 construct for 24h using 500ng/mL of doxycycline. Cells were co-treated with IFNβ (1000U/mL) and Remdesivir (20μM). To corroborate the MS results, we immunoprecipitated endogenous p62 and detected its ADP-ribosylation using the engineered macrodomain eAf1521 54 . Basal levels of p62 ADP-ribosylation were minimal, whereas IFNβ treatment significantly increased the amount of modified p62 ( Fig. 1C ). The IFNβ-dependent induction of p62 ADP-ribosylation after treatment suggests that the modification is catalyzed by one of the IFN-inducible mono-ARTs, which are strongly upregulated upon IFNβ treatment ( Fig. S1A ). This possibility is further supported by the observation that p62 ADP-ribosylation levels do not increase immediately but start to appear after 8 hours ( Fig. S1B ). Notably, this modification was induced not only by IFNβ but also by the type II interferon IFNγ and poly(I:C) ( Fig. S1C ), indicating that it has broad functional consequences given its induction by different stimuli. To confirm the ADPr-amino acid acceptor sites of p62 identified by mass spectrometry, we mutated the corresponding cysteine residues and complemented A549 cells with a doxycycline-inducible siRNA-resistant HA-tagged ADP-ribosylation-deficient p62 mutant (ΔADPR). Expression of the p62 mutant, while simultaneously depleting endogenous p62, revealed a loss of the p62 ADP-ribosylation signal upon overexpression of the ADP-ribosylation-deficient mutant compared to wildtype p62 ( Fig. S1E ). This indicates that the identified p62 ADP-ribosylation sites are indeed the major modification sites of p62. PARP14 modifies SQSTM1/p62 independently of PARP9 or DTX3L To identify the specific writer responsible for p62 modification, we focused on PARP14 despite the upregulation of other IFN-induced mono-ARTs (such as PARP9, PARP10, and PARP12) because it has been suggested to be a key enzyme in antiviral innate immunity 60 . Furthermore, the timing and magnitude of PARP14 up-regulation closely coincided with the observed p62 ADP-ribosylation ( Fig. S1B and S1D ). These findings were further strengthened by treating A549 cells with IFNβ alone and in combination with the selective PARP14 inhibitor RBN012759 61 (PARP14i). Immunoprecipitation of p62 followed by ADP-ribosylation detection using immunoblotting and eAf1521, revealed a complete loss of p62 modification upon combinatorial treatment with IFNβ and PARP14i ( Fig. 1D ), indicating that PARP14 is directly or indirectly (e.g., through another ART) responsible for the modification of p62. To investigate these possibilities, we performed a knockdown of PARP9 and its binding partner DTX3L, which has recently been described to form a complex with PARP14 and to cross-regulate each other’s protein levels 33 . Neither the knockdown of PARP9 nor DTX3L affected the ADP-ribosylation levels of p62. Loss of the modification could only be achieved by siRNA-mediated depletion of p62, PARP14 or by PARP14 inhibition ( Fig. 1E and S1F ). Taken together, these results indicate that PARP14 is likely to be the primary enzyme responsible for the ADP-ribosylation of p62. The ADP-ribosylation of SQSTM1/p62 is counteracted by the SARS-CoV-2 macrodomain To date, no human eraser of cysteine-modified ADP-ribosylation has been identified 62 . Based on recent reports that PARP9/DTX3L-associated ADP-ribosylation is reversed upon SARS-COV-2 overexpression 51 , we speculated that the ADP-ribosylation of p62 might be a target of this macrodomain. Overexpression of the nsP3 macrodomain1 of SARS-COV-2 in IFNβ treated A549 cells, indeed resulted in a reduction of p62 ADP-ribosylation ( Fig. 1F ). Co-treatment with Remdesivir, a SARS-CoV-2 macrodomain inhibitor 63 , rescued the loss of p62-ADP-ribosylation. These results suggest that the macrodomain targets p62 either directly by hydrolyzing its cysteine ADPr or indirectly by acting on PARP14. Cytoplasmic SQSTM1/p62 foci are ADP-ribosylated by PARP14 To determine whether