Small Molecule Targets Env for Endoplasmic Reticulum-Associated Protein Degradation and Inhibits Human Immunodeficiency Virus Type 1 Propagation

Human immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH₂), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH₂ was uncovered by examining Env expression and maturation in treated cells. GPG-NH₂ treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH₂ negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH₂, which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy.The endoplasmic reticulum (ER) contains a number of molecular chaperones and folding factors that aid in the maturation of proteins that traverse the secretory pathway. This process is strictly monitored by the ER quality control system, which selects properly folded proteins for export to the Golgi (16) and targets misfolded proteins for destruction through the ER-associated protein degradation pathway (ERAD) (4, 28). Once an ER protein is selected as a substrate for ERAD, it is translocated from the ER lumen to the cytosol through an ER translocon. This retrotranslocation process is thought to be driven by either the cytosolic AAA-ATPase p97 (39) or the 19S proteasome cap (23). Upon entrance into the cytosol, the ERAD substrate is ubquitinated, and its glycans are removed by an N-glycanase to prepare it for proteasomal degradation (11, 28).Viral envelope glycoproteins utilize the host cell secretory pathway for their proper maturation and trafficking to the site of viral assembly. The human immunodeficiency virus type 1 (HIV-1) encodes the envelope glycoprotein (Env), which initiates HIV-1 infections by mediating attachment and fusion of the viral envelope with the host cell membrane (17). Therefore, infectious HIV-1 particle production relies on the ability of Env to pass the rigorous ER quality control system.Env is initially synthesized as a type I membrane precursor glycoprotein termed gp160, which is cotranslationally targeted to the ER by its 30-amino-acid N-terminal signal sequence (24). Within the ER, gp160 receives ∼30 N-linked glycans and is assisted in its maturation by the chaperones BiP, calnexin, and calreticulin as it undergoes extensive disulfide bond formations (15, 21, 31). Once gp160 has reached its native state with ten disulfide bonds and its signal sequence has been cleaved posttranslationally (21, 25), it assembles into trimers (26) and is exported to the Golgi. Within the Golgi, gp160 is cleaved by cellular endoproteases, yielding the transmembrane protein gp41 and the noncovalently associated surface protein gp120 (27). Thereafter, this complex is transported to the plasma membrane, where it is incorporated into the envelope of assembling HIV-1 particles.We have previously shown that a tripeptide amide corresponding to a conserved motif of the HIV-1 Env, glycyl-prolyl-glycine amide (GPG-NH₂), suppressed the replication of all 47 HIV-1 laboratory strains and clinical isolates examined with a 50% inhibitory concentration of ∼10 μM, a concentration that is 200- to 2,000-fold less than what affected cell growth or had other toxic effects on peripheral blood mononuclear cells (35). However, this suppression was not, as we had anticipated, due to interactions of the peptide with the early events of the HIV-1 replication cycle, such as attachment or entry (36). In the present study, we demonstrate that GPG-NH₂ reduced Env incorporation into HIV-1 particles during replication by targeting Env toward the ERAD pathway. The ability of GPG-NH₂ to target Env for degradation was dependent on the presence of functional proteasomes and required the full-length Env signal sequence. These findings illustrate that small molecules may be utilized therapeutically to specifically target unwanted pathogenic proteins for degradation by the existing cellular machinery.     

Fluc‐EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age‐related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP‐fused firefly luciferase (Fluc‐EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc‐EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc‐EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington’s disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc‐EGFP reporter mice enable new insights into proteostasis alterations in different diseases.     

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. However, we recently discovered that MAN1B1 localizes to the Golgi complex in human cells and uncovered its participation in ERAD substrate retention, retrieval to the ER, and subsequent degradation from this organelle. The objective of the current study was to further characterize the contribution of MAN1B1 as part of a Golgi-based quality control network. Multiple lines of experimental evidence support a model in which neither the mannosidase activity nor catalytic domain is essential for the retention or degradation of the misfolded ERAD substrate Null Hong Kong. Instead, a highly conserved, vertebrate-specific non-enzymatic decapeptide sequence in the luminal stem domain plays a significant role in controlling the fate of overexpressed Null Hong Kong. Together, these findings define a new functional paradigm in which Golgi-localized MAN1B1 can play a mannosidase-independent gatekeeper role in the proteostasis network of higher eukaryotes.     

Remodeling the Proteostasis Network to Rescue Glucocerebrosidase Variants by Inhibiting ER-Associated Degradation and Enhancing ER Folding

Gaucher’s disease (GD) is characterized by loss of lysosomal glucocerebrosidase (GC) activity. Mutations in the gene encoding GC destabilize the protein’s native folding leading to ER-associated degradation (ERAD) of the misfolded enzyme. Enhancing the cellular folding capacity by remodeling the proteostasis network promotes native folding and lysosomal activity of mutated GC variants. However, proteostasis modulators reported so far, including ERAD inhibitors, trigger cellular stress and lead to induction of apoptosis. We show herein that lacidipine, an L-type Ca²⁺ channel blocker that also inhibits ryanodine receptors on the ER membrane, enhances folding, trafficking and lysosomal activity of the most severely destabilized GC variant achieved via ERAD inhibition in fibroblasts derived from patients with GD. Interestingly, reprogramming the proteostasis network by combining modulation of Ca²⁺ homeostasis and ERAD inhibition remodels the unfolded protein response and dramatically lowers apoptosis induction typically associated with ERAD inhibition.     

