Chemical chaperones in metabolic fitness beyond protein folding.

Chemical chaperones are small molecules that improve protein folding, alleviating aberrant pathological phenotypes due to protein misfolding. Recent reports suggest that, in parallel with their role in relieving endoplasmic reticulum (ER) stress, chemical chaperones rescue mitochondrial function and insulin signaling. These effects may underlie their pharmacological action on metabolically demanding tissues.

Combined Presence in Heterozygosis of Two Variant Usher Syndrome Genes in Two Siblings Affected by Isolated Profound Age-Related Hearing Loss.

Sensorineural age-related hearing loss affects a large proportion of the elderly population, and has both environmental and genetic causes. Notwithstanding increasing interest in this debilitating condition, the genetic risk factors remain largely unknown. Here, we report the case of two sisters affected by isolated profound sensorineural hearing loss after the age of seventy. Genomic DNA sequencing revealed that the siblings shared two monoallelic variants in two genes linked to Usher Syndrome (USH genes), a recessive disorder of the ear and the retina: a rare pathogenic truncating variant in USH1G and a previously unreported missense variant in ADGRV1. Structure predictions suggest a negative effect on protein stability of the latter variant, allowing its classification as likely pathogenic according to American College of Medical Genetics criteria. Thus, the presence in heterozygosis of two recessive alleles, which each cause syndromic deafness, may underlie digenic inheritance of the age-related non-syndromic hearing loss of the siblings, a hypothesis that is strengthened by the knowledge that the two genes are integrated in the same functional network, which underlies stereocilium development and organization. These results enlarge the spectrum and complexity of the phenotypic consequences of USH gene mutations beyond the simple Mendelian inheritance of classical Usher syndrome.

ERO1 alpha deficiency impairs angiogenesis by increasing N-glycosylation of a proangiogenic VEGFA.

N-glycosylation and disulfide bond formation are two essential steps in protein folding that occur in the endoplasmic reticulum (ER) and reciprocally influence each other. Here, to analyze crosstalk between N-glycosylation and oxidation, we investigated how the protein disulfide oxidase ERO1-alpha affects glycosylation of the angiogenic VEGF121, a key regulator of vascular homeostasis. ERO1 deficiency, while retarding disulfide bond formation in VEGF121, increased utilization of its single N-glycosylation sequon, which lies close to an intra-polypeptide disulfide bridge, and concomitantly slowed its secretion. Unbiased mass-spectrometric analysis revealed interactions between VEGF121 and N-glycosylation pathway proteins in ERO1-knockout (KO), but not wild-type cells. Notably, MAGT1, a thioredoxin-containing component of the post-translational oligosaccharyltransferase complex, was a major hit exclusive to ERO1-deficient cells. Thus, both a reduced rate of formation of disulfide bridges, and the increased trapping potential of MAGT1 may increase N-glycosylation of VEGF121. Extending our investigation to tissues, we observed altered lectin staining of ERO1 KO breast tumor xenografts, implicating ERO1 as a physiologic regulator of protein N-glycosylation. Our study, highlighting the effect of ERO1 loss on N-glycosylation of proteins, is particularly relevant not only to angiogenesis but also to other cancer patho-mechanisms in light of recent findings suggesting a close causal link between alterations in protein glycosylation and cancer development.

The Link between VAPB Loss of Function and Amyotrophic Lateral Sclerosis.

The VAP proteins are integral adaptor proteins of the endoplasmic reticulum (ER) membrane that recruit a myriad of interacting partners to the ER surface. Through these interactions, the VAPs mediate a large number of processes, notably the generation of membrane contact sites between the ER and essentially all other cellular membranes. In 2004, it was discovered that a mutation (p.P56S) in the VAPB paralogue causes a rare form of dominantly inherited familial amyotrophic lateral sclerosis (ALS8). The mutant protein is aggregation-prone, non-functional and unstable, and its expression from a single allele appears to be insufficient to support toxic gain-of-function effects within motor neurons. Instead, loss-of-function of the single wild-type allele is required for pathological effects, and VAPB haploinsufficiency may be the main driver of the disease. In this article, we review the studies on the effects of VAPB deficit in cellular and animal models. Several basic cell physiological processes are affected by downregulation or complete depletion of VAPB, impinging on phosphoinositide homeostasis, Ca2+ signalling, ion transport, neurite extension, and ER stress. In the future, the distinction between the roles of the two VAP paralogues (A and B), as well as studies on motor neurons generated from induced pluripotent stem cells (iPSC) of ALS8 patients will further elucidate the pathogenic basis of p.P56S familial ALS, as well as of other more common forms of the disease.