the ADP-ribosylation of p62 would affect its cellular distribution and characteristics of p62 foci, cells were treated with IFNβ, both alone and in combination with the PARP14i. Our immunofluorescence results revealed that p62 exclusively localized to the cytoplasm, forming two distinct pools: phase-separated foci and a dispersed form throughout the cytoplasm ( Fig. 2A ). Notably, neither the amount of p62 nor the number of p62 foci showed any discernible change in response to different treatment conditions ( Fig. S2A and S2B ) indicating that IFNβ-induced ADP-ribosylation does not affect the formation of p62 foci. However, we observed an increased number of ADP-ribosylation foci upon IFNβ treatment, which colocalized with p62 foci ( Fig. 2A and 2B ). This effect was reversed by PARP14i treatment. Based on the amount of ADP-ribosylated p62 observed in whole cell lysates by immunoblotting ( Fig. 1E ), we concluded that these ADP-ribosylation foci consisted primarily of modified p62. Download figure Open in new tab Figure 2: SQSTM1/p62 forms cytoplasmic foci which co-localize with ADP-ribosylation, PARP14 and Ubiquitin (A/B) A549 cells were treated with IFNβ and RBN012759 (20nM) for 24h, and p62 localization and their colocalization with ADP-ribosylation foci were analyzed by immunofluorescence. Representative images (A) and quantification of p62 and ADP-ribosylation foci colocalization (B) are shown. White arrows indicate colocalizing foci and magnified areas of IFNβ treated cells are marked by yellow squares and depicted adjacent to Fig. 1A . (C) Representative immunofluorescence images of A549 cells treated according to Fig. 2A/B . For the last 4h of the treatment, co-treatment with leptomycin B (LepB) (30nM) was performed. (D) PARP14 and ADPr foci colocalization was analyzed by immunofluorescence in A549 cells treated according to Fig. 2A/B . Representative images are shown. One-way ANOVA determined P values for experiments with more than three conditions and multiple comparisons analysis using Tukey’s multiple comparison test. * p<0.05, ** p<0.005, *** p<0.0005, ns not significant. The scale bar represents 20μm. p62 is capable of shuttling between the cytoplasm and nucleus, thereby facilitating the degradation of ubiquitinated proteins in both compartments 64 . Given the proximity of the ADP-ribosylation sites C289/290 to the nuclear localization signal (NLS) and the nuclear export signal (NES) ( Fig. 1B ), we investigated whether ADP-ribosylation affects the localization of p62. Cells were thus treated with the nuclear export inhibitor leptomycin B, which traps p62 in the nucleus 64 . After 4 hours of treatment, p62 was found exclusively in the nucleus, unaffected by previous treatment with IFNβ and PARP14i ( Fig. 2C ). These findings indicate that IFNβ-induced ADP-ribosylation does not affect p62 shuttling behavior. PARP14 and ubiquitin colocalize with ADP-ribosylated p62 foci To further characterize the function of the detected p62 foci, we co-stained IFNβ-treated A549 cells for several markers. These experiments revealed that neither markers for mitochondria, autophagy, nor the endoplasmic reticulum (ER) were enriched in p62 foci ( Fig. S2C-E ), indicating that the ADP-ribosylated p62 foci represent a distinct p62 pool within the cell. To investigate whether PARP14 not only ADP-ribosylates p62 but also colocalizes with the observed p62 foci, we repeated the treatment of A549 cells with IFNβ alone and in combination with the PARP14i and stained the cells for PARP14 and ADPr ( Fig. 2D ). The modified foci colocalize with PARP14 in an IFNβ-dependent manner, supporting its role as the primary writer of p62 ADP-ribosylation. Interestingly, treatment of cells with PARP14i dissociated PARP14 from the foci, suggesting that the enzymatic activity of PARP14 is required for its recruitment to the p62 foci. Colocalization studies with ubiquitin revealed that the p62 foci are ubiquitinated ( Fig. 2E ) but