Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.

Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.

Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors.

Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABAA receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABAA receptors. We carried out a quantitative immunoprecipitation-tandem mass spectrometry analysis utilizing stable isotope labeling by amino acids in cell culture. Furthermore, we performed comparative proteomics by using both WT α1 subunit and a misfolding-prone α1 subunit carrying the A322D variant as the bait proteins. We identified 125 interactors for WT α1-containing receptors, 105 proteins for α1(A322D)-containing receptors, and 54 overlapping proteins within these two interactomes. Our bioinformatics analysis identified potential GABAA receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors, and we assembled a model of their potential involvement in the cellular folding, degradation, and trafficking pathways for GABAA receptors. In addition, we verified endogenous interactions between α1 subunits and selected interactors by using coimmunoprecipitation in mouse brain homogenates. Moreover, we showed that TRIM21 (tripartite motif containing-21), an E3 ubiquitin ligase, positively regulated the degradation of misfolding-prone α1(A322D) subunits selectively. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABAA receptor proteostasis to ameliorate related neurological diseases such as epilepsy.

Thermochemical Recycling of Oily Sludge by Catalytic Pyrolysis: A Review.

The main methods of treating oily sludge at home and abroad and the current research status of oily sludge pyrolysis technology are briefly described, and four commonly used catalysts are introduced: metals, metal compounds, molecular sieves, metal-supported molecular sieves, and biomass catalysts for oily sludge. The influence of pyrolysis, the pyrolysis mechanism, and the product composition of oily sludge with the addition of different catalysts are also discussed. Finally, the development direction of preparing new catalysts and the mixed use of multiple catalysts is proposed as a theory to provide for the efficient and reasonable utilization of oily sludge.

Progress in Catalytic Pyrolysis of Oil Shale.

This paper briefly describes the research status of oil shale pyrolysis technology and the main factors affecting oil shale pyrolysis, with emphasis on four kinds of commonly used catalysts: The effects of natural minerals, metal compounds, molecular sixes, and supported catalysts on the pyrolysis of oil shale were discussed. The changes of the pyrolysis mechanism and product composition of oil shale with the addition of different catalysts were discussed. Finally, the development direction of preparation of new catalysts was discussed, in order to provide a prospect for the development and utilization of unconventional and strategic alternative energy resources around the world.

Proteostasis Regulators Restore Function of Epilepsy-Associated GABAA Receptors.

Proteostasis deficiency in mutated ion channels leads to a variety of ion channel diseases that are caused by excessive endoplasmic reticulum-associated degradation (ERAD) and inefficient membrane trafficking. We investigated proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors, the primary mediators of neuronal inhibition in the mammalian central nervous system. We screened a structurally diverse, Food and Drug Administration-approved drug library and identified dinoprost (DNP) and dihydroergocristine (DHEC) as highly efficacious enhancers of surface expression of four epilepsy-causing trafficking-deficient mutant receptors. Furthermore, DNP and DHEC restore whole-cell and synaptic currents by incorporating mutated subunits into functional receptors. Mechanistic studies revealed that both drugs reduce subunit degradation by attenuating the Grp94/Hrd1/Sel1L/VCP-mediated ERAD pathway and enhance the subunit folding by promoting subunit interactions with major GABAA receptors-interacting chaperones, BiP and calnexin. In summary, we report that DNP and DHEC remodel the endoplasmic reticulum proteostasis network to restore the functional surface expression of mutant GABAA receptors.

Effect of Double Transition Metal Salt Catalyst on Fushun Oil Shale Pyrolysis.

Shale ash (SA) as the carrier, the ratio of Cu to Ni in the Cu-Ni transition metal salt being, respectively, 1 : 0, 2 : 1, 1 : 1, 1 : 2, 0 : 1, the double transition metal salt catalyst (CumNin/SA) was prepared to explore the effect of such catalysts on the pyrolysis behavior and characteristics of Fushun OS. The research results show that the temperature (T max) corresponding to the maximum weight loss rate decreased by 12.9°C, 4.0°C, and 3.6°C; and the apparent activation energy decreased by 35.2%, 33.9%, and 29.6%, respectively, after adding catalysts Cu0Ni1/SA in pyrolysis. The addition of Cu0Ni1/SA and Cu2Ni1/SA further improves the shale oil (SO) yield of 3.5% and 3.1%, respectively. Cu0Ni1/SA produces more aromatic hydrocarbons, which, however, weakens the stability of SO and is of toxicity in use. After analyzing the pyrolysis product-semicoke (SC) and SO-with ATR-FTIR and GC-MS methods, CumNin/SA promotes the secondary cracking and aromatization of OS pyrolysis, increasing the content of the compound of olefins and aromatics in SO, and hastening the decomposition of long-chain aliphatic hydrocarbons to short-chain aliphatic hydrocarbons.

Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors.

Biogenesis of membrane proteins is controlled by the protein homeostasis (proteostasis) network. We have been focusing on protein quality control of γ-aminobutyric acid type A (GABAA) receptors, the major inhibitory neurotransmitter-gated ion channels in mammalian central nervous system. Proteostasis deficiency in GABAA receptors causes loss of their surface expression and thus function on the plasma membrane, leading to epilepsy and other neurological diseases. One well-characterized example is the A322D mutation in the α1 subunit that causes its extensive misfolding and expedited degradation in the endoplasmic reticulum (ER), resulting in autosomal dominant juvenile myoclonic epilepsy. We aimed to correct misfolding of the α1(A322D) subunits in the ER as an approach to restore their functional surface expression. Here, we showed that application of BIX, a specific, potent ER resident HSP70 family protein BiP activator, significantly increases the surface expression of the mutant receptors in human HEK293T cells and neuronal SH-SY5Y cells. BIX attenuates the degradation of α1(A322D) and enhances their forward trafficking and function. Furthermore, because BiP is one major target of the two unfolded protein response (UPR) pathways: ATF6 and IRE1, we continued to demonstrate that modest activations of the ATF6 pathway and IRE1 pathway genetically enhance the plasma membrane trafficking of the α1(A322D) protein in HEK293T cells. Our results underlie the potential of regulating the ER proteostasis network to correct loss-of-function protein conformational diseases.

GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells.

GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR) pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER)-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome.

Grp94 Protein Delivers γ-Aminobutyric Acid Type A (GABAA) Receptors to Hrd1 Protein-mediated Endoplasmic Reticulum-associated Degradation.

Proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors dictates their function in controlling neuronal inhibition in mammalian central nervous systems. However, as a multisubunit, multispan, integral membrane protein, even wild type subunits of GABAA receptors fold and assemble inefficiently in the endoplasmic reticulum (ER). Unassembled and misfolded subunits undergo ER-associated degradation (ERAD), but this degradation process remains poorly understood for GABAA receptors. Here, using the α1 subunits of GABAA receptors as a model substrate, we demonstrated that Grp94, a metazoan-specific Hsp90 in the ER lumen, uses its middle domain to interact with the α1 subunits and positively regulates their ERAD. OS-9, an ER-resident lectin, acts downstream of Grp94 to further recognize misfolded α1 subunits in a glycan-dependent manner. This delivers misfolded α1 subunits to the Hrd1-mediated ubiquitination and the valosin-containing protein-mediated extraction pathway. Repressing the initial ERAD recognition step by inhibiting Grp94 enhances the functional surface expression of misfolding-prone α1(A322D) subunits, which causes autosomal dominant juvenile myoclonic epilepsy. This study clarifies a Grp94-mediated ERAD pathway for GABAA receptors, which provides a novel way to finely tune their function in physiological and pathophysiological conditions.

Combining valosin-containing protein (VCP) inhibition and suberanilohydroxamic acid (SAHA) treatment additively enhances the folding, trafficking, and function of epilepsy-associated γ-aminobutyric acid, type A (GABAA) receptors.

GABAA receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the α1 subunit results in its excessive endoplasmic reticulum-associated degradation at the expense of plasma membrane trafficking, leading to autosomal dominant juvenile myoclonic epilepsy. Presumably, valosin-containing protein (VCP)/p97 extracts misfolded subunits from the endoplasmic reticulum membrane to the cytosolic proteasome for degradation. Here we showed that inhibiting VCP using Eeyarestatin I reduces the endoplasmic reticulum-associated degradation of the α1(A322D) subunit without an apparent effect on its dynamin-1 dependent endocytosis and that this treatment enhances its trafficking. Furthermore, coapplication of Eeyarestatin I and suberanilohydroxamic acid, a known small molecule that promotes chaperone-assisted folding, yields an additive restoration of surface expression of α1(A322D) subunits in HEK293 cells and neuronal SH-SY5Y cells. Consequently, this combination significantly increases GABA-induced chloride currents in whole-cell patch clamping experiments than either chemical compound alone in HEK293 cells. Our findings suggest that VCP inhibition without stress induction, together with folding enhancement, represents a new strategy to restore proteostasis of misfolding-prone GABAA receptors and, therefore, a potential remedy for idiopathic epilepsy.

