Gwenaël Rabut
email: gwenael.rabut@univ-rennes1.fr

Gaëlle Le Dez

Rati Verma

Taras Makhnevych

Axel Knebel

Thimo Kurz

Charles Boone

Raymond J Deshaies

Matthias Peter
email: matthias.peter@bc.biol.ethz.ch

The TFIIH subunit Tfb3 regulates cullin neddylation

Cullin proteins are scaffolds for the assembly of multi-subunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely-varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING-domain of Hrt1 nor did it require the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient neddylation involves the RING-domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 as well as the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING-domain protein Tfb3 controls activation of a subset of cullins.

Introduction

Nedd8 (Rub1 in yeast) is the closest relative to ubiquitin and functions as a posttranslational protein modifier [START_REF] Rabut | Protein modifications: beyond the usual suspects' review series[END_REF]. The best-characterized Nedd8 substrates are cullins, a family of proteins that serve as scaffolds for the assembly of multi-subunit ubiquitin ligases (E3s). Cullin-based E3s interact with various substrate specificity factors to regulate diverse cellular processes including cell cycle progression, genome integrity and signal transduction [START_REF] Petroski | Function and regulation of cullin-RING ubiquitin ligases[END_REF][START_REF] Pintard | Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family[END_REF]. Neddylation of cullins occurs on a conserved lysine residue in their C-terminal domain (CTD), which triggers a structural reorganization that not only initiates the assembly of E3 complexes but also activates ubiquitin-transfer to the bound substrate [START_REF] Duda | Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation[END_REF][START_REF] Saha | Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation[END_REF][START_REF] Yamoah | Autoinhibitory regulation of SCFmediated ubiquitination by human cullin 1's C-terminal tail[END_REF].

Owing to the importance of neddylation in the control of cullin activity, it is critical to understand how this modification is regulated. The conserved CTD of cullins tightly interacts with a RING domain protein termed Hrt1 or Rbx1, which in turn recruits ubiquitinloaded conjugating enzymes (E2s) and constitutes the catalytic core of cullin-based E3s [START_REF] Seol | Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34[END_REF][START_REF] Skowyra | Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1[END_REF]. Hrt1/Rbx1 also functions as an E3 towards the Nedd8-conjugating enzyme Ubc12 and mediates cullin nedylation in vitro [START_REF] Scott | A dual E3 mechanism for Rub1 ligation to Cdc53[END_REF] and references therein). However, Hrt1/Rbx1 is not sufficient to regulate cullin neddylation in vivo. For instance, the yeast Dcn1 protein and its homologues are required for efficient neddylation of various cullins in different organisms [START_REF] Kim | SCCRO (DCUN1D1) is an essential component of the E3 complex for neddylation[END_REF][START_REF] Kurz | The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae[END_REF]. Dcn1 directly interacts with the CTD of the Cul1 orthologue Cdc53 [START_REF] Kurz | Dcn1 functions as a scaffold-type E3 ligase for cullin neddylation[END_REF] and orients Ubc12 to promote Hrt1-mediated Cdc53 neddylation [START_REF] Scott | A dual E3 mechanism for Rub1 ligation to Cdc53[END_REF]. Moreover, the human Dcn1 homologue DCNL3 recruits Cul3 to the plasma membrane and stimulates its neddylation [START_REF] Meyer-Schaller | The human Dcn1-like protein DCNL3 promotes Cul3 neddylation at membranes[END_REF], suggesting that Dcn1 family proteins promote cullin neddylation and activity at specific cellular localizations or in response to specific signals.

Here, we further characterized the mechanisms regulating cullin neddylation. Unlike Cdc53, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated in vivo on the same lysine residue. Surprisingly, these modifications do not require Dcn1 or fully functional Hrt1, but depend on the RING domain of Tfb3, a conserved subunit of the TFIIH complex involved in transcription initiation and nucleotide excision repair. In addition to Rtt101, Tfb3 also regulates neddylation and activity of Cul3, but not Cdc53. These results indicate that multiple pathways cooperate to regulate the activity of distinct cullins.

Results

Neddylation and ubiquitylation activate Rtt101

Rtt101 function is required for yeast survival upon DNA-replication stress induced by genotoxic drugs such as camptothecin (CPT, an inhibitor of topoisomerase 1) or methyl methanesulfonate (MMS, a DNA methylating agent) [START_REF] Luke | The cullin Rtt101p promotes replication fork progression through damaged DNA and natural pause sites[END_REF]. To dissect how neddylation of its conserved C-terminal lysine 791 (K791) contributes to Rtt101 function, we assayed the resistance of yeast mutants to CPT and MMS (Fig. 1A). In contrast to wild type, a Rtt101 mutant in which K791 was changed to an arginine (K791R) was only partially able to complement the phenotype of RTT101 inactivation. Surprisingly however, the CPT and MMS sensitivity of rub1Δ cells was comparable to wild type controls, indicating that K791, but not neddylation, is required for proper Rtt101 function. Indeed, while Rtt101(K791R) appeared as a single band by western blot, two forms of Rtt101 could readily be detected even in the absence of Rub1 or Ubc12 (Fig. 1B), implying that Rtt101 is not only neddylated but also subject to another modification which similarly depends on K791 [START_REF] Laplaza | Saccharomyces cerevisiae ubiquitin-like protein Rub1 conjugates to cullin proteins Rtt101 and Cul3 in vivo[END_REF]. Interestingly, the second modification of Rtt101 is drastically (but not fully) reduced in strains deleted for the ubiquitin-E2 Ubc4, but not its closest homologue Ubc5 (Fig. 1B; Suppl. Information, Fig. S1), suggesting that Rtt101 may also be ubiquitylated in vivo. To determine whether ubiquitin can indeed be conjugated to K791, we used plasmids expressing 9Myc-tagged ubiquitin (9Myc-Ubq) or control 9Myc-Rub1. Both constructs were conjugated to wild type Rtt101 but not to Rtt101(K791R) (Fig. 1C). Conjugation of 9Myc-Ubq to Rtt101 was observed in wild type cells, although to a lesser extent than in rub1Δ cells (Fig. 1C), indicating that Rtt101 ubiquitylation occurs even in the presence of Rub1. Altogether, these results imply that Rtt101-K791 can be specifically ubiquitylated in a Ubc4-dependent manner.

To determine whether ubiquitylation activates Rtt101, we assayed the involvement of Ubc4 in DNA-replication stress. While both rub1Δ and ubc4Δ cells are resistant to CPT (Fig. 1D), rub1Δ ubc4Δ double mutants were almost as sensitive to replication stress as Rtt101(K791R) expressing cells. This sensitivity phenotype is most likely due to the loss of Rtt101 modification as the CPT sensitivity of rub1Δ ubc4Δ rtt101(K791R) triple mutants was comparable to Rtt101(K791R)-expressing cells. These results indicate that either neddylation or ubiquitylation of Rtt101 can activate its function in vivo.

