Pharmacological management of disease by overcoming structural adversity.

In this issue of Cell Chemical Biology, Sun et al. utilize computational and protein expression analyses, plus pharmacological proteostasis network activation, to simultaneously correct two genetic diseases linked to a single protein rather than modify the responsible DNA.

The cytoplasmic tail of human mannosidase Man1b1 contributes to catalysis-independent quality control of misfolded alpha1-antitrypsin.

The failure of polypeptides to achieve conformational maturation following biosynthesis can result in the formation of protein aggregates capable of disrupting essential cellular functions. In the secretory pathway, misfolded asparagine (N)-linked glycoproteins are selectively sorted for endoplasmic reticulum-associated degradation (ERAD) in response to the catalytic removal of terminal alpha-linked mannose units. Remarkably, ER mannosidase I/Man1b1, the first alpha-mannosidase implicated in this conventional N-glycan-mediated process, can also contribute to ERAD in an unconventional, catalysis-independent manner.

A CLN6-CLN8 complex recruits lysosomal enzymes at the ER for Golgi transfer.

Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8 (ceroid lipofuscinosis, neuronal, 8). Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system), which recruits lysosomal enzymes at the ER to promote their Golgi transfer.

A kinome-wide RNAi screen identifies ERK2 as a druggable regulator of Shank3 stability.

Neurons are sensitive to changes in the dosage of many genes, especially those regulating synaptic functions. Haploinsufficiency of SHANK3 causes Phelan-McDermid syndrome and autism, whereas duplication of the same gene leads to SHANK3 duplication syndrome, a disorder characterized by neuropsychiatric phenotypes including hyperactivity and bipolar disorder as well as epilepsy. We recently demonstrated the functional modularity of Shank3, which suggests that normalizing levels of Shank3 itself might be more fruitful than correcting pathways that function downstream of it for treatment of disorders caused by alterations in SHANK3 dosage.

CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis.

Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease).

Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution.

Chronic endoplasmic reticulum (ER) stress results in toxicity that contributes to multiple human disorders. We report a stress resolution pathway initiated by the nuclear receptor LRH-1 that is independent of known unfolded protein response (UPR) pathways. Like mice lacking primary UPR components, hepatic Lrh-1-null mice cannot resolve ER stress, despite a functional UPR.

A Golgi-localized mannosidase (MAN1B1) plays a non-enzymatic gatekeeper role in protein biosynthetic quality control.

Conformation-based disorders are manifested at the level of protein structure, necessitating an accurate understanding of how misfolded proteins are processed by the cellular proteostasis network. Asparagine-linked glycosylation plays important roles for protein quality control within the secretory pathway. The suspected role for the MAN1B1 gene product MAN1B1, also known as ER mannosidase I, is to function within the ER similar to the yeast ortholog Mns1p, which removes a terminal mannose unit to initiate a glycan-based ER-associated degradation (ERAD) signal.

ERManI is a target of miR-125b and promotes transformation phenotypes in hepatocellular carcinoma (HCC).

The MAN1B1 gene product, designated ER alpha-1, 2-mannosidase (ERManI), is an enzyme localized in the Golgi complex of mammalian cells. By functioning as a "gate keeper" to prevent the inappropriate secretion of misfolded glycoproteins, it plays a critical role in maintaining protein homeostasis in the mammalian secretory pathway. In the present study, we identified that a conserved motif within the 3'UTR of ERManI is a target of miR-125b, a microRNA frequently down-regulated in numerous types of cancers, including hepatocellular carcinoma (HCC).

Resurrecting the protein fold for disease intervention.

Because proteostasis networks manage the cellular proteome, their pharmacological manipulation might correct pathologies associated with numerous protein misfolding diseases. In this issue of Chemistry & Biology, Tong Ong and colleagues identify a novel biosynthetic juncture for glucocerebrosidase as a site for therapeutic intervention in Gaucher's disease.

Golgi-situated endoplasmic reticulum α-1, 2-mannosidase contributes to the retrieval of ERAD substrates through a direct interaction with γ-COP.

Endoplasmic reticulum (ER) α-1, 2-mannosidase (ERManI) contributes to ER-associated protein degradation (ERAD) by initiating the formation of degradation signals on misfolded N-linked glycoproteins. Despite its inferred intracellular location, we recently discovered that the mammalian homologue is actually localized to the Golgi complex. In the present study, the functional role of Golgi-situated ERManI was investigated.

β-N-Acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3.

O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood.

Golgi localization of ERManI defines spatial separation of the mammalian glycoprotein quality control system.

