Folliculin variants linked to Birt-Hogg-Dubé syndrome are targeted for proteasomal degradation

Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dubé (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present an integrated computational and experimental study that reveals that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant (ΔE510) forms perinuclear protein aggregates. A yeast-based stability screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature led to a stabilization of two FLCN missense proteins, and for one (R362C), function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization and resulting restoration of function may hold therapeutic potential of certain disease-linked variants. Our computational saturation scan encompassing both missense variants and single site deletions in FLCN may allow classification of rare FLCN variants of uncertain clinical significance.


Introduction
In order to function, most proteins require some conformational flexibility and are therefore not completely rigid in their native environment. In addition, since nature selects for function most proteins are not overly stable, and stress conditions or mutations may cause proteins to lose their native conformation and misfold. Because misfolded proteins can be toxic and form insoluble aggregates, they must be quickly eliminated from the cell via the so-called protein quality control (PQC) network [1][2][3]. Typically, PQC depends on molecular chaperones to associate with the misfolded proteins, and refold them or guide them for degradation via the ubiquitin-proteasome system (UPS) or autophagy [4][5][6][7][8]. Despite a number of recent efforts [9,10], it is not completely understood how cells discern misfolded from native proteins but it is likely to involve recognition of exposed hydrophobic regions that are normally buried in the native protein.
Recently, we have shown for several disease-related proteins that the degree of protein destabilization correlates with the turnover rate [11][12][13], and a structural destabilization of as little as 3 kcal/mol is enough to trigger degradation. This is in agreement with genetic studies in yeast that have shown that the PQC system operates by following a better-safe-than-sorry principle and is thus highly diligent and prone to target proteins that are only slightly structurally perturbed but still functional [14][15][16]. Likewise, seminal studies in human cells have shown that some cystic fibrosis patients carry a deletion in the CFTR gene that result in a protein that retains biochemical function. This protein variant fails to conduct its function not because it is intrinsically inactive, but because it is targeted by the PQC system for proteasomal degradation, which in turn leads to an insufficient amount of protein and ultimately disease [17,18]. Here, we show that a similar PQC mechanism is responsible for targeting certain variants of the folliculin protein (FLCN) linked to Birt-Hogg-Dubé (BHD) syndrome (OMIM: 607273) and that, similar to results for multiple other proteins, structural calculations predict these effects [11][12][13][19][20][21][22][23][24][25][26][27][28][29][30].
The autosomal dominantly inherited BHD syndrome is caused by variants in the FLCN gene [31][32][33]. BHD syndrome predisposes patients to develop hair follicle hamartomas (fibrofolliculomas), lung cysts, and renal neoplasia with an age-dependent and variable penetrance [34][35][36][37]. A locus for BHD syndrome was mapped to chromosome 17p11.2 by linkage analysis in BHD families, and subsequently germline mutations in the FLCN gene were identified [31,38,39]. Further analysis of tumors from patients with BHD syndrome revealed somatic FLCN second hit inactivating mutations in the wild-type allele [40,41], in line with FLCN having a tumor suppressor function. The tumor suppressor activity of FLCN is also supported by data from BHD animal models [42][43][44].
The widely expressed FLCN gene encodes the 64 kDa cytosolic and nuclear FLCN protein, which does not display high sequence similarity to any other human protein but is highly conserved across species [33]. Functional studies have linked FLCN to diverse metabolic pathways and cellular processes [33], including the mTOR signalling pathway [45], regulation of PGC1α and mitochondrial biogenesis [46], TFE3/TFEB transcriptional regulation [47,48], RhoA activation, cell adhesion [49], and amino acid-dependent activation of mTORC1 through Rag GTPases [50,51]. Intriguingly, recent data have shown that wild-type FLCN is a HSP90 target protein and FNIP1/2 act as co-chaperones to facilitate FLCN folding [52,53], thus directly linking FLCN to PQC.
A high-resolution crystal structure of the C-terminal domain of FLCN has been solved [54] and shows a remarkable structural similarity to the DENN domain in the DENN1B protein [55]. The DENN domain proteins have been found to function as guanine exchange factors (GEFs) for Rab-type GTPases and thus activate the vesicle transport function of these small GTPases [56]. Recently, a full-length structure of FLCN was obtained by cryo-EM [57,58], showing that the N-terminal region of FLCN contains a Longin domain which forms contacts with the Longin domain in its binding partner folliculin-interacting protein (FNIP1), while the C-terminal DENN domain interacts with the DENNc domain in FNIP1. In turn, FNIP1 mediates interaction to RagA, RagC and Ragulator to control mTORC activation [57].
In addition to FNIP1, FLCN also associates with the related protein FNIP2 and the AMPactivated protein kinase AMPK [59][60][61], which in cells serves as an energy sensor and regulator of the mTOR signalling pathway. The FNIP1 and FNIP2 proteins display sequence similarity (49% identity) and similar expression patterns [60], suggesting that they may overlap in function. The link between FLCN function and the FNIP1/2 proteins is also supported by genetic data. Thus, mice inactivated in both Fnip1 and Fnip2 develop cystic kidneys similar to the phenotype observed in kidney-targeted Flcn knock-out mice [45,62,63].
The vast majority of BHD-linked FLCN mutations are frameshift variants leading to truncations and splice site aberrations predicted to cause loss of function [33]. Truncated variants of the FLCN protein are likely to be structurally destabilized, engage with chaperones and eventually be degraded by the proteasome, as recently shown for the L460QsX25 variant [64]. On the other hand, the consequences of the more rare BHD-linked FLCN missense variants and short in-frame deletions are not well characterized, although some are likely to destabilize the FLCN protein [65].
Here, we show that disease-causing FLCN missense mutations, rather than directly ablating FLCN protein function, instead render the FLCN proteins to become PQC targets. Rapid proteasomal degradation of the FLCN variants results in strongly reduced steady-state FLCN levels in cells. Several of the variants are also insoluble and fail to associate with the FLCN binding partners FNIP1 and FNIP2. This lack of FLCN binding in turn triggers proteasomal degradation of FNIP1 and FNIP2. Some of the FLCN variants are mislocalized in cells and one variant (ΔE510) forms perinuclear protein aggregates. The USP7 deubiquitylating enzyme (DUB) regulates FLCN turnover. In agreement with the disease-linked FLCN variants being structurally destabilized, lowering of the temperature leads to a stabilization of the missense proteins, and for one variant function was re-established at low temperature. In conclusion, we propose that BHD-linked missense mutations operate by causing misfolding and degradation of the FLCN protein, and structural stabilization of certain disease-linked variants may hold therapeutic potential.