Modification of Cdc53 and Rtt101 in vivo do not require fully active Hrt1

The observation that Rtt101 can be ubiquitylated in vivo suggests that the modification of this cullin involves specific mechanisms. Indeed, while Dcn1 is required for efficient neddylation of Cdc53 [START_REF] Kurz | The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae[END_REF] (Fig. 2A), we observed that neither neddylation nor ubiquitylation of Rtt101 were altered in dcn1Δ cells (Fig. 2A). As Dcn1 is required to position Hrt1 to promote Cdc53 neddylation [START_REF] Scott | A dual E3 mechanism for Rub1 ligation to Cdc53[END_REF], we next tested whether Hrt1 is required for Rtt101 modification in vivo. As HRT1 is essential, we used three distinct point mutants of Hrt1 that differentially affect its activity (Fig. 2B). First, we mutated a conserved isoleucine (I57A) located in the RING interface with E2s. Corresponding mutations in Hrt1 orthologues considerably reduce neddylation of cullins in vitro [START_REF] Huang | E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification[END_REF]. Second, we mutated a conserved cysteine (C81Y) that coordinates one of the zinc ions that stabilize the RING fold, thereby greatly reducing the turnover of several Cdc53 substrates such as Cln2 [START_REF] Blondel | Isolation and characterization of HRT1 using a genetic screen for mutants unable to degrade Gic2p in saccharomyces cerevisiae[END_REF]. Third, we deleted an alanine (A51Δ) that lies within a loop at the junction between the RING domain of Hrt1 and the beta-strand that anchors Hrt1 to cullin CTD. While this mutation should not affect the interaction between the Hrt1 RING domain and E2s, it alters the conformation of the cullin/Hrt1 dimer. The corresponding mutation in human Rbx1 severely inhibits in vitro neddylation of Cul1 CTD, but does not affect in vitro ubiquitylation of model substrates [START_REF] Duda | Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation[END_REF]. Consistent with published observations, the efficiency of Rtt101 neddylation in vitro in crude protein extracts prepared from hrt1(I57A) and hrt1(C81Y) strains was strongly reduced (Fig. 2E). We also examined the effect of these mutations on Cdc4, an F-box protein that interacts with Cdc53 and is constitutively ubiquitylated and degraded throughout the cell cycle [START_REF] Galan | Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism[END_REF]. In contrast to the A51Δ loop mutant, I57A and C81Y RING mutants strongly impaired the turnover of Cdc4, resulting in its accumulation (Fig. 2C, Suppl. Information Fig. S2). Similar results were observed with Cln2 (Suppl. Information Fig. S2), confirming that the I57A and C81Y mutations strongly impair Hrt1 catalytic activity. Surprisingly however, Rtt101 and Cdc53 accumulated in their modified forms in these mutants (Fig. 2D). In contrast, the catalytically active A51Δ mutant was barely able to modify Rtt101, and Cdc53 neddylation was reduced (Fig. 2D). Thus, while Hrt1 is required for cullin neddylation in vitro, the steady state level of Rtt101 and Cdc53 neddylation in intact cells is not correlated with Hrt1 catalytic activity. This implies that additional mechanisms must exist to ensure efficient cullin neddylation in vivo.

Rtt101 and Cul3 modifications are regulated by the RING-domain protein Tfb3

As neither Dcn1 nor Hrt1 catalytic activity are sufficient to control Rtt101 modification in vivo, we decided to search for other regulator(s) of Rtt101 ubiquitylation and/or neddylation. A genetic screen did not identify such factors in a collection of 4308 non-essential yeast genes (see Suppl. Information online). We therefore examined whether essential genes with characteristic E3 features would regulate the modifications of Rtt101. Interestingly, we found that a mutation in the RING domain of Tfb3 (C16Y) significantly reduced Rtt101 neddylation and, to a lesser extent, ubiquitylation in vivo (Fig. 3A). Likewise, Rtt101 neddylation in vitro was strongly impaired in protein extracts prepared from tfb3(C16Y) mutant cells (Fig. 3B). While neddylation of Cdc53 was only slightly diminished in tfb3(C16Y) cells (Fig. 3A), neddylation of Cul3 required both Dcn1 and intact Tfb3, both in vivo and in vitro (Fig. 3D and3E). Interestingly, residual modification of Cul3 was detectable in both dcn1Δ and tfb3(C16Y) strains but not in the double mutant (Fig. 3D). In contrast, the residual modification of Rtt101 in the tfb3(C16Y) strain was not due to Dcn1 and, conversely, the residual neddylation of Cdc53 in the dcn1Δ strain was not dependent on Tfb3 (Suppl. Information, Fig. S3). Taken together, these results demonstrate that Tfb3 is a regulator of cullin neddylation and imply that different cullins are activated by distinct mechanisms in vivo.

We next asked whether Tfb3 is functionally linked to Rtt101 and Cul3. Interestingly, deletion of RUB1 strongly sensitized tfb3(C16Y) cells to CPT (Fig. 3C), indicating that, like Rtt101, Tfb3 contributes to the cellular resistance against DNA-replication stress. Following UV damage, Cul3 ubiquitylates the RNA polymerase II subunit Rpb1, which triggers its degradation by 26S proteasomes ( [START_REF] Verma | Cdc48/p97 mediates UV-dependent turnover of RNA Pol II[END_REF] and references therein). As expected, Rbp1 degradation was compromised in cells harboring non-neddylatable Cul3 (Cul3(K688R)) as well as in rub1Δ cells (Fig. 3F), demonstrating that Cul3 neddylation stimulates Rpb1 ubiquitylation. Interestingly, Rpb1 degradation upon UV damage was strongly impaired in tfb3(C16Y) cells, while deletion of DCN1 had little or no effect (Fig. 3F). Together, these results suggest that Tfb3 promotes Rtt101 neddylation to regulate DNA-replication stress, and that Tfb3 is needed for Cul3 neddylation, which promotes Rpb1 degradation upon UV damage.

Tfb3 Probably Regulates the Conjugation Step of Cullin Neddylation

Tfb3 is part of TFIIH, a well characterized complex involved in transcription initiation and nucleotide excision repair (NER) [START_REF] Mydlikova | Transcription factor IIH -the protein complex with multiple functions[END_REF]. To determine whether these functions of TFIIH are involved in Rtt101 regulation, we examined Rtt101 modification in various TFIIH mutants with impaired transcription or NER activities. For instance, ccl1-ts4, kin28-ts, rad3-14 and ssl2-ts mutants severely reduce general transcription activity [START_REF] Guzder | DNA repair gene RAD3 of S. cerevisiae is essential for transcription by RNA polymerase II[END_REF][START_REF] Lee | Transcriptional activation independent of TFIIH kinase and the RNA polymerase II mediator in vivo[END_REF][START_REF] Qiu | The Saccharomyces cerevisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II[END_REF][START_REF] Valay | Ccl1, a cyclin associated with protein kinase Kin28, controls the phosphorylation of RNA polymerase II largest subunit and mRNA transcription[END_REF][START_REF] Valay | The KIN28 gene is required both for RNA polymerase II mediated transcription and phosphorylation of the Rpb1p CTD[END_REF], while tfb1-1, tfb5Δ or rad2Δ mutants exhibit NER defects [START_REF] Habraken | Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome[END_REF][START_REF] Matsui | An interaction between the Tfb1 and Ssl1 subunits of yeast TFIIH correlates with DNA repair activity[END_REF][START_REF] Ranish | Identification of TFB5, a new component of general transcription and DNA repair factor IIH[END_REF]. However, none of these mutations affected the total level of K791 modification or ubiquitylation (Fig. 4A and Suppl. Information Fig. S4A. Furthermore, previously published microarray experiments showed that general transcription is not impaired in tfb3(C16Y) cells [START_REF] Jona | Mutations in the RING domain of TFB3, a subunit of yeast transcription factor IIH, reveal a role in cell cycle progression[END_REF]. Together this indicates that the defect of Rtt101 modification in tfb3(C16Y) cells is unlikely to be caused by general transcription or NER defects.