The Golgi complex has been implicated as a possible component of endoplasmic reticulum (ER) glycoprotein quality control, although the elucidation of its exact role is lacking. ERManI, a putative ER resident mannosidase, plays a rate-limiting role in generating a signal that targets misfolded N-linked glycoproteins for ER-associated degradation (ERAD). Herein we demonstrate that the endogenous human homologue predominantly resides in the Golgi complex, where it is subjected to O-glycosylation.

Analysis of serpin secretion, misfolding, and surveillance in the endoplasmic reticulum.

Biological checkpoints are known to function in the cellular nucleus to monitor the integrity of inherited genetic information. It is now understood that posttranslational checkpoint systems operate in numerous biosynthetic compartments where they orchestrate the surveillance of encoded protein structures. This is particularly true for the serpins where opposing, but complementary, systems operate in the early secretory pathway to initially facilitate protein folding and then selectively target the misfolded proteins for proteolytic elimination.

Intracellular processing of alpha1-antitrypsin.

α(1)-Antitrypsin (AAT) secreted from hepatocytes is an inhibitor of neutrophil elastase. Its normal circulating concentration functions to maintain the elasticity of the lung by preventing the hydrolytic destruction of elastin fibers. Severely diminished circulating concentrations of AAT, resulting from the impaired secretion of genetic variants that exhibit distinct polypeptide folding defects, can function as an etiologic agent for the development of chronic obstructive pulmonary disease.

Single nucleotide polymorphism-mediated translational suppression of endoplasmic reticulum mannosidase I modifies the onset of end-stage liver disease in alpha1-antitrypsin deficiency.

Unlabelled: Inappropriate accumulation of the misfolded Z variant of alpha1-antitrypsin in the hepatocyte endoplasmic reticulum (ER) is a risk factor for the development of end-stage liver disease. However, the genetic and environmental factors that contribute to its etiology are poorly understood. ER mannosidase I (ERManI) is a quality control factor that plays a critical role in the sorting and targeting of misfolded glycoproteins for proteasome-mediated degradation.

The mammalian UPR boosts glycoprotein ERAD by suppressing the proteolytic downregulation of ER mannosidase I.

The secretory pathway provides a physical route through which only correctly folded gene products are delivered to the eukaryotic cell surface. The efficiency of endoplasmic reticulum (ER)-associated degradation (ERAD), which orchestrates the clearance of structurally aberrant proteins under basal conditions, is boosted by the unfolded protein response (UPR) as one of several means to relieve ER stress. However, the underlying mechanism that links the two systems in higher eukaryotes has remained elusive.

Extracellular Alix regulates integrin-mediated cell adhesions and extracellular matrix assembly.

Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts.

Small molecules block the polymerization of Z alpha1-antitrypsin and increase the clearance of intracellular aggregates.

The Z mutant of alpha1-antitrypsin (Glu342Lys) causes a domain swap and the formation of intrahepatic polymers that aggregate as inclusions and predispose the homozygote to cirrhosis. We have identified an allosteric cavity that is distinct from the interface involved in polymerization for rational structure-based drug design to block polymer formation. Virtual ligand screening was performed on 1.

Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis.

In the early secretory pathway, opportunistic cleavage of asparagine-linked oligosaccharides by endoplasmic reticulum (ER) mannosidase I targets misfolded glycoproteins for dislocation into the cytosol and destruction by 26 S proteasomes. The low basal concentration of the glycosidase is believed to coordinate the glycan cleavage with prolonged conformation-based ER retention, ensuring that terminally misfolded glycoproteins are preferentially targeted for destruction. Herein the intracellular fate of human ER mannosidase I was monitored to determine whether a post-translational process might contribute to the regulation of its intracellular concentration.

Alpha1-antitrypsin as model to assess glycan function in endoplasmic reticulum.

It is now understood that a cohesive series of quality control checkpoints ensures the accuracy of gene expression in eukaryotic cells. Although initiated in the nucleus to monitor the integrity of inherited genetic information, the quality control program encompasses post-translational events that facilitate the structural maturation of encoded proteins or target them for degradation if unable to adopt native structure. Given the fact that many genetic mutations actually manifest themselves at the level of aberrant protein structure, a current challenge in the post-genomics era is to elucidate how post-translational checkpoints can modify the severity of numerous loss-of-function and gain-of-toxic-function diseases, possibly influencing an individual's susceptibility toward the development of the associated pathologies.

Human EDEM2, a novel homolog of family 47 glycosidases, is involved in ER-associated degradation of glycoproteins.

In the endoplasmic reticulum (ER), misfolded proteins are retrotranslocated to the cytosol and degraded by the proteasome in a process known as ER-associated degradation (ERAD). Early in this pathway, a proposed lumenal ER lectin, EDEM, recognizes misfolded glycoproteins in the ER, disengages the nascent molecules from the folding pathway, and facilitates their targeting for disposal. In humans there are a total of three EDEM homologs.