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

Abstract

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.

Mapping the degradation pathway of a disease-linked aspartoacylase variant

“…Although targeting Hsp110 to stabilize ASPA C152W resulted in the accumulation of insoluble protein, it might still be a relevant target for other ASPA variants that are at least partially soluble and contain residual activity. Accordingly, Hsp110 should be explored further as a potential therapeutic target to decrease the degradation of ASPA and other misfolded proteins, possibly using a recently developed inhibitor of Hsp110 [ 81 ], to potentially avoid or diminish the development of Canavan disease and other protein misfolding disorders such as Lynch syndrome or Birt-Hogg-Dubé syndrome [ 82 , 83 ].…”

Disease-linked mutations trigger exposure of a protein quality control degron in the DHFR protein

“…Currently, it is well established that certain missense mutations may cause protein destabilization and degradation (Stein et al, 2019), and that this is the underlying molecular mechanism for a number of hereditary diseases, including cystic fibrosis, phenylketonuria and various cancer predisposition disorders (Abildgaard et al, 2019; Ahner et al, 2007; Arlow et al, 2013; Canaff et al, 2012; Clausen et al, 2020; Gersing et al, 2021; Pey et al, 2007; Scheller et al, 2019). Here, we add hereditary megaloblastic anemia, linked to germline DHFR variants, to this list.…”

Folliculin: A Regulator of Transcription Through AMPK and mTOR Signaling Pathways

“…Rarer BHD-linked FLCN mutations result in the expression of missense and short in-frame deletions variants. Recent studies have shown that the loss-of-function associated with these variants is due to proteasomal degradation of FLCN proteins ( Nahorski et al, 2011 ; Clausen et al, 2020 ). FLCN is considered a tumor suppressor, following the Knudson “second hit” model, since somatic mutation or loss of heterozygosity of the non-affected allele has been observed in BHD-associated renal tumors and in animal models ( Schmidt and Linehan, 2018 ).…”