Disease-associated FLCN variants display reduced steady-state levels due to proteasomal degradation
Previous studies indicate that BHD-linked FLCN missense protein variants are destabilized [65], which, in turn, suggest that they might be subject to PQC-dependent clearance. To test this hypothesis, we first analyzed the steady-state level of wild-type FLCN and eight different FLCN variants that have been found in BHD patients ( Table 1). The proteins were produced untagged from the pIRES2-GFP expression vector in transiently transfected U2OS cells. In agreement with the previous report [65], we found that several of the FLCN variants were present at strongly reduced steady-state levels (Fig 1A and 1B). The reduction in the FLCN protein levels was not caused by changes in transfection efficiency or in transcription, since the level of GFP, produced from the internal ribosomal entry site (IRES) in the same mRNA as FLCN, was unchanged ( Fig 1A). The most severe reduction in steady-state level was observed for the ΔE510 FLCN variant, while the levels of the V400I and K508R variants were hardly affected, compared to the wild-type FLCN control (Fig 1A and 1B). Indeed, although both V400I and K508R have been found in BHD patients, these relatively conservative substitutions occurred together with other putative pathogenic variants and their link to the disease is unclear (Table 1).
To test directly if the reduced protein levels were caused by degradation, we followed the turnover of the FLCN variants in cultures treated with the translation inhibitor cycloheximide (CHX). The wild type, V400I and K508R variants were stable, while the other variants were degraded ( Fig 1C). By densitometry of Western blots, we estimate the half-life of wild-type FLCN to be >>8 hours, while the short-lived variants displayed half-lives between 3 and 8 hours (Fig 1D).
Most intracellular proteins are degraded via the proteasome or via the autophagy system. To analyze whether the observed FLCN turnover was caused by proteasomal degradation or via autophagy, we analyzed the level of the FLCN variants in cultures treated with the proteasome inhibitor bortezomib (BZ) or chloroquine (CQ), which inhibits autophagy. In case of wild-type FLCN and the stable variants, V400I and K508R, there was no effect of BZ (Fig 1E), since these variants are not rapidly degraded. Similar to overexpressed wild type FLCN, endogenous FLCN in the U2OS cells also appears stable (S1 Fig). However, for all other variants the levels were increased in response to proteasome inhibition (Fig 1E), suggesting that these variants are proteasome targets. We did not observe stabilization in the presence of CQ for any of the variants (S2 Fig).  [54]. For missense variants, it is well-established that many have neutral or slightly destabilizing effects, and only a fraction of the possible single amino acid changes are severely detrimental [66][67][68]. In contrast, there is far less data on protein stability for in-frame deletions, though existing studies on certain model proteins show that a number of both insertions and deletions are functional [69][70][71][72]. All three deletions in our initial dataset, however, are severely destabilized (Fig 1). Thus, we aimed to identify putative non-deleterious deletion variants and validate those experimentally.
We calculated ΔΔGs for missense variants with Rosetta [12,73,74] (Fig 2B and 2C). For inframe deletions, we modified a previously described protocol [75]. Briefly, this consists of creating the sequence of the deletion variant of interest, creating a homology model using the original crystal structure (PDB: 3V42) as the template, relaxing this model with Rosetta, and calculating the energy difference (ΔΔG) to the original structure (see Methods). This approach models both the effect of losing the side chain and backbone, but also the strain on the overall structure from the deletion ( Fig 2D). All data are included in the supplemental material (S1 Dataset). Data for the selected variants are included in Table 1.
Similar to previous studies [11-13, 66, 76] we observe good correlation between predicted ΔΔGs and experimentally determined protein levels. Notably, both missense variants and inframe deletions can be well-tolerated but can also lead to substantial loss of stability and degradation ( Fig 2B). In addition to the disease-associated variants in our dataset, we tested several additional deletion and missense variants. These additional variants (Fig 2E), have, as far as we know, not been observed in patients or in population sequencing studies [77] and were only included here to test the in silico stability predictions. They include two predicted and experimentally confirmed stable in-frame deletions ΔE455 and ΔA474 (Fig 2E). In contrast, K508A shows substantially reduced stability, confirming the importance of the Lys side chain at position 508 (Fig 2D). The ΔE509 variant was predicted to be as detrimental as its neighbours ΔK508 and ΔE510, however, it is only moderately destabilized ( Fig 2E). We speculate that this may be because the side chain points into solvent, where it is less critical than in the core of the folded protein, although this is not perfectly captured by our computational model. Lastly, we compared the predicted ΔΔGs with the variant allele frequency as reported in gnomAD [77]. Similar to previously analysed proteins [11][12][13] this shows a "fishtail" distribution where the more common variants such as V400I and K508R have near-neutral stability changes, while some of the rarest and pathogenic variants are destabilized ( Fig 2F).