independent of their ADP-ribosylation status, suggesting that the ubiquitination is not induced by the ADP-ribosylation of p62 by PARP14. ADP-ribosylated SQSTM1/p62 can participate in autophagy Despite the lack of colocalized p62 and LC3, selective autophagy is considered to be the primary function of p62 41 . Given the existing literature suggesting that the sites identified as ADP-ribosylated may have implications for autophagy 65 , 66 , we investigated the role of p62 in autophagy. To investigate whether ADP-ribosylation of p62 would prevent its involvement and degradation by autophagy, we blocked autophagy using the lysosomotropic agent chloroquine (CQ), which inhibits the fusion and maturation of endosomes and lysosomes with autophagosomes 67 . Co-treatment of A549 cells with IFNβ and CQ resulted in an accumulation of both total p62 and its ADP-ribosylated form ( Fig. 3A ). This observation suggests that despite the absence of LC3 accumulation at p62 foci, ADP-ribosylation of p62 does not prevent its interaction with LC3 or its degradation along with selected cargo. Using a GFP-Trap to pull down overexpressed LC3-EGFP, we demonstrated that both p62 and its ADP-ribosylated form do indeed interact with LC3 ( Fig. 3B ). Download figure Open in new tab Figure 3: Autophagic turnover of ADP-ribosylated p62 is prevented by TRIM21 (A) A549 cells were treated with IFNβ (1000U/mL) and 10μM of chloroquine (CQ) for 24h, followed by immunoblot analysis. (B) GFP-Trap co-immunoprecipitation of A549 cells with stable overexpression of a doxycycline-inducible LC3-EGFP construct and siRNA knockdown of p62 (48h). For the last 24h of the experiment, cells were treated with 500ng/mL doxycycline to induce LC3-GFP expression, IFNβ (1000U/mL), and RBN012759 (20nM). (C) Immunoblot of A549 cells after TRIM21 knockdown for 48h. Cells were co-treated with IFNβ (1000U/mL) and chloroquine (CQ) (10μM) for the last 24h of the experiment. ADP-ribosylated SQSTM1/p62 foci are prevented from autophagic degradation by TRIM21 Contrary to our expectations, we observed no significant changes in p62 protein levels upon treatment with IFNβ ( Fig. 3A ), although the existing literature suggests that type I interferons enhance autophagy to increase viral clearance and antigen presentation 68 . This could be explained by a factor(s) that actively prevent p62 from undergoing autophagic clearance, even when ADP-ribosylated (e.g., by ubiquitination). The tripartite motif-containing protein 21 (TRIM21), which is induced by IFNβ treatment in A549 cells 69 , has been shown to induce p62 ubiquitination within its PB1 domain, preventing self-oligomerization and autophagic targeting 69 . We therefore depleted TRIM21 to confirm that ADP-ribosylated p62 is being degraded by autophagy. Indeed, siRNA-mediated TRIM21 knockdown reduced both total p62 levels and the ADP-ribosylated form of p62 ( Fig. 3C ). The loss of ADP-ribosylated p62 was rescued by co-treatment of the cells with CQ, thereby supporting the evidence that modified p62 can participate in autophagy, but it appears to be reserved for autophagy-independent functions. SQSTM/p62 ADP-ribosylation does not affect its signaling functions in A549 cells Since IFN induced the ADP-ribosylation of p62, we investigated the signaling functions of p62 by analyzing the expression of NF-κB, NRF2, and ISG target genes. We chose CCL5 as an NF-κB target gene and a set of different ISGs that were reported to be upregulated in A549 upon IFNβ treatment 70 . p62 has multifaceted involvement in NF-κB signaling by interacting with aPKCs via its PB1 domain, RIP1 via its ZZ domain, and TRAF6 via its TB domain, all of which promote NF-κB signaling 71 , 72 . The crosstalk between NF-κB signaling and STAT1 signaling, which acts downstream of the interferon response, makes the study of target genes of both pathways critically crucial 73 . However, both p62 knockdown and PARP14 inhibition did not alter the inflammatory gene