Mutant VAPB: Culprit or Innocent Bystander of Amyotrophic Lateral Sclerosis?

Nearly twenty years ago a mutation in the VAPB gene, resulting in a proline to serine substitution (p.P56S), was identified as the cause of a rare, slowly progressing, familial form of the motor neuron degenerative disease Amyotrophic Lateral Sclerosis (ALS). Since then, progress in unravelling the mechanistic basis of this mutation has proceeded in parallel with research on the VAP proteins and on their role in establishing membrane contact sites between the ER and other organelles. Analysis of the literature on cellular and animal models reviewed here supports the conclusion that P56S-VAPB, which is aggregation-prone, non-functional and unstable, is expressed at levels that are insufficient to support toxic gain-of-function or dominant negative effects within motor neurons. Instead, insufficient levels of the product of the single wild-type allele appear to be required for pathological effects, and may be the main driver of the disease. In light of the multiple interactions of the VAP proteins, we address the consequences of specific VAPB depletion and highlight various affected processes that could contribute to motor neuron degeneration. In the future, distinction of specific roles of each of the two VAP paralogues should help to further elucidate the basis of p.P56S familial ALS, as well as of other more common forms of the disease.

Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity.

The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.

Searching for remote homologs of CAML among eukaryotes.

The tryptophan rich basic protein/calcium signal-modulating cyclophilin ligand (WRB/CAML) and Get1p/Get2p complexes, in vertebrates and yeast, respectively, mediate the final step of tail-anchored protein insertion into the endoplasmic reticulum membrane via the Get pathway. While WRB appears to exist in all eukaryotes, CAML homologs were previously recognized only among chordates, raising the question as to how CAML's function is performed in other phyla. Furthermore, whereas WRB was recognized as the metazoan homolog of Get1, CAML and Get2, although functionally equivalent, were not considered to be homologous. CAML contains an N-terminal basic, TRC40/Get3-interacting, region, three transmembrane segments near the C-terminus, and a poorly conserved region between these domains. Here, I searched the NCBI protein database for remote CAML homologs in all eukaryotes, using position-specific iterated-basic local alignment search tool, with the C-terminal, the N-terminal or the full-length sequence of human CAML as query. The N-terminal basic region and full-length CAML retrieved homologs among metazoa, plants and fungi. In the latter group several hits were annotated as GET2. The C-terminal query did not return entries outside of the animal kingdom, but did retrieve over one hundred invertebrate metazoan CAML-like proteins, which all conserved the N-terminal TRC40-binding domain. The results indicate that CAML homologs exist throughout the eukaryotic domain of life, and suggest that metazoan CAML and yeast GET2 share a common evolutionary origin. They further reveal a tight link between the particular features of the metazoan membrane-anchoring domain and the TRC40-interacting region. The list of sequences presented here should provide a useful resource for future studies addressing structure-function relationships in CAML proteins.

The WRB Subunit of the Get3 Receptor is Required for the Correct Integration of its Partner CAML into the ER.

Calcium-modulating cyclophilin ligand (CAML), together with Tryptophan rich basic protein (WRB, Get1 in yeast), constitutes the mammalian receptor for the Transmembrane Recognition Complex subunit of 40 kDa (TRC40, Get3 in yeast), a cytosolic ATPase with a central role in the post-translational targeting pathway of tail-anchored (TA) proteins to the endoplasmic reticulum (ER) membrane. CAML has also been implicated in other cell-specific processes, notably in immune cell survival, and has been found in molar excess over WRB in different cell types. Notwithstanding the stoichiometric imbalance, WRB and CAML depend strictly on each other for expression. Here, we investigated the mechanism by which WRB impacts CAML levels. We demonstrate that CAML, generated in the presence of sufficient WRB levels, is inserted into the ER membrane with three transmembrane segments (TMs) in its C-terminal region. By contrast, without sufficient levels of WRB, CAML fails to adopt this topology, and is instead incompletely integrated to generate two aberrant topoforms; these congregate in ER-associated clusters and are degraded by the proteasome. Our results suggest that WRB, a member of the recently proposed Oxa1 superfamily, acts catalytically to assist the topogenesis of CAML and may have wider functions in membrane biogenesis than previously appreciated.

The Ways of Tails: the GET Pathway and more.

Due to their topology tail-anchored (TA) proteins must target to the membrane independently of the co-translational route defined by the signal sequence recognition particle (SRP), its receptor and the translocon Sec61. More than a decade of work has extensively characterized a highly conserved pathway, the yeast GET or mammalian TRC40 pathway, which is capable of countering the biogenetic challenge posed by the C-terminal TA anchor. In this review we briefly summarize current models of this targeting route and focus on emerging aspects such as the intricate interplay with the proteostatic network of cells and with other targeting pathways. Importantly, we consider the lessons provided by the in vivo analysis of the pathway in different model organisms and by the consideration of its full client spectrum in more recent studies. This analysis of the state of the field highlights directions in which the current models may be experimentally probed and conceptually extended.