Loss of Rtt101 neddylation in tfb3(C16Y) cells could be due to decreased activity of neddylation activator(s) or due to increased activity of neddylation inhibitor(s). Currently, the only known inhibitors of cullin neddylation in yeast are the COP9 signalosome (CSN), a protein complex that catalyzes cullin deneddylation [START_REF] Cope | Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1[END_REF], and Lag2, an inhibitor of Cdc53 that blocks access to its neddylation site [START_REF] Liu | A longevity protein, Lag2, interacts with SCF complex and regulates SCF function[END_REF][START_REF] Siergiejuk | Cullin neddylation and substrate-adaptors counteract SCF inhibition by the CAND1-like protein Lag2 in Saccharomyces cerevisiae[END_REF]. If one of these factors was activated in tfb3(C16Y) cells, its inactivation should restore Rtt101 neddylation. However, neither deletion of RRI1, the catalytic subunit of the CSN, nor deletion of LAG2 rescued Rtt101 modification in tfb3(C16Y) cells (Fig. 4B). Furthermore in vitro neddylation of tagged Rtt101 was not significantly inhibited by a 4-fold excess of protein extract prepared from tfb3(C16Y) cells (Suppl. Information Fig S4B ), excluding the possibility that tfb3(C16Y) cells contain a trans-inhibitor of Rtt101 neddylation. We conclude that Tfb3 directly or indirectly activates the Rtt101 neddylation and ubiquitylation.

The fact that Cdc53 neddylation was almost normal in tfb3(C16Y) cells (see Fig. 3A, 4B and Suppl. Information, Fig. S3) indicates that Tfb3 functions downstream of Ubc12. Indeed, Ubc12 was correctly expressed and efficiently charged with Rub1 in tfb3(C16Y) cells (Fig. 4C). To examine whether the RING domain of Tfb3 could function as a Rub1 or ubiquitin E3, we probed its interaction with E2s using recombinant proteins. Both GST-tagged Ubc12 and Ubc4, but not Rad6, were able to bind Hrt1 and the RING domain of Tfb3 (Fig. 4D). In contrast, the RING domain of Ssl1 -another TFIIH subunit previously reported to function as a ubiquitin E3 [START_REF] Takagi | Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage[END_REF]-was preferentially precipitated by Ubc4 (Fig. 4D). To determine whether Tfb3 also physically interacts with the Rtt101/Hrt1 complex, we performed GST pull-down experiments. As shown in Fig. 4E, recombinant GST-Hrt1, either alone or in complex with the Rtt101 CTD, was able to specifically retain tagged Tfb3 in yeast extracts. Interestingly, a 4-fold excess of protein extract containing wild type Tfb3 was not able to significantly rescue neddylation of tagged Rtt101 from tfb3(C16Y) extracts (Suppl. Information Fig. S4C). Together, these results suggest that the RING-finger protein Tfb3 regulates Rtt101 modification in "cis" and may promote Rtt101 neddylation and ubiquitylation through the interaction of its RING domain with E2 conjugating enzymes.

Discussion

Our finding that ubiquitylation of lysine 791 activates the yeast cullin Rtt101 in vivo reveals an unexpected mode of cullin regulation. As ubiquitylation of this residue is functionally redundant with its neddylation, both modifications may share a common activation mechanism. The recently solved structure of the neddylated CTD of Cul5 demonstrated that neddylation triggers a conformational switch that favors substrate ubiquitylation [START_REF] Duda | Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation[END_REF][START_REF] Saha | Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation[END_REF][START_REF] Yamoah | Autoinhibitory regulation of SCFmediated ubiquitination by human cullin 1's C-terminal tail[END_REF]. In this structure, Cul5 interacts with several residues of Nedd8 that are conserved in ubiquitin [START_REF] Duda | Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation[END_REF], including Leu8 and Ile44 that are important to promote Cul1-activity in vitro [START_REF] Sakata | Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullinbased E3 ligase activity[END_REF]. It is therefore likely that ubiquitylation of Rtt101 triggers a similar conformational switch as neddylation, and ubiquitylation of Cul1 can indeed activate substrate ubiquitylation in vitro [START_REF] Duda | Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation[END_REF]. It will therefore be important to determine whether cullins other than Rtt101 can also be regulated by ubiquitylation in vivo and whether cullin ubiquitylation and neddylation can be independently regulated.

Since Hrt1/Rbx1 neddylates cullins in vitro, it is generally assumed that this protein also governs cullin neddylation in vivo. Surprisingly, our results show that catalytic mutants of Hrt1 greatly impair cullin neddylation in vitro but not in vivo. To explain this discrepancy, we propose that Hrt1-dependent neddylation observed in vitro may reflect a basal activity of Hrt1, while intact cells require additional factors such as Dcn1 and Tfb3 to ensure efficient neddylation of specific cullins. Indeed, Tfb3 is needed for efficient neddylation of Cul3 and for neddylation and ubiquitylation of Rtt101, two yeast cullins regulating DNA-associated processes. Tfb3 is an essential subunit of the TFIIH complex, which mediates transcription initiation and is also involved in nucleotide excision repair (NER) [START_REF] Mydlikova | Transcription factor IIH -the protein complex with multiple functions[END_REF]). Because we have not found other mutations in TFIIH subunits that affect Rtt101 modification, it is unlikely that activation of Rtt101 and Cul3 neddylation is linked to TFIIH function in transcription initiation or NER. However, we currently do not know whether TFIIH integrity is needed for modifying Rtt101 and Cul3 in vivo. As Tfb3 functions downstream of Ubc12 and contains a RING domain that is characteristic of E3s, it is tempting to speculate that Tfb3 itself may function as an E3 for a subset of cullins. In agreement with this model, we observed that Tfb3 interacts with Ubc12 and Ubc4, and also binds Rtt101 in complex with Hrt1. Alternatively, Tfb3 may promote cullin modification like Dcn1 by stimulating Hrt1-mediated cullin neddylation. Finally, it is possible that Tfb3 does not act at the modification step per se, but may rather alter the cullin conformation to a "neddylatable" state. Consistent with this mechanism, we were unable to rescue Rtt101 neddylation in extracts prepared from tfb3(C16Y) cells with wild type Tfb3.

Irrespective of the precise molecular mechanism of Tfb3 function, our data demonstrate that Tfb3 and Dcn1 differentially regulate cullin modification. While efficient Rtt101 modification only requires Tfb3, Cul3 neddylation requires both Tfb3 and Dcn1, and Cdc53 neddylation primarily depends on Dcn1. Interestingly, all three cullins maintained low level of modification in either DCN1 or TFB3 mutated cells. The remaining modification of Rtt101 in the tfb3(C16Y) strain may be due to residual activity of Tfb3 in this strain or it may reflect the existence of additional activation mechanisms.