signature after IFNβ treatment ( Fig. S3A ). Therefore, we also tested the antioxidant NRF2 signaling pathway, which is known to be suppressed during SARS-CoV-2 infection 74 . p62 binds to NRF2s inhibitory subunit KEAP1 via its KEAP1-interaction region, leading to the degradation of KEAP1 and release of NRF2 71 . Again, we were unable to show any significant changes in response to either p62 knockdown or PARP14 inhibition on NRF2-dependent gene expression in A549 ( Fig. S3B ), suggesting that p62 ADP-ribosylation may be involved in other signaling pathways or some other unidentified function. DISCUSSION In this study, we have shown that the selective autophagy receptor SQSTM/p62 is ADP-ribosylated in different NSCLC cell lines. We showed that in A549 cells, this modification is absent under basal conditions but is induced by various inflammatory stimuli and is dependent on the catalytic activity of PARP14. The p62 modification occurred exclusively at cysteine residues, suggesting that PARP14 is a cysteine modifier. In addition, p62 was found to be a substrate for the SARS-CoV-2 macrodomain, demonstrating some selectivity as other interferon-induced ADP-ribosylated proteins remained unaffected. Furthermore, we showed that p62 foci are ADP-ribosylated and colocalize with PARP14 and ubiquitin but not with autophagy markers. Despite this modification, the autophagy function of p62 is unaffected. Interestingly, autophagy-mediated degradation of ADP-ribosylated p62 is prevented by TRIM21, suggesting that modified p62 has autophagy-independent functions. We validated the cysteine ADP-ribose acceptor sites by mutagenesis, which resulted in the loss of modification without affecting the physiological behavior of p62 (localization and ability to form foci) ( Fig. S2F ). In contrast to our results, the current literature suggests that PARP14 typically modifies substrates on acidic amino acid residues such as glutamic and aspartic acids. However, these previous studies were either based on in vitro modification of recombinant proteins 75 , or have been questioned due to potential confounding factors, such as nearby modifications by different posttranslational modifications (PTMs) 76 , 77 . Despite the strong evidence for the cysteine sites identified in this study, we cannot completely exclude modifications at other acceptor sites, particularly acidic residues. This is due to the labile nature of ADP-ribosylation on acidic residues 78 , which may be lost using current mass spectrometry enrichment strategies. Recent advances in detection methods are promising and may improve the identification of these labile modifications, potentially leading to the discovery of additional acceptor sites on p62 79 . Our results suggest that the SARS-CoV-2 macrodomain can either directly reverse cysteine modifications of p62 or remove ADP-ribosylation from PARP14, which may be necessary for its activity. In vitro de-modification assays would be essential to assess this possibility. Regardless of whether the macrodomain acts by a direct or indirect mechanism to prevent p62 modifications, it highlights an ongoing host-virus conflict and suggests a potential antiviral role for p62 ADP-ribosylation. The formation of p62 foci is well documented in the literature and is typically associated with autophagic turnover, as p62 aggregation is a prerequisite for this process 80 . It was therefore not surprising to observe both p62 foci and a dispersed form of p62 in the cytoplasm. However, the foci stained negative for the autophagosome marker LC3, ruling out their direct involvement in autophagy. Interestingly, we were able to colocalize these foci with ADP-ribosylation, which was highly enriched. However, we cannot exclude the possibility that the cytoplasmic pool of p62 (not associated with foci) is also ADP-ribosylated but undetectable due to the sensitivity limitations of our detection tools. Initially, we assumed that the ADP-ribosylation