VAPB depletion alters neuritogenesis and phosphoinositide balance in motoneuron-like cells: relevance to VAPB-linked amyotrophic lateral sclerosis.

VAPB and VAPA are ubiquitously expressed endoplasmic reticulum membrane proteins that play key roles in lipid exchange at membrane contact sites. A mutant, aggregation-prone, form of VAPB (P56S) is linked to a dominantly inherited form of amyotrophic lateral sclerosis; however, it has been unclear whether its pathogenicity is due to toxic gain of function, to negative dominance, or simply to insufficient levels of the wild-type protein produced from a single allele (haploinsufficiency). To investigate whether reduced levels of functional VAPB, independently from the presence of the mutant form, affect the physiology of mammalian motoneuron-like cells, we generated NSC34 clones, from which VAPB was partially or nearly completely depleted. VAPA levels, determined to be over fourfold higher than those of VAPB in untransfected cells, were unaffected. Nonetheless, cells with even partially depleted VAPB showed an increase in Golgi- and acidic vesicle-localized phosphatidylinositol-4-phosphate (PI4P) and reduced neurite extension when induced to differentiate. Conversely, the PI4 kinase inhibitors PIK93 and IN-10 increased neurite elongation. Thus, for long-term survival, motoneurons might require the full dose of functional VAPB, which may have unique function(s) that VAPA cannot perform.

The GET pathway can increase the risk of mitochondrial outer membrane proteins to be mistargeted to the ER.

Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It has been widely observed that some mitochondrial outer membrane (MOM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked. However, the mechanism of this erroneous sorting is currently unknown. In this study, we demonstrate the involvement of the GET machinery in the mistargeting of suboptimal MOM proteins to the ER. These findings suggest that the GET machinery can, in principle, recognize and guide mitochondrial and non-canonical TA proteins. Hence, under normal conditions, an active mitochondrial targeting pathway must exist that dominates the kinetic competition against other pathways.

The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.

The α3ß4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 ß4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the ß4 and α3 subunit sequences, with different monomeric subunits. With this strategy, which allowed the formation of single populations of receptors with unique stoichiometry, we demonstrated with immunofluorescence and biochemical and functional assays that only the receptors with 3 ß4 subunits are efficiently expressed at the plasma membrane. Moreover, the LFM export motif of ß4 subunit in the fifth position exerts a unique function in the regulation of the intracellular trafficking of the receptors, their exposure at the cell surface, and consequently, their function, whereas the same export motif present in the ß4 subunits forming the acetylcholine binding site is dispensable.-Crespi, A., Plutino, S., Sciaccaluga, M., Righi, M., Borgese, N., Fucile, S., Gotti, C., Colombo, S. F. The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.

Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail-anchored proteins.

Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell-free and in cellula results, we analyzed targeting in digitonin-permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C-terminal negative charge of b5-ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ESI ) reduced insertion of b5-ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ESI , Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and reveal a novel, substrate-specific ER-targeting pathway.

CAML mediates survival of Myc-induced lymphoma cells independent of tail-anchored protein insertion.

Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum (ER) protein that functions, along with WRB and TRC40, to mediate tail-anchored (TA) protein insertion into the ER membrane. Physiologic roles for CAML include endocytic trafficking, intracellular calcium signaling, and the survival and proliferation of specialized immune cells, recently attributed to its requirement for TA protein insertion. To identify a possible role for CAML in cancer cells, we generated Eµ-Myc transgenic mice that carry a tamoxifen-inducible deletion allele of Caml. In multiple B-cell lymphoma cell lines derived from these mice, homozygous loss of Caml activated apoptosis. Cell death was blocked by Bcl-2/Bcl-xL overexpression; however, rescue from apoptosis was insufficient to restore proliferation. Tumors established from an Eµ-Myc lymphoma cell line completely regressed after tamoxifen administration, suggesting that CAML is also required for these cancer cells to survive and grow in vivo. Cell cycle analyses of Caml-deleted lymphoma cells revealed an arrest in G2/M, accompanied by low expression of the mitotic marker, phospho-histone H3 (Ser10). Surprisingly, lymphoma cell viability did not depend on the domain of CAML required for its interaction with TRC40. Furthermore, a small protein fragment consisting of the C-terminal 111 amino acid residues of CAML, encompassing the WRB-binding domain, was sufficient to rescue growth and survival of Caml-deleted lymphoma cells. Critically, this minimal region of CAML did not restore TA protein insertion in knockout cells. Taken together, these data reveal an essential role for CAML in supporting survival and mitotic progression in Myc-driven lymphomas that is independent of its TA protein insertion function.