Several FLCN variants are insoluble and mislocalized
Supported by the thermodynamic predictions, the reduced steady-state levels and increased proteasomal turnover suggest that the FLCN variants are misfolded and become PQC targets. In some cases, protein misfolding results in the formation of insoluble aggregates [76,78,79]. To test this for the FLCN variants, we separated crude cell lysates into soluble (supernatant) and insoluble (pellet) fractions by centrifugation and analyzed the abundance of the FLCN variants in these fractions by Western blotting. Wild-type FLCN was found both in the soluble fraction and, presumably by interaction with membranes, in the pellet (S3 Fig). Several of the FLCN variants, particularly the R239C, ΔK508 and ΔE510 variants, were mostly insoluble (S3 Fig), suggesting that they are misfolded and may form aggregates.
Next, we proceeded to directly assess the subcellular localization of the FLCN variants by fluorescence microscopy. Consistent with other reports [45,60,61], wild-type FLCN was evenly distributed throughout the cytosol and nucleus ( Fig 3A). This was also the case for the R239C, R362C, V400I and K508R variants. However, the nuclear localization of the other FLCN variants was strongly reduced, and in case of the ΔE510 variant, most of the cells (~70%) displayed several perinuclear aggregate-like structures ( Fig 3A). Accordingly, retention of the ΔE510 variant was also apparent in filter trap assays (Fig 3B and 3C), where aggregated proteins fail to migrate through a nitrocellulose membrane [80].
The absence of nuclear-localized FLCN has been observed for an unstable FLCN splice variant before [81], and considering the unstable nature of these proteins, this could suggest that the observed increased protein turnover is restricted to the pool of nuclear FLCN. However, repeating the localization experiments in the presence of bortezomib did not result in any increased nuclear localization of the unstable variants or did otherwise affect the subcellular distribution of the FLCN variants (S4 Fig). This indicates that the lack of nuclear signal is caused primarily by a failure in nuclear import of FLCN rather than increased specific degradation of nuclear FLCN.