signal in these foci was entirely due to ADP-ribosylated p62, as it appeared to be highly modified based on immunoblot analysis ( Fig. 1E ). However, mutagenesis of the ADPr acceptor sites revealed that ADP-ribosylation enrichment persisted within these foci ( Fig. S2F ), suggesting the presence of additional ADP-ribosylated proteins. These could include DTX3L or PARP12, which were identified in our ADP-ribosylome ( Fig. 1A ), or PARP14, which is ADP-ribosylated according to immunoblot analysis ( Fig. 1E ). Furthermore, the foci were enriched for PARP14, confirming its role as the ADP-ribosylation writer, and ubiquitin. Ubiquitinated proteins aggregate within p62 foci 41 . However, p62 itself is also targeted by several ubiquitin ligases 81 . The signal identified by immunofluorescence could therefore be attributed either to aggregated ubiquitinated proteins or to the ubiquitination of p62 itself. Interestingly, the association of PARP14 with ADP-ribosylated foci was dependent on its catalytic activity ( Fig. 2D ), suggesting that either auto-modification of PARP14 or modifications of currently unknown proteins are essential for the recruitment of PARP14 to the foci. The cysteine sites identified, particularly C113 and C289/90, have previously been characterized to alter the autophagic functions of p62 65 , 66 . While the oxidation of redox sensitive C113 has been described to promote autophagy through the formation of disulfide-linked conjugates 65 , acylation at C289/290 has recently been reported to enhance autophagy by fostering the interaction with LC3 66 . Based on these findings, we postulated that ADP-ribosylation at these sites may regulate the autophagic turnover of p62 by potentially preventing its interaction with LC3, which may explain the observed lack of colocalization. Although LC3 was not enriched in the foci, p62 and its ADP-ribosylated form still participated in autophagic degradation ( Fig. 3A ), suggesting that autophagy occurs at a slow rate and, thus, the enrichment of LC3 is likely not detectable within the foci. This is confirmed by both our LC3-EGFP pulldown experiment and TRIM21 knockdown ( Fig. 3B and 3C ), both of which show that the involvement of p62 in autophagy is unaffected. Interestingly, LC3-EGFP pulldown revealed the co-immunoprecipitation of PARP9 and, to some extent, PARP14, suggesting that these proteins may also be degraded by autophagy, in addition to their described degradation by the proteasome 33 . However, the interaction of these proteins was independent of p62, suggesting another selective autophagy receptor that facilitates their degradation. We were particularly intrigued that TRIM21 appeared to prevent the autophagic turnover of ADP-ribosylated p62, suggesting that foci-bound p62 has a role independent of its receptor function. To explore this, we tested whether the associated NF-κB and NRF2 signaling functions of p62 were affected. We also assessed type I interferon-associated expression of ISGs. So far, we have not detected any gene expression changes within these signaling cascades, suggesting that p62 is either not involved in these signaling pathways or that the stimuli used in A549 were not optimal to reveal the potential modulatory role of p62. Alternatively, p62 may be involved in novel signaling pathways by providing a docking platform for proteins with ADP-ribosylation recognition domains, such as PARP9 and PARP14 themselves. In summary, these findings highlight p62 as a novel ADP-ribosylated protein that is dependent on the catalytic activity of PARP14. Understanding how these modifications affect p62’s functions will be crucial to elucidating potential host-virus conflicts and the mechanisms underlying the antiviral role of mono-ARTs. AUTHOR CONTRIBUTIONS Project conceptualization and administration: D.K. and M.O.H. (lead) Data curation and Formal analysis: D.K. (lead), D.M.L.P. (lead mass spectrometry) Investigation (specific