Tail-anchored Protein Insertion in Mammals: FUNCTION AND RECIPROCAL INTERACTIONS OF THE TWO SUBUNITS OF THE TRC40 RECEPTOR.

The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in ∼5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER.

Getting membrane proteins on and off the shuttle bus between the endoplasmic reticulum and the Golgi complex.

Secretory proteins exit the endoplasmic reticulum (ER) in coat protein complex II (COPII)-coated vesicles and then progress through the Golgi complex before delivery to their final destination. Soluble cargo can be recruited to ER exit sites by signal-mediated processes (cargo capture) or by bulk flow. For membrane proteins, a third mechanism, based on the interaction of their transmembrane domain (TMD) with lipid microdomains, must also be considered. In this Commentary, I review evidence in favor of the idea that partitioning of TMDs into bilayer domains that are endowed with distinct physico-chemical properties plays a pivotal role in the transport of membrane proteins within the early secretory pathway. The combination of such self-organizational phenomena with canonical intermolecular interactions is most likely to control the release of membrane proteins from the ER into the secretory pathway.

Autophagy and Neurodegeneration: Insights from a Cultured Cell Model of ALS.

Autophagy plays a major role in the elimination of cellular waste components, the renewal of intracellular proteins and the prevention of the build-up of redundant or defective material. It is fundamental for the maintenance of homeostasis and especially important in post-mitotic neuronal cells, which, without competent autophagy, accumulate protein aggregates and degenerate. Many neurodegenerative diseases are associated with defective autophagy; however, whether altered protein turnover or accumulation of misfolded, aggregate-prone proteins is the primary insult in neurodegeneration has long been a matter of debate. Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by selective degeneration of motor neurons. Most of the ALS cases occur in sporadic forms (SALS), while 10%-15% of the cases have a positive familial history (FALS). The accumulation in the cell of misfolded/abnormal proteins is a hallmark of both SALS and FALS, and altered protein degradation due to autophagy dysregulation has been proposed to contribute to ALS pathogenesis. In this review, we focus on the main molecular features of autophagy to provide a framework for discussion of our recent findings about the role in disease pathogenesis of the ALS-linked form of the VAPB gene product, a mutant protein that drives the generation of unusual cytoplasmic inclusions.

Amyotrophic lateral sclerosis-linked mutant VAPB inclusions do not interfere with protein degradation pathways or intracellular transport in a cultured cell model.

VAPB is a ubiquitously expressed, ER-resident adaptor protein involved in interorganellar lipid exchange, membrane contact site formation, and membrane trafficking. Its mutant form, P56S-VAPB, which has been linked to a dominantly inherited form of Amyotrophic Lateral Sclerosis (ALS8), generates intracellular inclusions consisting in restructured ER domains whose role in ALS pathogenesis has not been elucidated. P56S-VAPB is less stable than the wild-type protein and, at variance with most pathological aggregates, its inclusions are cleared by the proteasome. Based on studies with cultured cells overexpressing the mutant protein, it has been suggested that VAPB inclusions may exert a pathogenic effect either by sequestering the wild-type protein and other interactors (loss-of-function by a dominant negative effect) or by a more general proteotoxic action (gain-of-function). To investigate P56S-VAPB degradation and the effect of the inclusions on proteostasis and on ER-to-plasma membrane protein transport in a more physiological setting, we used stable HeLa and NSC34 Tet-Off cell lines inducibly expressing moderate levels of P56S-VAPB. Under basal conditions, P56S-VAPB degradation was mediated exclusively by the proteasome in both cell lines, however, it could be targeted also by starvation-stimulated autophagy. To assess possible proteasome impairment, the HeLa cell line was transiently transfected with the ERAD (ER Associated Degradation) substrate CD3δ, while autophagic flow was investigated in cells either starved or treated with an autophagy-stimulating drug. Secretory pathway functionality was evaluated by analyzing the transport of transfected Vesicular Stomatitis Virus Glycoprotein (VSVG). P56S-VAPB expression had no effect either on the degradation of CD3δ or on the levels of autophagic markers, or on the rate of transport of VSVG to the cell surface. We conclude that P56S-VAPB inclusions expressed at moderate levels do not interfere with protein degradation pathways or protein transport, suggesting that the dominant inheritance of the mutant gene may be due mainly to haploinsufficiency.