In conclusion, our findings imply that cells use distinct pathways to activate cullins by covalent modification of their C-terminus. As each cullin regulates different cellular processes, this separation allows activating a specific subset of cullin complexes in response to different signals. For example, it is tempting to speculate that DNA/chromatin-associated functions of cullins may be controlled by a Tfb3-dependent neddylation mechanism. Additional mechanisms that affect particular cullin complexes include sequestration by the assembly inhibitor CAND1/Lag2, and stable binding of some members to the CSN complex [START_REF] Olma | An interaction network of the mammalian COP9 signalosome identifies Dda1 as a core subunit of multiple Cul4-based E3 ligases[END_REF]. Together with regulated binding of substrates and substrate-specific adaptor modules, these mechanisms collectively orchestrate the spatial and temporal ubiquitylation of substrates.

Experimental procedures

Yeast strains and plasmids

All yeast strains used in this study are haploid derivatives of either BY4743 (ATCC 201390) or W303, and are listed in Suppl. Information Table S1. They were grown at 30°C and manipulated using standard techniques. For the spotting experiments (Fig. 1 and3) photographs of the plates were taken after an incubation of ~60h at 30°C. A list of all plasmids used in this study is provided in Suppl. Information Table S2.

Cullin neddylation in crude yeast protein extracts

Concentrated yeast protein extracts were prepared as described [START_REF] Kong | Incision of a 1,3-intrastrand d(GpTpG)-cisplatin adduct by nucleotide excision repair proteins from yeast[END_REF]. These extracts were then diluted in neddylation buffer (20mM Hepes pH 7.5, 100mM NaCl, 10mM MgCl 2 ) to a final protein concentration of 25μg/μL. Neddylation of cullins present in the extract was started by addition of 1μM recombinant Rub1 and 2mM ATP. The reactions were incubated at 30°C for 15min to 1h, stopped with 2× Laemmli loading buffer, and boiled for 5min at 95°C.

Interaction of E2 conjugating enzymes with RING domain containing proteins

Plasmids expressing GST, GST-Ubc12, GST-Ubc4 or GST-Rad6 were co-transformed with plasmids expressing Protein A (PA)-tagged Hrt1, PA-Tfb3(RING) or PA-Ssl1(RING) into E. coli BL21(DE3)-RIL cells. Proteins were expressed using the auto-induction method. After an overnight incubation at 25°C, cells were harvested, resuspended in 125mM Tris pH 7.5, 50mM NaCl, 5mM mercaptoethanol and 2mM PMSF, and lysed by sonication. The extracts were clarified and diluted to contain similar concentration of GST-tagged E2 conjugating enzymes. Diluted extracts were then incubated with glutathione beads for 1h at room temperature. The beads were then washed extensively in a buffer containing 50mM Tris pH 7.5, 200mM NaCl, 0.1% NP40 and 5mM mercaptoethanol, resuspended in sample buffer, and processed for immunoblotting.

GST-binding assays of Tfb3 from yeast extracts

GST, GST-Hrt1 and GST-Hrt1 co-expressed with the Rtt101 C-terminal domain were produced in E. coli BL21(DE3)-RIL cells using the auto-induction method. After an overnight incubation at 18°C, cells were harvested, resuspended in lysis buffer (50 mM Tris pH 7.5, 200mM NaCl, 10mM mercaptoethanol, and 2mM PMSF) and lysed by sonication. The extracts were clarified and incubated with glutathione beads for 1h at room temperature. The beads were washed extensively with lysis buffer and resuspended in 50mM Tris pH 7.5, 150mM NaCl, 0.5% Triton X100, and 5mM mercaptoethanol. A fraction of the beads was then used to pull-down Protein A-Tfb3 from a yeast protein extract. Approximately 10 8 cells expressing Tfb3-TAP were harvested, resuspended in 300μL of yeast lysis buffer (50mM Tris pH 7.5, 150mM NaCl, 0.5% Triton X100, 10mM mercaptoethanol, and a protease inhibitor cocktail (Complete EDTA-free, Roche)), and lysed with a FastPrep® instrument (QBiogene) in the presence of 1mL of 0.5mm glass beads (20s, speed 4.5). After 1h rotation at room temperature, the glutathione beads were extensively washed with the yeast lysis buffer, resuspended in sample buffer, and further processed for immunoblotting.

Mol Cell. Author manuscript. (E) Hrt1 and the Rtt101/Hrt1 complex physically interact with Tfb3. Recombinant GST, GST-Hrt1 and a complex of GST-Hrt1 and the Rtt101 CTD was immobilized on glutathione beads (GSH-beads), and incubated with extracts prepared from yeast cells expressing protein A-tagged Tfb3. Proteins bound to GSH beads were detected by Coomassie staining (upper panel) and immunoblotting (bottom panel).

HHMI Author Manuscript

HHMI Author Manuscript

HHMI Author Manuscript

Highlights•

  Ubiquitylation and neddylation of Rtt101 activate its function in vivo • Rtt101 modifications neither require Dcn1 nor fully active Hrt1 • Tfb3 regulates the modifications and activities of Rtt101 and Cul3

Figure 1 .

 1 Figure 1. Neddylation and ubiquitylation of lysine 791 activate Rtt101 (A) Rtt101 lysine 791 but not neddylation is required for yeast resistance to replication stress. Equivalent amounts of the indicated yeast strains were spotted in 5-fold serial dilutions onto YPD plates supplemented as indicated with MMS or CPT. More details about the yeast strains used in each figure panel can be found in the Suppl. Information online. (B) The Rub1-independent modification of Rtt101 depends on Ubc4. Immunoblot of total extracts prepared from yeast strains with either wild type (wt) or the K791R mutant of tagged Rtt101 expressed from the endogenous promoter. See also Suppl. Information, Figure S1. (C) Rtt101 is ubiquitylated in a K791-dependent manner. Immunoblot showing the modification of Rtt101 in cells expressing 9Myc-tagged ubiquitin or 9Myc-tagged Rub1. (D) Neddylation and ubiquitylation redundantly activate Rtt101. Equivalent amounts of the indicated yeast strains were spotted in 5-fold serial dilutions onto YPD plates, supplemented with 20μg/mL CPT.

Figure 2 .

 2 Figure 2. Rtt101 modification in DCN1 and HRT1 mutant cells (A) Rtt101 neddylation and ubiquitylation is not impaired in dcn1Δ cells. Immunoblots showing the modification of Rtt101 and Cdc53 in the indicated yeast strains. (B) Position of I57A, C81Y and A51Δ mutations depicted on the structure of Rbx1 (red) in complex with neddylated Cul5 CTD (green) (DOI:10.2210/pdb3dqv/pdb). (C) Substrate ubiquitylation is impaired in hrt1(C81Y) and hrt1(I57A) cells. Immunoblot showing the steady state-level of Cdc4 in the indicated yeast strains. Actin was used to control equal loading. See also Suppl. Information, Fig. S2. (D) Rtt101 and Cdc53 modification is not impaired in hrt1(C81Y) and hrt1(I57A) cells. Immunoblot showing the modification of Rtt101 and Cdc53 in the indicated yeast strains. Actin was used to control equal loading. (E) Kinetics of Rtt101 neddylation in vitro. Immunoblots showing Rtt101 modification in crude extracts after addition of recombinant Rub1 and ATP.

Figure 3 .