experiments) : D.K. (lead) Visualization and validation : D.K. (lead) Methodology: D.K. (lead), D.M.L.P. (lead mass spectrometry) Writing, review & editing of MS : D.K. and M.O.H. (lead), D.M.L.P. (supporting) Declaration of interests: The authors declare no conflict of interest. Download figure Open in new tab Figure S1: SQSTM1/p62 is ADP-ribosylated at cysteine residues in a PARP14 dependent manner (A) Heatmap showing qPCR analysis in A549 cells treated with IFNβ (1000U/mL) for 24h. (B) Immunoblot analysis of A549 cells treated with IFNβ (1000U/mL) for up to 48h. Cells were harvested at the indicated time points post IFNβ treatment. (C) Immunoblot of A549 cells after p62 knockdown for 48h. For the last 24h of the experiment, cells were either treated with IFNβ (1000U/mL) or IFNγ (100U/mL). Poly(I:C) (10ng/mL) was transfected in the last 8h of the experiment. (D) qPCR analysis of A549 cells treated with IFNβ (1000U/mL) for up to 24h. Cells were harvested and analyzed at the indicated time points. (E) Immunoblot of stable A549 p62-HA Wildtype (WT) and A549 p62-HA ADPr-deficient mutant (ΔADPR) cell lines after knockdown of endogenous p62 for 48h. The ΔADPr mutant contains the following mutations: C113A, C289A, C290A and C331A. Cells were treated with IFNβ (1000U/mL) for the last 24h. Expression of the respective p62-HA construct was induced with 1μg/mL doxycycline for the last 6h of the experiment. (F) Knockdown of DTX3L shown in Fig. 1E was confirmed by qPCR analysis. P values were determined by a paired, two-tailed Student’s t-test. Download figure Open in new tab Figure S2: SQSTM1/p62 forms cytoplasmic foci which co-localize with ADP-ribosylation, PARP14 and Ubiquitin (A/B) Quantification of p62 foci number (A) and mean fluorescence intensity (MFI) of p62 foci (B) from the data sets shown in Fig. 2A . (C/D) Immunofluorescence images of A549 cells treated with IFNβ (1000U/mL) and RBN012759 (20nM) for 24h. (E) A549 LC3-EGFP cells were treated with 500ng/mL doxycycline for 24h to induce LC3-GFP expression. Cells were additionally treated with IFNβ (1000U/mL) and RBN012759 (20nM) for 24h. (F) Stable A549 p62-HA Wildtype (WT) and A549 p62-HA ADPr-deficient mutant (ΔADPR) cell lines after knockdown of endogenous p62 for 48h. The ΔADPr mutant contains the following mutations: C113A, C289A, C290A and C331A. Cells were treated with IFNβ (1000U/mL) for the last 24h. Expression of the respective p62-HA construct was induced with 1μg/mL doxycycline for the last 4h of the experiment. Download figure Open in new tab Figure S3: Autophagic turnover of ADP-ribosylated p62 is prevented by TRIM21 (A/B) Heatmap showing qPCR analysis of A549 cells treated with IFNβ (1000U/mL) and RBN012759 (20nM) for 24h. MATERIALS and METHODS Cell Culture Wildtype A549 cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS) and 5% (v/v) penicillin/streptomycin (P/S). Genetically engineered cell lines (A549 p62-HA, A549 p62-HA C113A, C289A, C290A, C331A mutan, A549 LC3-EGFP and A549 nsP3 Mac1) were grown in high-glucose DMEM medium supplemented with 10% FCS, 5% P/S and 30μg/mL Blasticidin (Invivogen). Transgene expression of genetically engineered cell lines was induced using doxycycline. Treatment time and concentration is indicated in the figure legends. All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO 2 . Generation of genetically engineered cell lines Inducible overexpression cell lines were generated by transposase-mediated integration. Therefore, the coding sequences of p62-HA (Addgene #28027), LC3-EGFP (Addgene #11546) and SARS-CoV-2 nsP3 Mac1 (Kind gift by Sarah Knapp, RWTH Aachen) were subcloned into a Sleeping Beauty vector (Addgene #60506). The Sleeping Beauty vector was co-transfected with a Sleeping Beauty Transposase (Addgene #34879) using Lipofectamine3000 (Thermo Fisher). 