FLCN binding protects FNIP1 and FNIP2 from proteasomal degradation
Genetic and structural studies have shown that FLCN function is intimately linked to its binding partners FNIP1 and FNIP2 [33,57], and we therefore investigated the interplay between the FLCN variants and FNIP1/2. Upon introducing HA-tagged FNIP1 or FNIP2 expression constructs, we observed very low FNIP1/2 levels (Fig 4A and 4B). Since the FNIP1/2 levels increased in response to the proteasome inhibitor bortezomib (S5 Fig), the low steady-state levels of FNIP1/2 are caused by proteasomal degradation (Fig 4A and 4B, S5 Fig). However, upon co-expression of wild-type FLCN, the FNIP1/2 proteins were dramatically stabilized ( Fig  4A and 4B), suggesting that FLCN protects FNIP1/2 from degradation. Indeed, when (C) Heat map for the segment from position 490-520 visualizing both missense and deletion (del, top row) ΔΔGs. A large part of this sequence forms an alpha helix, and positions that point into the domain core (arrowheads) are more constrained than those pointing towards the solvent. (D) Structural model for the deletion of K508. Deletion model in solid tracing, wild-type transparent. The helix is strained (magenta) and the hydrogen bond which K508 forms with the backbone of T393 is lost. (E) The steadystate levels of the indicated FLCN variants in whole cell lysates were compared by blotting. β-actin served as a loading control. These additional variants were tested based on stability calculations. K508A leads to loss of hydrophobic interaction and the side-chain-backbone hydrogen bond described in (D) and is thus predicted to be less stable. ΔE445 and ΔA474 are in linker regions and predicted to not substantially destabilize the structure. ΔE509 was predicted to be as destabilizing as its neighbors ΔK508 and ΔE510, however, experimentally it is only mildly destabilized. This may be because its side chain points into solvent, whereas the side chains of K508 and E510 make important contacts with other parts of the C-terminal domain of FLCN. (F) "Fishtail plot" illustrating the gnomAD allele frequency of missense and deletion variants vs. the Rosetta ΔΔG. Empty blue triangles represent benign ClinVar entries. Common and benign variants have near-neutral effects on stability, while several pathogenic variants are predicted to lose stability. https://doi.org/10.1371/journal.pgen.1009187.g002
As a further test, we also analyzed the FLCN disease-linked frameshift variant H429Pfs. This variant is missing the C-terminal part of FLCN, including a region involved in FNIP binding (S6 Fig). Accordingly, this variant is also not able to stabilize FNIP1/2 and displays strongly reduced binding to FNIP1/2 (S6 Fig).
Based on these data, we reasoned that FNIP1 and FNIP2 are protected from proteasomal degradation upon FLCN binding. Accordingly, in co-precipitation experiments with FNIP1/2 the V400I, K508R, R239C, and R362C FLCN variants all appeared adept at FLCN interaction, while the FNIP1/2 binding was strongly reduced for the other FLCN variants (Fig 5A and 5B). Co-expression of FNIP1 and FNIP2 did not lead to any mutual stabilization (Fig 5C), excluding that FNIP stabilization by FLCN occurs indirectly e.g. via a FNIP1-FNIP2 interaction.

A yeast screen links FLCN turnover to chaperones and the deubiquitylating enzyme Ubp15/USP7
To further analyze the degradation of the unstable FLCN variants we took advantage of a yeast selection system for protein stability [9]. Specifically, we expressed wild-type FLCN (as a control) and the unstable ΔE510 variant as fusion proteins with the orotidine-5'-phosphate (OMP) decarboxylase Ura3 enzyme in a strain carrying a deletion of the endogenous URA3 gene (ura3Δ) (Fig 6A). Since proteasomal protein degradation is processive [82], degradation of the ΔE510 fusion protein should lead to reduced amounts of the Ura3 fusion partner. Accordingly, we observed the FLCN ΔE510 fusion protein was rapidly degraded via the proteasome (Fig 6B), and the ΔE510 strain displayed a reduced growth in media lacking uracil ( Fig 6C). As expected the growth defect was completely suppressed by addition of sublethal amounts of the proteasome inhibitor bortezomib (Fig 6C), and we therefore continued to screen for components targeting the unstable FLCN variants for degradation. To this end, the ΔE510 yeast expression construct was introduced into a library of 4992 individual gene deletion mutants [83], one-by-one, by mating. After sporulation and selection of haploid cells with the gene deletion (G418 resistant) and the Ura3 reporter fusion construct, the resulting haploids were scored for growth by monitoring the colony sizes on solid media lacking uracil. In total 27 mutants caused significantly increased growth, suggesting a stabilization of the ΔE510 variant, while 135 mutants displayed a significantly reduced growth, indicating that the ΔE510 variant was less abundant in these cells. All 162 significant hits from this screen are included in the supplemental material (S2 Dataset). To test the validity of the screen we selected two hits, ydj1Δ and ubp15Δ, for further analyses. In agreement with the results from the high-throughput screen, introducing the ΔE510 fusion in ydj1Δ cells led to a visibly increased growth, while

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the ubp15Δ cells appeared to grow more poorly (Fig 6D). This difference was also apparent in the steady-state levels of the ΔE510 fusion. Hence, in ydj1Δ cells we observed a higher level of ΔE510 fusion protein, while in the ubp15Δ strain, the ΔE510 protein level was reduced ( Fig 6E).