 3 Figure 3. Tfb3 controls Cul3 and Rtt101 modification, and contributes to their function (A) Rtt101 modification is impaired in tfb3(C16Y) cells. Immunoblots showing the modification of Rtt101 and Cdc53 in the indicated yeast strains. See also Suppl. Information, Fig. S3. (B) Kinetics of Rtt101 neddylation in tfb3(C16Y) extracts. Immunoblots showing Rtt101 modification in crude extracts after addition of recombinant Rub1 and ATP. (C) Tfb3 contributes to the cellular resistance to replication stress. Equivalent amounts of the indicated yeast strains were spotted in 5-fold serial dilutions onto YPD plates, supplemented with 10μg/mL CPT. (D) Cul3 neddylation is impaired in dcn1Δ and tfb3(C16Y) cells. Immunoblot showing Cul3 neddylation in the indicated yeast strains. (E) Cul3 neddylation in vitro. Immunoblot showing Cul3 modification in crude extracts 1h after addition of recombinant Rub1 and ATP. (F) Tfb3 is needed for Rpb1 degradation after UV irradiation. Immunoblots showing Rpb1 levels following UV irradiation in the indicated yeast strains. Tubulin was used to control equal loading.

Figure 4 .

 4 Figure 4. Tfb3 functions downstream of Ubc12 and interacts with Ubc12, Ubc4 and the Rtt101/Hrt1 complex (A) Mutations in TFIIH subunits other than Tfb3 do not impair Rtt101 modification. Immunoblots showing the modification of Rtt101 in the indicated yeast strains. See also Suppl. Information, Fig. S4A. (B) Rri1 and Lag2 do not inhibit Rtt101 modification in tfb3(C16Y) cells. Immunoblots showing the modification of Rtt101 and Cdc53 in the indicated yeast strains. (C) Ubc12 is properly charged with Rub1 in tfb3(C16Y) cells. Immunoblots of DTT-or TCEP-treated total extracts showing the amount of Rub1 charged Ubc12 (Ubc12~Rub1) in the indicated yeast strains. Contrary to TCEP, DTT hydrolyses the thioester bond of Ubc12 charged with Rub1. Actin was used to control equal loading. (D) The RING domain of Tfb3 interacts with Ubc12 and Ubc4. Protein A-tagged Hrt1 (PA-Hrt1), and the RING-domains of Tfb3 (PA-Tfb3(RING)) or Ssl1 (PA-Ssl1(RING)) were expressed in E. coli together with GST, GST-Ubc12, GST-Ubc4 or GST-Rad6. Protein extracts prepared from these cells were incubated with glutathione beads (GSH beads) to precipitate GST-tagged E2 as well their binding partners. Proteins bound to GSH beads were visualized by Coomassie staining (upper panel) and immunoblotting (middle panel). An

Mol Cell. Author manuscript.

Acknowledgments

We thank C. Rupp, C. Zbinden and R. Oania for technical assistance, and B. Schulman and members of the Peter laboratory for helpful discussions. G. Rabut was recipient of an HFSP Long-Term fellowship and is currently supported by INSERM and Région Bretagne. Work in the Deshaies and Peter laboratories is supported by the Howard Hughes Medical Institute, and by research grants from Oncosuisse, the Swiss National Science Foundation and the ETHZ, respectively.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


Rodolphe ; Anty

Sophie Marjoux

Bekri Soumeya

Laurianne De Galleani

Raffaella Dainese

Eve Gelsi

Faredj Cherikh

Albert Tran

Thierry Piche
email: piche.t@chu-nice.fr

Degalleani

Cherikh

Piche

Plasma carnitine is associated with fatigue in chronic hepatitis C but not in irritable bowel syndrome

Keywords: Irritable bowel syndrome < Disease-based, Hepatitis C < Hepatology, Liver < Organ-based, Functional GI diseases Hepatitis C, IBS, fatigue, carnitine, leptin IBS: irritable bowel syndrome

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Fatigue is one of the most common complain reported by patients in primary care settings [START_REF] Kroenke | Chronic fatigue in primary care[END_REF] . This persistent feeling of exhaustion and decrease capacity for physical and mental work is a ground state for many diseases and common symptom of various chronic digestive illness such as chronic hepatitis C (CHC) [START_REF] Piche | Fatigue is associated with high circulating leptin levels in chronic hepatitis C[END_REF] , primary biliary cirrhosis 3 1 , and also functional bowel disorders, namely the irritable bowel syndrome (IBS) [START_REF] Piche | Fatigue in irritable bowel syndrome: characterization and putative role of leptin[END_REF] . To date, no proper fatigue-related biochemical changes have been established, therefore identifying alterations of biochemical parameters would be helpful to understand the complexity of fatigue in the hope for future therapies.

As described by Swain and Maric 5 6 , three major central changes have been implicated; (i) neuroendocrine causes, with abnormal function of the hypothalamic-pituitary-adrenal (HPA) axis; (ii) abnormalities in neurotransmission, particularly in the central serotonin pathway and (iii) alteration in immune activation and cytokines release, in particular the adipokine leptin.

For example, in patients with chronic liver diseases, we have shown that the severity of fatigue was associated with high leptin levels in both primary biliary cirrhosis and CHC, in the latter, with a concomitant increase of the tumor necrosis factor α (TNF-α) levels, one major secretagogue of leptin [START_REF] Piche | Fatigue is associated with high circulating leptin levels in chronic hepatitis C[END_REF] . We have also subsequently found a close association between the severity of fatigue and plasma leptin levels adjusted for fat-mass in a cohort of patients with IBS, suggesting a non-specific contribution of leptin in the complexity of fatigue associated with digestive and liver diseases [START_REF] Piche | Fatigue in irritable bowel syndrome: characterization and putative role of leptin[END_REF] . F o r P e e r R e v i e w 5 L-carnitine is an endogenous compound absorbed in the intestine by a combination of active transport and passive diffusion [START_REF] Li | The effect of enteral carnitine administration in humans[END_REF] . The heart, skeletal muscle, liver, kidneys and epididymis have specific transport system for carnitine that concentrate carnitine within these tissues.

Carnitine's primary mechanism of action is apparently attributable to its role as a cofactor in the transformation of free long-chain fatty acids into acyl carnitine for subsequent transport into the mitochondrial matrix [START_REF] Jogl | Structure and function of carnitine acyltransferases[END_REF] . The well-established roles in energy production and modulation of the mitochondrial coenzyme A (CoA)/acyl-Coa ratio in the skeletal muscle, suggests that carnitine supplementation might increase fatty acid oxidation, thus making more ATP available for mechanical work and delaying fatigue development [START_REF] Swart | The effect of L-carnitine supplementation on plasma carnitine levels and various performance parameters of male marathon athletes[END_REF] . In human, a serum acylcarnitine deficiency has been observed in certain types of diseases such as patients with chronic fatigue syndrome 10 and CHC [START_REF] Kuratsune | Low levels of serum acylcarnitine in chronic fatigue syndrome and chronic hepatitis type C, but not seen in other diseases[END_REF] . However, plasma carnitine levels have never been evaluated in patients having IBS and fatigue. Interestingly, administration of carnitine to healthy elderly subjects has been shown to exert a favorable effect on fatigue and serum lipids [START_REF] Pistone | Levocarnitine administration in elderly subjects with rapid muscle fatigue: effect on body composition, lipid profile and fatigue[END_REF] . Moreover, carnitine supplementation is relevant for the fatigue treatment of cancer chemotherapy [START_REF] Graziano | Potential role of levocarnitine supplementation for the treatment of chemotherapy-induced fatigue in non-anaemic cancer patients[END_REF] , multiple sclerosis [START_REF] Tomassini | Comparison of the effects of acetyl L-carnitine and amantadine for the treatment of fatigue in multiple sclerosis: results of a pilot, randomised, double-blind, crossover trial[END_REF] , fibromyalgia [START_REF] Rossini | Doubleblind, multicenter trial comparing acetyl l-carnitine with placebo in the treatment of fibromyalgia patients[END_REF] haemodialysis [START_REF] Bellinghieri | Correlation between increased serum and tissue L-carnitine levels and improved muscle symptoms in hemodialyzed patients[END_REF] , celiac disease [START_REF] Ciacci | L-Carnitine in the treatment of fatigue in adult celiac disease patients: a pilot study[END_REF] and also in patients with CHC who received interferon therapy [START_REF] Neri | L-carnitine decreases severity and type of fatigue induced by interferon-alpha in the treatment of patients with hepatitis C[END_REF] . Interestingly, experimental findings support that carnitine palmitotransferase-1 linked fatty acid oxidation is a key modulator of leptin expression [START_REF] Wolkowicz | Activation of leptin expression by an inhibitor of carnitine palmitoyltransferase-1[END_REF] . In healthy ponies, carnitine supplementation has been associated with enhanced glucose tolerance and increase in leptin concentration [START_REF] Van Weyenberg | Increased plasma leptin through l-carnitine supplementation is associated with an enhanced glucose tolerance in healthy ponies[END_REF] .