72h post-transfection, cells were subjected to Blasticidin selection. ADP-ribosylation-deficient p62-HA mutant (C113A, C289A, C290A, C331A) was generated by standard site-directed mutagenesis PCR. Drug treatment PARP14 was inhibited using 20nM of RBN012759 (MedChemExpress LLC, Monmouth Junctions, NJ). If not stated otherwise, RBN012759 was co-treated with the following p62-ADPr inducing agents at the following concentrations by direct addition to the culture medium IFNβ (1000U/mL, Peprotech), IFNγ (100U/mL, Peprotech). The p62-ADPr inducing agent Poly(I:C) LMW (Invivogen) was transfected using Lipofectamine3000 at a concentration of 10ng/mL. Treatment durations are indicated in the figure legends. siRNA transfection siRNA-mediated knockdowns were performed via reverse transfection using Lipofectamine RNAiMAX (Thermo Fisher). 10nM of siRNA were mixed with 3μL lipofectamine in 500μL serum-free OptiMEM and incubated for 20min at room temperature (RT). The transfection mix was added dropwise to the cells and incubated for 48h. A non-targeting siRNA (siMOCK) was used as a control for each experiment. Immunoblotting Cells were lysed with LDS lysis buffer (60mM Tris/HCL pH=6.5, 2% LDS, and 10% Glycerol) and denatured at 70°C for 3min. The samples were sonicated, and proteins were separated on 10% Bis-Tris gels at 100V. A wet transfer was performed onto PVDF membranes at 100V for 1.5h at room temperature (RT), and membranes were blocked with 5% non-fat milk in TBS-T for 1h at RT. Primary antibodies were diluted in 5% non-fat milk in TBS-T and incubated with the membrane at 4°C overnight. Membranes were then washed three times in TBS-T before being incubated with a secondary antibody diluted in TBS-T for 1h at RT. After three washes in TBS-T, specific proteins/bands were detected with the Odyssey infrared imaging system (LI-COR). The following primary and secondary antibodies were used at the indicated dilutions: rabbit anti-SQSTM1/p62 (ab155686, Abcam, 1:1000), mouse anti-SQSTM1/p62 (ab56416, Abcam, 1:1000), mouse anti-Tubulin (T6199, Sigma, 1:5000), rabbit anti-PARP9 (ab53796, Abcam, 1:250), rabbit anti-PARP14 (ab224352, Abcam, 1:500), rabbit anti-TRIM21 (12108-1-AP, Proteintech), rabbit anti-HA (ab9110, Abcam, 1:5000), mouse anti-FLAG (F3165, Sigma, 1:500), mouse anti-GFP (66002-1-Ig, Proteintech, 1:20000), mouse eAf1521 macrodomain (Hottiger laboratory, final concentration 0.4 ng/µL),), IRDye 800 CW goat anti-rabbit IgG (LI-COR, 1:10000) and IRDye 680RD goat anti-mouse IgG (LI-COR, 1:10000). Immunofluorescence (IF) Cells were grown on glass coverslips prior to the treatments. Following the treatments, cells were fixed with 4% formaldehyde in PBS for 20min at RT and permeabilized with 0.2% Triton-X in PBS for 10min. DMEM supplemented with FCS and 0.1% Triton-X was used as a blocking solution to block the cells for 1h at RT. Next, coverslips were incubated in primary antibody diluted in blocking buffer at 4°C overnight. Coverslips were washed three times with PBS and incubated with a secondary antibody diluted in a blocking buffer for 1h at RT. After three washes, coverslips were stained with a 0.1µg/mL DAPI solution in PBS for 20min at RT. Coverslips were again washed three times with PBS and mounted on microscope slides using Mowiol® 4-88. Images were acquired on an Olympus ScanR HCS and Leica Thunder microscope. Contrast and brightness adjustments were performed with Fiji(ImageJ) 82 , and the same settings were applied to the entire image set within one experiment. Quantifications of immunofluorescence colocalization and mean fluorescence intensity were performed with a custom CellProfiler 83 image analysis pipeline. The following primary and secondary antibodies were used for IF: rabbit anti-SQSTM1/p62 (ab155686, Abcam, 1:250), mouse anti-SQSTM1/p62 (ab56416, Abcam, 1:250), mouse anti-HA (901502, Biolegend, 1:500), rabbit anti-HA (ab9110, Abcam, 1:250), rabbit anti-PARP14 (ab 229756, Abcam 1:250), Mono- and polyubiquitinylated conjugates FK2 (BML-PW8810-0100, Enzo Life Sciences, 1:200), rabbit anti-Calnexin (2679, CellSignaling, 1:100), goat anti-rabbit