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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., Author summary Birt-Hogg-Dubé (BHD) syndrome is a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. The disease is linked to germline variants in the folliculin (FLCN) tumor suppressor gene. Here, we present a combined computational and experimental study, focusing on rare BHD-linked missense and single amino acid deletion variants. Our data show that many disease-causing FLCN variants lead to structural destabilization and rapid proteasomal degradation of the FLCN protein. The reduced level of FLCN, in turn, leads to degradation of the FLCN binding partners FNIP1 and FNIP2. Additional results show that the turnover of FLCN is regulated by the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones. We propose that for some missense variants, stabilization and resulting restoration of function may hold therapeutic potential, and that 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.

Mammalian glycan-binding receptors, sometimes known as lectins, interact with glycans, the oligosaccharide portions of endogenous mammalian glycoproteins and glycolipids as well as sugars on the surfaces of microbes. These receptors guide glycoproteins out of and back into cells, facilitate communication between cells through both adhesion and signaling, and allow the innate immune system to respond quickly to viral, fungal, bacterial, and parasitic pathogens. For many of the roughly 100 glycan-binding receptors that are known in humans, there are good descriptions of what types of glycans they bind and how selectivity for these ligands is achieved at the molecular level. In some cases, there is also comprehensive evidence for the roles that the receptors play at the cellular and organismal levels. In addition to highlighting these well-understood paradigms for glycan-binding receptors, this review will suggest where gaps remain in our understanding of the physiological functions that they can serve.

Amyotrophic lateral sclerosis (ALS) is a motoneuron disease characterized by misfolded proteins aggregation in affected motoneurons. In mutant SOD1 (mutSOD1) ALS models, aggregation correlates to impaired functions of proteasome and/or autophagy, both essential for the intracellular chaperone-mediated protein quality control (PQC), and to a reduced mutSOD1 clearance from motoneurons. Skeletal muscle cells are also sensitive to mutSOD1 toxicity, but no mutSOD1 aggregates are formed in these cells, that might better manage mutSOD1 than motoneurons. Thus, we analyzed in spinal cord and in muscle of transgenic (tg) G93A-SOD1 mice at presymptomatic (PS, 8 weeks) and symptomatic (S, 16 weeks) stages, and in age-matched control mice, whether mutSOD1 differentially modulates relevant PQC players, such as HSPB8, BAG3, and BAG1. Possible sex differences we real so considered. No changes of HSPB8, BAG3, and BAG1 at PS stage (8 weeks) were seen in all tissues examined in tg G93A-SOD1 and control mice. At S stage (16 weeks), HSPB8 dramatically increased in skeletal muscle of tg G93A-SOD1 mice, while a minor increase occurred in spinal cord of male, but not female tg G93A-SOD1 mice. BAG3 expression increased both in muscle and spinal cord of tg G93A-SOD1 mice at S stage, BAG1 expression increased only in muscle of the same mice. Since, HSPB8-BAG3 complex assists mutSOD1 autophagic removal, we analyzed two well-known autophagic markers, LC3 and p62. Both LC3 and p62 mRNAs were significantly up-regulated in skeletal muscle of tg G93A-SOD1 mice at S stage (16 weeks). This suggests that mutSOD1 expression induces a robust autophagic response specifically in muscle. Together these results demonstrate that, in muscle mutSOD1-induced autophagic response is much higher than in spinal cord. In addition, if mutSOD1 exerts toxicity in muscle, this may not be mediated by misfolded proteins accumulation. It remains unclear whether in muscle mutSOD1 toxicity is related to aberrant autophagy activation.

In eukaryotes, proteins that are secreted into the ER are mostly modified by N-glycans on consensus NxS/T sites. The N-linked glycan subsequently undergoes varying degrees of processing by enzymes which are spatially distributed over the ER and the Golgi apparatus. The post-ER N-glycan processing to complex glycans differs between animals and plants, with consequences for N-glycan and glycopeptide isolation and characterization of plant glycoproteins. Here we describe some recent developments in plant glycoproteomics and illustrate how general and plant specific technologies may be used to address different important biological questions.

ER-phagy (reticulophagy) defines the degradation of portions of the endoplasmic reticulum (ER) within lysosomes or vacuoles. It is part of the self-digestion (i.e., autophagic) programs recycling cytoplasmic material and organelles, which rapidly mobilize metabolites in cells confronted with nutrient shortage. Moreover, selective clearance of ER subdomains participates in the control of ER size and activity during ER stress, the reestablishment of ER homeostasis after ER stress resolution, and the removal of ER parts in which aberrant and potentially cytotoxic material has been segregated. ER-phagy relies on the individual and/or concerted activation of the ER-phagy receptors, ER peripheral or integral membrane proteins that share the presence of LC3/Atg8-binding motifs in their cytosolic domains. ER-phagy involves the physical separation of portions of the ER from the bulk ER network and their delivery to the endolysosomal/vacuolar catabolic district. This last step is accomplished by a variety of mechanisms including macro-ER-phagy (in which ER fragments are sequestered by double-membrane autophagosomes that eventually fuse with lysosomes/vacuoles), micro-ER-phagy (in which ER fragments are directly engulfed by endosomes/lysosomes/vacuoles), or direct fusion of ER-derived vesicles with lysosomes/vacuoles. ER-phagy is dysfunctional in specific human diseases, and its regulators are subverted by pathogens, highlighting its crucial role for cell and organism life.