Using pharmacological chaperones to restore proteostasis.

Normal organismal physiology depends on the maintenance of proteostasis in each cellular compartment to achieve a delicate balance between protein synthesis, folding, trafficking, and degradation while minimizing misfolding and aggregation. Defective proteostasis leads to numerous protein misfolding diseases. Pharmacological chaperones are cell-permeant small molecules that promote the proper folding and trafficking of a protein via direct binding to that protein. They stabilize their target protein in a protein-pharmacological chaperone state, increasing the natively folded protein population that can effectively engage trafficking machinery for transport to the final destination for function. Here, as regards the application of pharmacological chaperones, we focus on their capability to promote the folding and trafficking of lysosomal enzymes, G protein coupled receptors (GPCRs), and ion channels, each of which is presently an important drug target. Pharmacological chaperones hold great promise as potential therapeutics to ameliorate a variety of protein misfolding diseases.

SAHA enhances Proteostasis of epilepsy-associated α1(A322D)ß2γ2 GABA(A) receptors.

GABA(A) receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the α1 subunit of GABA(A) receptors is known to result in its degradation and reduce its cell surface expression, leading to loss of GABAA receptor function in autosomal dominant juvenile myoclonic epilepsy. Here, we show that SAHA, a FDA-approved drug, increases the transcription of the α1(A322D) subunit, enhances its folding and trafficking posttranslationally, increases its cell surface level, and restores the GABA-induced maximal current in HEK293 cells expressing α1(A322D)ß2γ2 receptors to 10% of that for wild-type receptors. To enhance the trafficking efficiency of the α1(A322D) subunit, SAHA increases the BiP protein level and the interaction between the α1(A322D) subunit and calnexin. SAHA is a drug that enhances epilepsy-associated GABAA receptor proteostasis.

L-theanine inhibits nicotine-induced dependence via regulation of the nicotine acetylcholine receptor-dopamine reward pathway.

In this study, the inhibitory effect of L-theanine, an amino acid derivative of tea, on the rewarding effects of nicotine and its underlying mechanisms of action were studied. We found that L-theanine inhibited the rewarding effects of nicotine in a conditioned place preference (CPP) model of the mouse and reduced the excitatory status induced by nicotine in SH-SY5Y cells to the same extent as the nicotine receptor inhibitor dihydro-beta-erythroidine (DHßE). Further studies using high performance liquid chromatography, western blotting and immunofluorescence staining analyses showed that L-theanine significantly inhibited nicotine-induced tyrosine hydroxylase (TH) expression and dopamine production in the midbrain of mice. L-theanine treatment also reduced the upregulation of the α(4), ß(2) and α(7) nicotine acetylcholine receptor (nAChR) subunits induced by nicotine in mouse brain regions that related to the dopamine reward pathway, thus decreasing the number of cells that could react to nicotine. In addition, L-theanine treatment inhibited nicotine-induced c-Fos expression in the reward circuit related areas of the mouse brain. Knockdown of c-Fos by siRNA inhibited the excitatory status of cells but not the upregulation of TH induced by nicotine in SH-SY5Y cells. Overall, the present study showed that L-theanine reduced the nicotine-induced reward effects via inhibition of the nAChR-dopamine reward pathway. These results may offer new therapeutic strategies for treatment of tobacco addiction.

The cessation and detoxification effect of tea filters on cigarette smoke.

To treat tobacco addiction, a tea filter was developed and studied for smoking cessation. This work reports the smoking cessation effect of tea when it was used as a component of cigarette filters. In one trial it was found that after using the tea filters for 2 months, the volunteer smokers decreased their cigarette consumption by 56.5%, and 31.7% of them stopped smoking. This work identified a new method and material, tea filter and theanine, which inhibit tobacco and nicotine addiction and provide an effective strategy for treating tobacco addiction.