In this study, we aimed at determining the contributory role of plasma levels of carnitine on fatigue in patients with untreated CHC and IBS.

Patients and Methods

Participants

Forty two patients with IBS (34F, 8M, mean-age 50.7±13.6 yrs) were included prospectively in the study during a 6-month recruitment period. The presence of IBS was determined according to the Rome II criteria following exclusion of organic disease. These criteria required the presence of abdominal pain or discomfort relieved by defecation or associated with change in stool form or frequency, together with at least two of the following symptoms: altered stool frequency (> 3 per day or < 3 per week), altered stool passage (straining/urgency), passage of mucus, bloating and/or abdominal distension. These symptoms had to be present more than 25% of the time. Patients were diagnosed with predominant constipation (IBS-C) if they reported abdominal pain or discomfort associated with a primary alteration in bowel function consistent with constipation (i.e. infrequent hard stools or increased straining to pass stools). Patients were categorized with predominant diarrhea (IBS-D) if they reported abdominal pain or discomfort associated with primary alterations in bowel function consistent with diarrhea (i.e. frequent loose or watery stools and urgency). Patients with alternating symptoms (IBS-A) of constipation and diarrhea were also included with at least one of the criteria required for the diagnosis of IBS-C or IBS-D.

The following exams were performed: laboratory blood tests (including erythrocyte sedimentation rate, C reactive protein, hemoglobin level, leukocyte count, electrolytes, liver function tests, pancreatic amylase, Human imunodeficiency and hepatitis C and B viruses, and thyroid function tests); a gastroscopy with biopsy specimen of the duodenum; an abdominal ultrasound or a computed tomography; and finally a fecal culture (also looking for Eighty one patients admitted to our liver unit for compensated liver disease due to CHC (47F, 34M, mean age 43.7±12.3 years) were included in the study. The diagnosis of CHC was based on the association of: (a) elevation of serum alanine aminotransferase (ALT) above 40 U/l (upper normal limit) for six months or longer, (b) presence of anti-hepatitis C virus (HCV) antibodies, (c) presence of HCV viraemia, and (d) exclusion of other causes of chronic liver disease (alcoholism, chronic hepatitis B, Wilson's disease, hepatotoxic drugs, haemochromatosis, α1 antitrypsin deficiency, autoimmune chronic active hepatitis). A liver biopsy was performed in all patients, none of whom was cirrhotic. These patients had no evidence of dehydration or overhydration or any other acute or chronic disease suspected of causing hypermetabolism (including human immunodeficiency virus). Furthermore, none had renal failure, thyroid disease, or clinically apparent depression, and none was treated with beta blockers or had received antiviral therapy.

Forty-four healthy volunteers (34F, 8M, mean age 46.5±17.3 yrs) were recruited by advertisement during the same period and formed the control group. These subjects were considered normal on the basis of history, physical exam and biochemical blood screening and were enrolled if they did not have any digestive symptom. None of them experienced fatigue and depression that were excluded by a psychiatrist. All clinical studies were performed according to the declaration of Helsinki, and all participants gave their written informed consent. Participants were systematically asked for fatigue and were convened to complete a selfreported measure pertaining to the psychological state (e.g. fatigue). Fatigue was measured using the French version of the Fatigue Impact Scale (FIS) questionnaire which was used by us in patients with chronic hepatitis C 2 and IBS [START_REF] Piche | Mast cells and cellularity of the colonic mucosa correlated with fatigue and depression in irritable bowel syndrome[END_REF] . FIS is a self-report questionnaire consisting of 40 statements that describes possible manifestations of fatigue; these statements are divided in three categories (cognitive

Questionnaires

[n=10], physical [n=10], psychosocial [n=20] ).
Each item is rated on a five-point scale of distress, ranging from 0 ("no problem") to 4

("extreme problem") with a maximum of 160 points. In addition to the 40 items, the FIS asks for the frequency of fatigue in terms of days per month and the usual duration of fatigue per day. The FIS also includes a visual analog scale (VAS) allowing evaluation of the "disabling effect" of fatigue on a scale of 1 ("no disabling effect") to 10 ("severe disabling effect").

Bioelectrical impedance analysis

Body composition was measured in the morning, after a 12-hours overnight fast (including alcohol and drugs and no smoking) in the Functional Explorations Unit of the department of Gastroenterology. Body composition was measured by bipolar bioelectrical impedance analysis (BIA) with an alternating electric current (50 µA) at two frequencies: 1 MHz and 5 kHz, as previously described and validated by Boulier et al [START_REF] Boulier | Fat-free mass estimation by the two-electrode impedance method[END_REF] . Measurements were taken in the conditions described hereafter. The subjects were supine for 30 min, arms relaxed along their side but not touching the body. Body mass index (BMI) is expressed as weight per height squared (kg/m 2 ). All plasma samples were collected at 08:00 AM in both IBS and controls and were stored in duplicate at -80°C until assayed. Plasma cortisol, ACTH and TSH levels were used to evaluate basal HPA axis as well as thyroid functions. Serum leptin levels were measured using a specific radioimmunoassay for human leptin (Linco Research, St. Charles, MO, USA) with a limit of detection of 0.5 ng/mL. The intra-and inter-assay coefficients of variation were less than 8%. Acylcarnitines samples were prepared for analysis as following: 10µL of plasma sample is extracted with 100 µl of methanolic solution containing stable isotopelabeled acylcarnitines internal standards (Cambridge Isotope Laboratories, Inc. #NSK-B) for 20 min at ambient temperature.

Plasma biochemical analysis

The methanolic eluates were dried under nitrogen, and reconstituted with 100 µl of mobile phase (water/acetonitrile v/v, 0.02% formic acid) prior to analysis.

Electrospray MS/MS was performed on an API3000 model tandem mass spectrometer (Applied Bioystems, Foster City, CA). Acylcarnitine standards including free, acetyl, propionyl, butyryl, hexanoyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl, palmitoyl and stearoyl carnitines were purchased from Dr. HJ ten Brink (VU Medical Center, Amsterdam, The Netherlands) and allowed to establish standard curves for quantitation.