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Ydj1 is a DnaJ domain family co-chaperone linked to proteasomal degradation of misfolded chaperone clients [10], while Ubp15 is a deubiquitylating enzyme which counteracts E3s to block degradation. Whereas human cells encode a number of J-domain proteins that are homologous to the yeast Ydj1, the yeast Ubp15 is in human cells represented by a single orthologue called USP7. Next, we therefore co-transfected U2OS cells to express FLCN and either wild-type USP7 or, as a control, a catalytically dead USP7 variant carrying a C223A substitution of the active site cysteine residue. We note that the overexpression of USP7 did not affect the overall ubiquitylation of cell proteins (Fig 6F), suggesting that USP7 is specific for a subset of ubiquitylated proteins. In agreement with the results in yeast cells, overexpression of the wild-type USP7, but not of the catalytically dead USP7 variant, led to a slight stabilization of the FLCN ΔF157 and ΔE510 variants (Fig 6F), suggesting that USP7 deubiquitylates these proteins to subtly regulate their cellular abundance. However, in case of the frameshift FLCN H429Pfs we did not observe any effect of USP7 (S6 Fig).
Since we found that FLCN contains multiple potential TRAF-and UBL1/2-domain binding consensus motifs (S7 Fig, S8 Fig) that could mediate direct interaction to USP7 [84], we tested if FLCN and USP7 interact by co-precipitation. To achieve equal amounts of wild type and ΔE510, we transfected the U2OS cells with 20-fold less plasmid encoding wild-type FLCN compared to ΔE510, but equal amounts of a plasmid encoding myc-tagged USP7 C223A. We observed a clear interaction between USP7 and FLCN (Fig 6G), supporting further a role of USP7 in regulating FLCN turnover. Since USP7 is also associated with wild-type FLCN, it is possible that it also plays a regulatory role in FLCN function.

Effect of temperature on FLCN stability and function
In some cases, PQC targets can be stabilized structurally by lowering the temperature, which in turn leads to a reduced degradation and increased steady-state level [11]. To test this for the FLCN variants, we compared their steady-state levels at 37˚C and 29˚C. Importantly, lowering the temperature to 29˚C does not affect the ubiquitin-proteasome system in U2OS cells (S9 Fig) [11]. Several of the FLCN variants were stabilized at 29˚C (Fig 7A), indicating that these variants are indeed structurally stabilized at the lower temperature. This effect was most apparent for the R362C variant (Fig 7A), in line with our observation that this variant is borderline stable already at 37˚C, and interacts with and partially stabilizes FNIP1/2 (Table 1). Thus, a slight structural stabilization conveyed by lowering the temperature is enough for this variant to evade the PQC-mediated clearance. Accordingly, we reasoned that perhaps the tumor suppressor function of some FLCN variants could be restored upon lowering the temperature. We  FLCN), and, as a loading control, to α-tubulin. (F) FLCN variants were transfected into U2OS cells with an expression vector for either wild-type myc-tagged USP7 or the catalytically dead USP7 C223A variant. Whole cell lysates were analyzed by SDS-PAGE and Western blotting using antibodies to FLCN, myc (to detect the co-expressed USP7) and ubiquitin. β-actin served as a loading control. Quantification of the FLCN (short exposure) signals in fold compared to mock transfected is given below. (G) The wild-type and ΔE510 FLCN variants were analyzed for interaction with the catalytically dead USP7 C223A variant. Myc-tagged USP7 was immunoprecipitated using the myc-trap system and analyzed by blotting. Input samples (5%) were included as a control. To achieve equal amounts of the FLCN proteins, the cells were transfected with 20-fold less wild-type FLCN DNA compared to FLCN ΔE510 DNA. https://doi.org/10.1371/journal.pgen.1009187.g006

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therefore investigated the ability of the FLCN variants to suppress the growth of FLCN-negative FTC-133 cells suspended in soft agar at 37˚C and 29˚C. The FLCN expression constructs were transfected into FTC-133 cells as stable mixed cell populations, and after four weeks at 37˚C or 29˚C colonies were counted. Wild-type FLCN led to roughly 50% reduction in colony formation at both temperatures ( Fig 7B). The stable variants, V400I and K508R, both behaved like wild type and suppressed growth to a level similar to wild-type FLCN (Fig 7B). In line with a stabilization at low temperature, the R362C variant significantly reduced colony formation only at 29˚C (Fig. 7B), which was not apparent for any of the other FLCN variants. Presumably the other destabilized FLCN variants are subject to more dramatic structural effects causing FLCN misfolding to an extent where the proteins cannot attain their native conformation and will thus not be functional when the temperature is lowered or if the degradation was blocked.