IgG(H+L) Alexa Fluor 488 (A11008, Invitrogen, 1:250), goat anti-rabbit IgG(H+L) Alexa Fluor 488 (A11008, Invitrogen, 1:250), goat anti-mouse IgG (H+L) Cy3 (115-165-003, Jackson Immunoresearch, 1:250). Quantitative real-time PCR (qPCR) Isolation of RNA was performed with the NucleoSpin RNA II kit (Macherey-Nagel). RNA concentrations were determined by Nanodrop and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher/Applied Biosystems) according to the manufacturer’s instructions. cDNA was used for qPCR using the KAPA SYBR® FAST (Sigma Aldrich) master mix on a Rotor-Gene Q 2plex HRM system (Qiagen) or QuantStudio5 System (Applied Biosystems). qPCR data was normalized to RPS12, and ΔΔCt analysis was performed relative to the control sample. Primers are listed in Table 1 . View this table: View inline View popup Table 1: List of primers Immunoprecipitation (IP) Cells were lysed with non-denaturing lysis buffer to maintain protein-protein interactions (10mM Tris-HCL pH=7.5, 100mM NaCl, 0.5mM EDTA, 0.5% NP-40, 40μM PJ-34, 1μg Leupeptin, 1μg Pepstatin, 1μg Bestatin, 100μM PMSF) at 4°C for 30min on a head-over-tail rotor. Samples were subsequently centrifuged at 14000g for 15min at 4°C, and the supernatant was incubated with either 1μg of rabbit anti-SQSTM1/p62 antibody (ab155686, Abcam) or rabbit IgG antibody (PP64B, Millipore) as a control for 2h at 4°C. Protein A Dynabeads (Invitrogen) were equilibrated with wash buffer (NP-40 omitting lysis buffer), and 30μL of beads were subsequently added to the lysates and incubated for 1h at 4°C. Beads were then washed three times with wash buffer. 30μL of 2x LDS lysis buffer were added to the samples, boiled for 3min at 70°C, and analyzed by immunoblotting. GFP-Trap Immunoprecipitation Cells were harvested and processed as described for our standard immunoprecipitation (IP) protocol. For immunoprecipitation of GFP-tagged proteins, 25μL of GFP-Trap® Magnetic Particles M-270 (gtd, ChromoTek) were incubated with the lysate for 1h at 4°C. Samples were then again processed as previously described. ADPr-peptide enrichment coupled with liquid chromatography and mass spectrometry analysis Our peptide enrichment strategy and mass spectrometry workflow, previously detailed in another publication 84 , were consistently applied in the experiments conducted for this manuscript. Statistical Analysis Fluorescence intensity measurements were normalized, and the means of three independent experiments were compared. Pearson correlation was again calculated from the means of three independent experiments for statistical analysis. For all experiments with two groups, p values were determined by Student’s t-test with *, p<0.05; **, p <0.005; ***, p < 0.0005, **** p < 0.0001. For experiments with three or more groups, p values were determined by one-way ANOVA with multiple comparisons analysis. The exact statistical test is detailed in the figure legends. ACKNOWLEDGMENTS We thank Sudharshana Sundaresan, Sofia Andretta, Lukas Muskalla (University of Zurich) and Sarah Ochs (Charité Berlin) for providing editorial assistance. We thank the Center for Microscopy and Image Analysis (ZMB) of the University of Zurich (FGCZ) for their services and assistance. This work was supported by the Swiss National Science Foundation [31003A_176177 and 310030_205202 to M.O.H.]. REFERENCES ↵ Boehi , F. , Manetsch , P. & Hottiger , M. O . Interplay between ADP-ribosyltransferases and essential cell signaling pathways controls cellular responses . Cell Discovery 7 , 104 , doi: 10.1038/s41421-021-00323-9 ( 2021 ). OpenUrl CrossRef Zhu , H. , Tang , Y.-D. , Zhan , G. , Su , C. & Zheng , C . The Critical Role of PARPs in Regulating Innate Immune Responses . Frontiers in Immunology 12 , doi: 10.3389/fimmu.2021.712556 ( 2021 ). OpenUrl CrossRef ↵ Kunze , F. A. & Hottiger , M. O . Regulating Immunity via ADP-Ribosylation: Therapeutic Implications and Beyond . 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