Statistical analysis

Quantitative data were expressed as mean ± SD and were compared using the Mann-Whitney U-test. The Chi-2 test was used to compare qualitative data. The independent predictors of fatigue were determined using a multiple logistic regression model. A p value < 0.05 was considered significant.

Results

Characteristics of the study population

The characteristics of the study populations are presented in Table 1. The percentage of female was significantly (p=0.0002) more elevated in patients with IBS (80.9%) than in patients with CHC (42%) or controls (52,2%). Patients with CHC were significantly younger than IBS patients (43.7±12.3 vs 50.7±13.6, years p=0.03). Patients with IBS had significantly higher BMI than patients with CHC or controls (27±5 vs 24±4 and 23±4 kg/m 2 , p=0.008 vs CHC and p=0.007 vs controls, respectively).

Evaluation of fatigue

Overall, 46% of patients with CHC and 55% of patients with IBS verbally expressed fatigue and rated more than 4 on the visual analog scale. Among IBS patients, 20% considered that fatigue was the worst symptom of their disease whereas 45% observed that it was as disabling as their digestive symptoms. The total score of fatigue was significantly higher in IBS patients (68.4±32.9) and CHC patients (56.0±39.8) than in controls (15.5±11.6, p<0.0001 vs IBS and CHC). Fatigue scores were not different between IBS subtypes. Table 2 presents fatigue scores according to the sex of both patients and controls. There were no significant differences between patients with CHC or IBS among each subscale of fatigue (Figure 1). 3 in both male and female patients and controls.

Carnitine and leptin assessments

Absolute plasma leptin levels were significantly higher in IBS patients (32.8±26.2 ng/ml) than in patients with CHC (15.0±19.4 ng/ml, p<0.0001 vs IBS) and controls (7.4±5.2 ng/ml, p<0.0001 vs CHC). When adjusted for fat mass or BMI, leptin levels were significantly higher in IBS (1.8±2.8 ng/mL.Kg -1 and 11.6±9.4 ng/mL.Kg/m 2 respectively) than in CHC (0.6±0.6 ng/mL.Kg -1 and 5.9±7.0 ng/mL.Kg/m 2 p<0.0001 and p=0.0002 respectively) or in CHC than in controls (0.5±0.4 ng/mL.Kg -1 and 3.0±2.1 ng/mL.Kg/m 2 , p=NS and p=0.03 vs CHC respectively).

Absolute plasma levels of total carnitine were similar among patients with CHC (44.7±10.1 ng/mL), IBS (47.0+10.1ng/mL) and controls (47.1±10.1 ng/mL, p=NS). Similarly, absolute plasma levels of free carnitine were not significantly different among patients with CHC (36.0±8.5 ng/mL), IBS (36.7±8.9 ng/mL) and controls (36.7±8.9 ng/mL). When adjusted for fat mass, total and free carnitine levels were significantly lower in CHC (2.7±1.6 and 2.2±1.3 ng/mL.Kg -1 ) than in controls (4.5±4.3 and 3.5±3.6 ng/mL.Kg -1 , p=0.02 and p=0.02 respectively). In IBS patients, there were a statistical trend favor to lower total and free carnitine levels compared to controls (2.9±4.7 and 2.4±4.6 ng/mL.Kg -1 , p=0.08 and p=0.12 respectively) (Figure 2).

Correlation analysis

In patients with CHC, the total score of fatigue was significantly correlated with leptin (r=0.30, p=0.009), leptin adjusted for fat mass (r=0.31, p=0.01). The total score of fatigue 12 also correlated with total (r=-0.41, p=0.0003) and free (r=-0.43, p=0.0001) carnitine levels even when adjusted for fat mass (r=-0.23 p=0.03 and r=-0.22, p=0.04) (Figure 3). There were no significant relationship between plasma levels of carnitine and the degree of fibrosis and/or inflammation observed in liver biopsy specimens.

In patients with IBS, the total score of fatigue was significantly correlated with leptin (r=0.31, p=0.04), leptin adjusted for fat mass (r=0.25, p=0.03), but not with total or free carnitine levels even when adjusted for fat mass.

In CHC patients, the following parameters were included in a multiple logistic regression model: METAVIR score activity and fibrosis, leptin, BMI, fat mass, age, sex, and free carnitine levels. Only free carnitine levels were significantly and independently associated with the severity of fatigue (OR=2.019, p=0.02, CI 95% [1.01-1.23]).

Discussion

In the present study, we showed that fatigue, evaluated with the Fatigue Impact Scale questionnaire, altered equally patient's quality of life in both CHC and IBS. We found that plasma leptin was associated with the severity of fatigue in both CHC and IBS. Moreover, our findings revealed a carnitine deficiency in patients with CHC, but not with IBS, and that carnitine levels were negatively correlated with fatigue scores in CHC only. Whether carnitine is specifically related to fatigue in CHC remains to be further elucidated.

Because chronic fatigue is commonly reported in more than 20% of people [START_REF] Cullen | Prevalence of fatigue in general practice[END_REF] , it is necessary to study what cause fatigue and to develop treatments for it. To date, no specific fatigue related changes have been clearly established, leading to major limitations to determine the influence of fatigue on the disease process. Thus, identifying alterations of biochemical parameters would enable to understand the underlying mechanisms of fatigue. In the present study, the Fatigue Impact Scale questionnaire allowed an evaluation of the perceived impact of fatigue on patients' -lives, the factors that affect patients' perceptions of fatigue, and the affect of fatigue on the mental and general health of patients. As previously reported, we found that the severity of fatigue was markedly increased in both CHC and IBS patients, particularly in females, involving all three domains exploring quality of life (i.e. cognitive, physical, and psychosocial) 4 2 24 . We also retrieved significant positive correlations between plasma leptin levels adjusted for fat mass and the total score of fatigue in both patients with CHC and IBS further arguing for the role of the adipokine leptin in the sequence leading to fatigue. In the current study, patients with CHC, but not with IBS, had significant lower levels of total and free carnitine adjusted for fat mass as compared to healthy subjects. These findings are in accordance with those of Kuratsune et al. [START_REF] Kuratsune | Low levels of serum acylcarnitine in chronic fatigue syndrome and chronic hepatitis type C, but not seen in other diseases[END_REF] who found low levels of serum acylcarnitine (e.g. carnitine deficiency) in chronic fatigue syndrome and CHC, but not in other diseases. In our study, all patients were evaluated in same conditions, completing questionnaires in the morning and having no major changes of their daily activities (e.g. physical & psychosocial) and/or dietary habits in the past 4 weeks. Although the mechanism involved in the occurrence of carnitine deficiency in CHC has not been investigated in the present study, it could be cautiously speculated that virus C infection may alter the physiological process of carnitine biosynthesis in the liver.

In our study, we found significant negative correlations between circulating carnitine levels (e.g.total and free) and fatigue scores in patients with CHC but not in IBS. Moreover, in a logistic model of regression, free carnitine was significantly associated with fatigue, independently from age, sex, fat mass, METAVIR score (both activity and fibrosis), BMI and leptin. In patients with CHC, similar strength of associations between carnitine levels and each domain of fatigue were observed (data not showed), thus arguing for a non-specific contributory role of carnitine in the development of both mental and physical fatigue.