Discussion
According to the ClinVar database [85], most disease-linked FLCN variants in the coding regions are frameshift mutations leading to C-terminal truncations of the FLCN protein (S10 Fig.). In the gnomAD database containing more than 100,000 exome sequences, some FLCN frameshift variants are also reported, but as expected these are exceedingly rare in the general population (S10 Fig.). In this work, we focus on the rare FLCN missense and single codon deletion variants linked to BHD syndrome.
Certainly, point mutations may directly ablate protein function without grossly affecting protein structure, e.g. by disrupting the active site of an enzyme. More commonly, however, missense mutations or in-frame deletions result in a structural perturbation or destabilization of the encoded protein [68], and indeed a previous report has shown that several BHD-linked single site FLCN variants are destabilized [65]. The results presented here are perfectly in line with this observation, supported by structural modelling (Fig 2, S11 Fig.), and show that the structural destabilization, conferred by the mutations, leads to rapid proteasomal degradation

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of the FLCN variants, and in the rare cases that BHD syndrome is triggered by such single site mutations, the disease should be considered a protein misfolding disease.
We found that some of the FLCN variants are mislocalized in cells and one variant (ΔE510) formed perinuclear protein aggregates, suggesting that at least this variant is highly misfolded. Often, aggregated proteins are cleared by autophagy. However, similar to aggregation-prone variants of VHL [79], ataxin-3 [86] and DJ-1 [76], the FLCN ΔE510 protein appeared to be entirely degraded via the proteasome, which suggests that molecular chaperones may dissolve the ΔE510 aggregates prior to degradation.
Our screen in yeast cells revealed two oppositely acting regulators of FLCN stability. In agreement with chaperones playing a role in FLCN turnover, deletion of the Ydj1 co-chaperone led to a stabilization of the FLCN ΔE510 variant. Deletion of the Ubp15, on the other hand, destabilized the FLCN ΔE510 variant. This observation fits with Ubp15/USP7 possessing deubiquitylating activity, which by catalyzing the removal of ubiquitin chains from proteins destined for the proteasome counters the degradation. Previous studies have identified numerous binding partners and substrates of USP7 [84,87], including p53, p16 INK4a and WASH [88][89][90][91]. Typically, these interact with USP7 through its TRAF domain and/or the UBL1/2 domains, and consensus motifs in USP7-binding partners have been described [84]. We note that FLCN contains multiple of these potential TRAF-and UBL1/2-domain binding consensus motifs, which could mediate direct interaction to USP7. However, notably USP7 appears to preferentially interact with E3 ubiquitin-protein ligases [84], as exemplified by the effect of USP7 on p53. Hence, in case of p53, USP7 has been shown to deubiquitylate and stabilize the E3 MDM2, which in turn leads to an increased degradation of p53 [90][91][92][93]. Presumably since PQC E3s display overlapping substrate specificity and are highly redundant [94,95] our screen failed to uncover the E3(s) that target FLCN for degradation, and our results on USP7 suggest that USP7 directly deubiquitylates FLCN, rather than targeting an FLCN-specific E3.
In our experiments, we have relied on transient overexpression of FLCN variants. It is possible that different PQC system operate depending on the expression level of the misfolded protein, but to our knowledge this has not been systematically tested, and we cannot rule out that endogenous FLCN variants are targeted for degradation via a different PQC pathway. However, since we observe degradation of the FLCN variants that are predicted to be destabilized, it is unlikely that the overexpression is saturating the molecular chaperones or other PQC components in our experimental setup.
Classically, molecular chaperones are viewed as protein folding machines that capture nonnative proteins and, through cycles of binding and release, catalyze folding into the correct native state. However, chaperones also directly interact and collaborate with components of the ubiquitin-proteasome system [2,10,15,94,96,97]. For instance, HSP70 and HSP90 directly associate with the E3 ubiquitin ligase, CHIP, which in turn ubiquitylates the chaperone clients to ensure their degradation [4,98]. Recently, it was shown that also wild-type FLCN is a HSP90 client protein and the FNIP1/2 binding partners function as co-chaperones that facilitate FLCN folding and ensure FLCN stability [52,53]. Our finding that the yeast Hsp40 cochaperone Ydj1 regulates the stability of the FLCN ΔE510 variant could be relevant also for the stability of wild-type FLCN, and more detailed studies on the potential interplay between Hsp40s and FNIP1/2 in FLCN folding and stability are warranted. Since some co-chaperones, including Ydj1 [99], appear to play an active role in degradation, it is possible that the co-chaperones regulate whether FLCN is folded or degraded. In our assays, we observed that FLCN also functions to stabilize FNIP1/2, and this stabilization depends on FNIP1/2 interaction. Hence, for those FLCN variants that are misfolded and fail to interact with FNIP1/2, the FNIP1/2 levels are reduced, due to proteasomal degradation. Subunits in multiprotein assemblies are generally more stable once incorporated into a complex and unassembled (orphan) proteins, produced in excess, are targeted for degradation [100,101]. Hence, our observation that FLCN stabilizes FNIP1/2 are in line with earlier reports showing that FNIP1/2 stabilize FLCN [52,59,60]. The observation of such mutually stabilizing effects confirms that FLCN forms a stable complex with FNIP1/2. However, in case of FLCN and FNIP1/2, the balance in their stoichiometry might also be regulated by environmental cues. Indeed, it was recently shown that in the presence of nutrients, FNIP2 is phosphorylated by casein kinase 1 (CK1) and degraded by the proteasome [102]. The experiments, presented here, were also performed under nutrient-rich conditions, and it is possible that the observed degradation of FNIP1/2 is triggered by CK1 phosphorylation of orphan FNIP1/2.
Intriguingly, we observed a clear correlation between the degradation rate of the unstable variants and several of the other tested parameters. Thus, all the unstable variants displayed reduced solubility, and with the exception of R239C and R362C, the unstable FLCN variants were all exclusively localized in the cytosol and failed to associate with and stabilize FNIP1 and FNIP2 (Table 1).
Of the tested FLCN variants, only two, V400I and K508R, behaved like wild-type FLCN with regard to level, turnover, interaction, localization and cell proliferation (Table 1). Since valine/isoleucine and lysine/arginine, are, respectively, chemically similar amino acids, it is not surprising that these substitutions do not render FLCN structurally unstable. However, importantly, these findings do not preclude that the V400I and K508R variants are pathogenic. According to the gnomAD database [77], both of the V400I and K508R variants are rare (minor allele frequencies ca.~1:40,000 and 1:5,000, respectively). The estimated prevalence of BHD syndrome according to the Orphanet database is 1:200,000, suggesting that if the K508R mutation is pathogenic, it is likely to have a reduced penetrance. ClinVar lists K508R as likely benign [85]. However, a recent report has shown that although introducing the K508R variant in Flcn knock-out mice prolonged survival, the mice later developed cystic kidneys and succumbed to renal failure [103], indicating that K508R might be a weakly pathogenic mutation. It is possible that the slow onset, observed for the K508R mouse model, is connected with the K508R variant being only slightly more unstable than wild-type FLCN in vivo. However, it is more likely that any potential pathogenicity associated with the V400I and K508R variants, is not caused by structural destabilization, but instead associated with other properties of the FLCN protein that we did not test here.
Since we found that several of the variants were stabilized by lowering the temperature, these proteins are probably not highly destabilized or misfolded. Indeed, for the R362C variant, we observed an increased cellular function when the temperature was reduced. Thus, similar to other genetic diseases, including cystic fibrosis [104] and von Hippel-Lindau disease [105], as well as certain cancer-linked mutations in p53 [106], it may be possible to stabilize some misfolded FLCN protein variants using a small molecule that either blocks the PQC system or directly stabilizes the FLCN protein structure [107]. However, to achieve this it is essential with further, in particular structural and biophysical, studies of both wild-type and diseaselinked FLCN protein variants. Finally, the structural stability predictions that we describe here may be useful in diagnostics of BHD syndrome in the rare cases where missense variants or single residue deletions are observed.