Targeting of the unfolded protein response (UPR) as therapy for Parkinson's disease

Parkinson's disease is the second most common neurodegenerative disorder, leading to the progressive decline of motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta. At the molecular level, Parkinson's disease share common molecular signatures with most neurodegenerative diseases including the accumulation of misfolded proteins in the brain. Alteration in the buffering capacity of the proteostasis network during aging is proposed as one of the triggering steps leading to abnormal protein aggregation in this disease, highlighting disturbances in the function of the endoplasmic reticulum (ER). The ER is the main subcellular compartment involved in protein folding and quality control. ER stress triggers a signalling reaction known as the unfolded protein response (UPR), which aims restoring proteostasis through the induction of adaptive programs or the activation of cell death pathways when damage is chronic and cannot be repaired. Here, we overview most evidence linking ER stress to Parkinson's disease. Strategies to alleviate ER stress by targeting specific components of the UPR using small molecules and gene therapy are highlighted.     

A revised mechanism for how Plasmodium falciparum recruits and exports proteins into its erythrocytic host cell

Plasmodium falciparum exports ~10% of its proteome into its host erythrocyte to modify the host cell’s physiology. The Plasmodium export element (PEXEL) motif contained within the N-terminus of most exported proteins directs the trafficking of those proteins into the erythrocyte. To reach the host cell, the PEXEL motif of exported proteins is processed by the endoplasmic reticulum (ER) resident aspartyl protease plasmepsin V. Then, following secretion into the parasite-encasing parasitophorous vacuole, the mature exported protein must be unfolded and translocated across the parasitophorous vacuole membrane by the Plasmodium translocon of exported proteins (PTEX). PTEX is a protein-conducting channel consisting of the pore-forming protein EXP2, the protein unfoldase HSP101, and structural component PTEX150. The mechanism of how exported proteins are specifically trafficked from the parasite’s ER following PEXEL cleavage to PTEX complexes on the parasitophorous vacuole membrane is currently not understood. Here, we present evidence that EXP2 and PTEX150 form a stable subcomplex that facilitates HSP101 docking. We also demonstrate that HSP101 localises both within the parasitophorous vacuole and within the parasite’s ER throughout the ring and trophozoite stage of the parasite, coinciding with the timeframe of protein export. Interestingly, we found that HSP101 can form specific interactions with model PEXEL proteins in the parasite’s ER, irrespective of their PEXEL processing status. Collectively, our data suggest that HSP101 recognises and chaperones PEXEL proteins from the ER to the parasitophorous vacuole and given HSP101’s specificity for the EXP2-PTEX150 subcomplex, this provides a mechanism for how exported proteins are specifically targeted to PTEX for translocation into the erythrocyte.     

Roles of extracellular chaperones in amyloidosis

Extracellular protein misfolding and aggregation underlie many of the most serious amyloidoses including Alzheimer's disease, spongiform encephalopathies and type II diabetes. Despite this, protein homeostasis (proteostasis) research has largely focussed on characterising systems that function to monitor protein conformation and concentration within cells. We are now starting to identify elements of corresponding systems, including an expanding family of secreted chaperones, which exist in the extracellular space. Like their intracellular counterparts, extracellular chaperones are likely to play a central role in systems that maintain proteostasis; however, the precise details of how they participate are only just emerging. It is proposed that extracellular chaperones patrol biological fluids for misfolded proteins and facilitate their clearance via endocytic receptors. Importantly, many amyloidoses are associated with dysfunction in rates of protein clearance. This is consistent with a model in which disruption to, or overwhelming of, the systems responsible for extracellular proteostasis results in the accumulation of pathological protein aggregates and disease. Further characterisation of mechanisms that maintain extracellular proteostasis will shed light on why many serious diseases occur and provide us with much needed strategies to combat them.     

Inhibition of endoplasmic reticulum associated degradation reduces endoplasmic reticulum stress and alters lysosomal morphology and distribution

Disturbances in proteostasis are observed in many neurodegenerative diseases. This leads to activation of protein quality control to restore proteostasis, with a key role for the removal of aberrant proteins by proteolysis. The unfolded protein response (UPR) is a protein quality control mechanism of the endoplasmic reticulum (ER) that is activated in several neurodegenerative diseases. Recently we showed that the major proteolytic pathway during UPR activation is via the autophagy/lysosomal system. Here we investigate UPR induction if the other major proteolytic pathway of the ER -ER associated degradation (ERAD)-is inhibited. Surprisingly, impairment of ERAD results in decreased UPR activation and protects against ER stress toxicity. Autophagy induction is not affected under these conditions, however, a striking relocalization of the lysosomes is observed. Our data suggest that a protective UPR-modulating mechanism is activated if ERAD is inhibited, which involves lysosomes. Our data provide insight in the cross-talk between proteolytic pathways involved in ER proteostasis. This has implications for neurodegenerative diseases like Alzheimer’s disease where disturbed ER proteostasis and proteolytic impairment are early phenomena in the pathology.