Although our study cannot address further the mechanism underlying fatigue in CHC, it is tempting to speculate on the relationship between carnitine and liver inflammation. Indeed, Selimoglu et al. [START_REF] Selimoglu | Plasma and liver carnitine levels of children with chronic hepatitis B[END_REF] found that plasma carnitine levels were lower in children with chronic hepatitis B compared with healthy ones and that carnitine was inversely correlated with liver portal inflammation and fibrosis scores. In the present work, although significant lower levels of carnitine adjusted for fat mass were observed in CHC patients, we found no relationship between carnitine and the degree of liver fibrosis and/or inflammation. In fact, we believe that Our study has also other limitations. In the present work, we did not evaluate numerous other biological candidates that could play a role in the development of fatigue in human [START_REF] Nozaki | Mental and physical fatigue-related biochemical alterations[END_REF] .

Moreover, a more marked effect of carnitine on physical fatigue was not explored since all patients and controls were not evaluated while on physical tasks [START_REF] Nozaki | Mental and physical fatigue-related biochemical alterations[END_REF] . We did not perform specific dynamic challenges of the HPA axis and repeated circulating levels of both leptin and carnitine which would have been helpful to identify more subtle biological disturbances [START_REF] Cleare | The neuroendocrinology of chronic fatigue syndrome[END_REF] .

Moreover, we did not analyze liver biopsy specimens, which would have also been relevant to consider the relationship between immune activation, plasma cytokines and carnitine. Finally, increasing the sample size of patients would have probably unmask significant higher levels of carnitine in male patients in particular in the IBS group.

The present findings have important and practical clinical consequences in the hope to reduce fatigue and increase adherence to antiviral therapy in CHC. Indeed, there are few available effective therapies for fatigue associated with CHC. We have previously documented that the 5-hydroxytryptamine receptor type 3 antagonist ondansetron had a significant positive effect on fatigue in CHC. However, its use may be impinged by a relative high rate of constipation that occurred in patients who received active drug [START_REF] Piche | Effect of ondansetron, a 5-HT3 receptor antagonist, on fatigue in chronic hepatitis C: a randomised, double blind, placebo controlled study[END_REF] . Given the fact that emerging evidence from an uncontrolled study [START_REF] Neri | L-carnitine decreases severity and type of fatigue induced by interferon-alpha in the treatment of patients with hepatitis C[END_REF] has suggested that CHC patients treated with interferon in association with carnitine (2g daily) showed a marked and early significant reduction of fatigue levels, our findings provide good reasons to pursue larger trials on the possible benefit of carnitine supplementation in CHC patients reporting fatigue. In conclusion, we have shown that fatigue alters equally patient's quality of life in both CHC and IBS. Our findings suggest that carnitine may play a contributory role in the occurrence of fatigue associated with CHC but not in IBS. Whether the relationship between fatigue and carnitine involves a specific pathway in CHC requires further investigations. Finally, the benefit of carnitine supplementation may be evaluated in future randomized trials aimed at reducing fatigue and increasing adherence to available antiviral therapies.

Financial Support

This study was partly supported by a grant from Sigma Tau company.

  parasites). A colonoscopy was performed in all patients and biopsy specimens were taken if diarrhea was predominant.

  sex dependency of body composition, leptin and carnitine biological findings are presented in Table

  interpreted with caution since blood samples were not concomitant to liver biopsy (e.g. up to 8 months), liver contents of carnitine were not assessed and only 13% of CHC patients had a METAVIR score activity up to 2.

Figure 1 :Figure 2 :

 12 Figure legends:

Figure 3 :

 3 Figure 3: Correlation between the total score of fatigue and absolute plasma levels of total and free carnitine (left of the panel) and total and free carnitine adjusted for fat mass (right of the panel) in patients with chronic hepatitis C.

Table 1 :

 1 Characteristics of patients with hepatitis C, irritable bowel syndrome and controls.

			Hepatitis C	IBS		Controls	P value
	Sex (F/M)		34/47	34/8		23/21	<0.001
	Age (years)		43.7±12.3 a	50.7±13.6	46.5±17.3	a 0.03 vs IBS
	BMI (Kg/m 2 )	24±4	27±5	a	23±4	a	0.008	vs
							hepatitis C and
							0.007 vs controls
	Fat mass (Kg)	20.3±9.6 a	25.2±11.6 b	16.3±9.2	a 0.04 vs controls
							and 0.02 vs IBS
							b	0.0002	vs
							controls
	Fat-free	mass	49.4±11.3	46.9±9.1	50.8±9.4	NS
	(Kg)

Table 2 :

 2 Fatigue scores in patients with chronic hepatitis C, irritable bowel syndrome and controls according to the sex.

			CHC			IBS		Controls
		F	M	p	F	M	p	F	M	p
	Total score	77.1±38.4 42.6±36.2 0.001 71.1±34.1 56.0±26.3 0.2 15.9±10.7 15.9±12.6 0.8
	psychosocial	36.7±19.3 19.5±19.0 0.001 35.2±18.6 23.0±17.3 0.2 7.4±7.5	6.1±5.4	0.3
	physical	21.8±9.6	12.8±9.7	0.002 19.3±9.5	17.4±7.7	0.5 4.5±3.5	4.9±4.7	0.8
	cognitive	18.6±11.5 10.3±8.8	0.002 18.4±9.4	12.4±8.3	0.1 3.9±3.9	4.8±4.4	0.5

Table 3 :

 3 Plasma levels of leptin and carnitine in patients with chronic hepatitis C, irritable bowel syndrome and controls according to the sex.

			CHC			IBS		Controls
		F	M	p	F	M	p	F	M	p
	Leptin	24.6±23.9 7.8±10.9 0.0002	38.0±26.2 9.2±3.5	0.02 9.4±4.3	5.4±5.4	0.02
	(ng/mL)
	Leptin/BMI	9.0±6.7	2.9+3.7c 0.0001	13.8±9.6 3.4±1.5	0.02 4.1±2.0	2.0±1.7	0.005
	(ng/mL
	Kg/m 2 )
	Leptin/FM	1.0±0.6	0.3±0.4	0.0001	2.1±3.0	0.4±0.1	0.2	0.6±0.5	0.4±0.3	0.04
	(ng/mL.Kg -1 )
	Total	42.7±12.3 46.2±8.0 0.1	46.4±10.3 49.7±9.6 0.09 43.1±5.8 49.1±12.3 0.07
	carnitine
	(ng/mL)
	Free	33.8±9.9 37.7±6.9 0.06	37.2±7.7 41.7±6.3 0.2	33.6±5.5 39.8±10.5 0.04
	carnitine
	(ng/mL)
	Total	2.3±1.4	3.0±2.3	0.05	2.9±1.2	2.9±5.2	0.9	2.7±1.3	6.0±5.6	0.01
	carnitine/ FM
	(ng/mL.Kg -1 )

Free carnitine/FM (ng/mL.Kg -1 )

	1.8±1.1	2.4±1.3 F 0.05 o r 2.4±4.5 P e 2.5±1.1 e r 0.8 R e 2.1±1.1 v i 4.9±4.7 e F o r P e e r R e v i e F o r P e e r R e v i e F F o r F o r F o r o r P P P P e e e r e e r e e r e r R e v i e R e v i e w 0.01 R e v i e w R e v i e w
		w w w w