Electrophoresis and blotting
Unless otherwise stated whole cell extracts were prepared by lysing cells directly in SDS sample buffer. SDS-PAGE was performed on 12.5% acrylamide gels. For Western blotting 0.2 μm nitrocellulose membranes were used. For blocking, the membranes were incubated in 5% fatfree milk powder and 0.

Protein degradation experiments
Bortezomib (LC Laboratories) and chloroquine (Sigma) were used at 15 μM and 20 μM respectively, for 8 hours in serum-free media. Degradation was followed in cultures treated with cycloheximide (CHX) as described previously [11].

Co-precipitation experiments
Binding studies were performed as described before [11], using anti-HA resin (Sigma) or myctrap beads (Chromotek). Bound proteins were eluted directly in SDS sample buffer.

FLCN solubility
To estimate the solubility of the FLCN variants, a confluent 6-well dish of transfected U2OS cells was lysed in 200 μL buffer B by three 20 second rounds of sonication on ice. The lysate was then centrifuged at 15000 g for 30 minutes at 4˚C. Finally, 4x concentrated SDS sample buffer was added to the supernatant to obtain a 1x SDS sample buffer lysate. The pellet was resuspended in 1x SDS sample buffer to match the volume of the supernatant.

Filter trap assays
Filter trap assays were performed largely as described previously [80]. Briefly, to achieve equal cellular expression of wild-type FLCN and FLCN Δ510 protein, the plasmid encoding wildtype FLCN was diluted with empty control vector prior to transfection. Transfected cells were harvested in ice-cold PBS using a cell scraper and lysed by sonication for three times 10 seconds in ice-cold PBS containing Complete protease inhibitors (Roche). Unbroken cells were removed by centrifugation at 500 g for 2 minutes. SDS was added to a final concentration of 2% and three two-fold serial dilutions in 2% SDS in PBS were prepared. Samples of 200 μL were loaded onto a 0.2 μm nitrocellulose membrane affixed in a BioRad Bio-Dot apparatus (BioRad) pre-equilibrated with 100 μL PBS per well. The wells were then washed once with 400 μL 2% SDS in PBS and then once with 200 μL 2% SDS in PBS. The nitrocellulose membrane was then removed from the apparatus and washed briefly in PBS, before blocking and development as a Western blot using antibodies to FLCN and as a control GAPDH.

Soft agar colony formation assays
Soft agar colony formation assays were performed as described [65] using pools of FTC-133 cells stably transfected to express the FLCN variants. After four weeks, colonies greater than 100 μm in diameter were counted. The experiments were performed in triplicates.

Yeast strains and techniques
All yeast strains were from the Euroscarf collection and transformations were performed using lithium acetate [108]. For analyzing growth on solid media, exponential phase cultures were diluted to an OD 600nm of 0.40 and 5 μL of 5-fold serial dilutions were applied in spots on agar plates. Cell extracts for Western blotting were prepared with glass beads in trichloroacetic acid (Sigma) as described [109]. Cycloheximide (CHX) (Sigma) was used at a concentration of 100 μg/mL. Bortezomib (BZ) (LC Laboratories) was used at a concentration of 1 mM. The screening of the yeast gene deletion library [83] was performed by first transforming the FLCN-Ura3 reporter plasmid (pTR1412-FLCNΔE510) into the query strain Y7092 (MATα can1Δ::STE2pr-Sp_his5 lyp1Δ ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 geneX::KanMX6) and selecting transformants on SC-Leu medium. Next, the transformed strain was mated to the yeast gene deletion strain collection by automated pinning (RoToR, Singer Instruments, UK). Selection for diploids, sporulation, and selection for haploid progeny containing the gene deletion (G418 R ) and the FLCN-Ura3 reporter plasmid was performed essentially as described [110].

Calculation of ΔΔGs for missense variants
We used the Rosetta cartesian_ddg protocol [73] and the high-resolution structure of the Cterminal FLCN domain (PDB 3V42) [54] to calculate ΔΔGs for missense variants, as also previously described [12]. Three iterations were performed for each of the 20 possible amino acid variants at each position, including the wild type for reference. The value reported here is the difference between the average of the wild-type Rosetta energies and the average for the respective variant. All scores are divided by 2.9 to bring them to a kcal/mol scale (Frank DiMaio, personal communication).

ΔΔGs for in-frame deletion variants
First 25 homology models were created [111] for each single-amino-acid-deletion in the C-terminal domain, using PDB 3V42 [54] as the template. Each homology model was relaxed in two independent trajectories using the command line: /path/to/rosetta/source/bin/relax.linuxgccrelease / -database /path/to/rosetta/database \ -l list_w_models.lst -ignore_unrecognized_res -use_input_sc -constrain_relax_to_start_coords -flip_HNQ -no_optH false -relax:fast -nstruct 2 based on Jackson et al. [75]. The median of the resulting 50 scores was calculated to estimate the ΔG (in Rosetta Energy Units) of the respective deletion variant. To estimate ΔΔGs of inframe deletions, the 10 th percentile of the ΔG distribution was subtracted. Analogous to the missense variant ΔΔGs above, scores were divided by 2.9 to bring them to a scale that corresponds to kcal/mol.  Fig. Analyses of the FLCN H429Pfs variant. (A) The FLCN H429Pfs variant leads to addition of the shown residues before terminating (boxed sequence). The structure of the FLCN-FNIP2 complex is shown based on the recently resolved cryo-EM structure of FLCN (coloured) and FNIP2 (gray) (PDB 6ULG) (Shen et al., 2019). The purple region is missing in the H429Pfs variant. (B) U2OS cells were transiently transfected to express either wt FLCN or H429Pfs, with or without expression of HA-tagged FNIP1 as indicated. Then the levels of FLCN and FNIP1 in whole cell lysates were compared by SDS-PAGE and Western blotting with antibodies to FLCN or the HA-tag on FNIP1. β-actin served as a loading control. (C) U2OS cells were transiently transfected to express either wt FLCN or H429Pfs, with or without expression of HA-tagged FNIP2 as indicated. Then the levels of FLCN and FNIP2 in whole cell lysates were compared by SDS-PAGE and Western blotting with antibodies to FLCN or the HA-tag on FNIP2. β-actin served as a loading control. (D) U2OS cells were transiently transfected to express either vector, wt FLCN or H429Pfs, and HA-tagged FNIP1 or FNIP2 as indicated, and treated with bortezomib (+BZ) for 8 hours. Cleared extracts (input) were prepared and used for immunoprecipitation (IP) using antibodies to the HA-tag on FNIP1/2. Finally, the samples were resolved by SDS-PAGE and analyzed by Western blotting with antibodies to FLCN or the HA-tag on FNIP1/2, and as a control to β-actin. (E) The level of transfected H429Pfs in U2OS was compared upon co-transfection with myc-tagged USP7 and the catalytically dead USP7 variant (C223A) by Western blotting of whole cell lysates. FLCN was detected using the antibody to FLCN. USP7 was detected by using antibodies to the myc-tag. Probing for β-actin was included as a control.