Genetic and Pharmacological Modulation of Cellular Proteostasis Leads to Partial Functional Rescue of Homocystinuria-Causing Cystathionine-Beta Synthase Variants.

Homocystinuria (HCU), an inherited metabolic disorder caused by lack of cystathionine beta-synthase (CBS) activity, is chiefly caused by misfolding of single amino acid residue missense pathogenic variants. Previous studies showed that chemical, pharmacological chaperones or proteasome inhibitors could rescue function of multiple pathogenic CBS variants; however, the underlying mechanisms remain poorly understood. Using Chinese hamster DON fibroblasts devoid of CBS and stably overexpressing human WT or mutant CBS, we showed that expression of pathogenic CBS variant mostly dysregulates gene expression of small heat shock proteins HSPB3 and HSPB8 and members of HSP40 family. Endoplasmic reticulum stress sensor BiP was found upregulated with CBS I278T variant associated with proteasomes suggesting proteotoxic stress and degradation of misfolded CBS. Co-expression of the main effector HSP70 or master regulator HSF1 rescued steady-state levels of CBS I278T and R125Q variants with partial functional rescue of the latter. Pharmacological proteostasis modulators partially rescued expression and activity of CBS R125Q likely due to reduced proteotoxic stress as indicated by decreased BiP levels and promotion of refolding as indicated by induction of HSP70. In conclusion, targeted manipulation of cellular proteostasis may represent a viable therapeutic approach for the permissive pathogenic CBS variants causing HCU.

Multiomic screening of invasive GBM cells reveals targetable transsulfuration pathway alterations.

While the poor prognosis of glioblastoma arises from the invasion of a subset of tumor cells, little is known of the metabolic alterations within these cells that fuel invasion. We integrated spatially addressable hydrogel biomaterial platforms, patient site-directed biopsies, and multiomics analyses to define metabolic drivers of invasive glioblastoma cells. Metabolomics and lipidomics revealed elevations in the redox buffers cystathionine, hexosylceramides, and glucosyl ceramides in the invasive front of both hydrogel-cultured tumors and patient site-directed biopsies, with immunofluorescence indicating elevated reactive oxygen species (ROS) markers in invasive cells. Transcriptomics confirmed upregulation of ROS-producing and response genes at the invasive front in both hydrogel models and patient tumors. Among oncologic ROS, H2O2 specifically promoted glioblastoma invasion in 3D hydrogel spheroid cultures. A CRISPR metabolic gene screen revealed cystathionine γ-lyase (CTH), which converts cystathionine to the nonessential amino acid cysteine in the transsulfuration pathway, to be essential for glioblastoma invasion. Correspondingly, supplementing CTH knockdown cells with exogenous cysteine rescued invasion. Pharmacologic CTH inhibition suppressed glioblastoma invasion, while CTH knockdown slowed glioblastoma invasion in vivo. Our studies highlight the importance of ROS metabolism in invasive glioblastoma cells and support further exploration of the transsulfuration pathway as a mechanistic and therapeutic target.

Cystathionine-γ-lyase attenuates inflammatory response and pain of osteoarthritis.

The chronic articular disease osteoarthritis (OA) is characterized by osteophyte generation, subchondral bone remodeling, and cartilage deterioration. Low levels of H2S catalyzed by cystathionine-γ-lyase (CSE) encoded by Cthhas neuroprotective, cardioprotective, anti-apoptotic, and anti-inflammatory effects thus, Cth is being developed as a potential therapy for the management of the pathogenesis and symptoms of osteoarthritis. Single-cell RNA sequencing (scRNA-seq) and immunohistochemistry of human cartilage revealed that the expression of CTH was decreased in OA patients. We found that Cthoverexpression decrease IL-1ß-induced overactivation of the NF-κB signaling pathway. In vivo, Cthoverexpression relieved pain response and cartilage damage in the anterior cruciate ligament transection (ACLT) rat model. In vitro, CSE alleviated chondrocytes catabolism, inflammation, apoptosis, and senescence, and suppressed the NF-κB pathway. We postulate that CSE has therapeutic effects in suppressing inflammation and degeneration in OA and should be further investigated clinically.

Hydrogen sulfide donor GYY4137 attenuates vascular complications in mesenteric bed of streptozotocin-induced diabetic rats.

Hydrogen sulfide (H2S) has been reported to have beneficial effects in different pathological conditions. OBJECTIVES: the effects of chronic treatment of diabetic rats with GYY4137 (slow releasing H2S donor) or NaHS (fast releasing H2S donor) on the reactivity of the mesenteric bed to vasoactive agonists and the changes in its downstream effectors, ERK1/2 and p38 MAP Kinase have been investigated. In addition, the levels of nitric oxide (NO) and H2S in all groups were measured. METHODS: diabetes was induced by a single intraperitoneal (ip) injection of streptozotocin (STZ; 55 mg/kg). Sprague Dawley (SD; n = 10-12/group) rats were randomly divided into six groups: control, STZ-induced diabetic rats, GYY4137-treated control, NaHS-treated control, GYY4137-treated diabetic, and NaHS-treated diabetic. After 28 days of treatment, rats were sacrificed and mesenteric beds were isolated for functional or biochemical studies. The vascular reactivity of the perfused mesenteric bed to norepinephrine, carbachol and sodium nitroprusside were determined by measurement of changes in perfusion pressure. Western blotting was performed to measure the protein expression of ERK1/2, p38, eNOS, and H2S biosynthesizing enzymes cystathionine-ß-synthase and cystathionine-γ-lyase. NO and H2S levels were measured in all groups in isolated mesenteric tissues or plasma. RESULTS: diabetes resulted in a significant increase in vasoconstrictor responses to norepinephrine (e.g., 129.6 ± 6.77 mmHg in diabetic vs 89.3 ± 8.48 mmHg in control at 10-7 dose), and carbachol-induced vasodilation was significantly reduced in diabetic mesenteric bed (e.g., 68.9 ± 4.8 mmHg in diabetic vs 90.6 ± 2.2 mmHg in control at 10-7 dose). Chronic treatment of the diabetic rats with GYY4137 resulted in a significant improvement in the response to norepinephrine (e.g., 86.66 ± 8.04 mmHg in GYY4137-treated diabetic vs 129.6 ± 6.77 mmHg in untreated diabetic at 10-7 dose) or carbachol (e.g., 84.90 ± 2.48 mmHg in GYY4137-treated diabetic vs 68.9 ± 4.8 mmHg in untreated diabetic at 10-7 dose). The biochemical studies showed a marked reduction of the protein expression of ERK and p38 and a significant upregulation of the expression of eNOS and H2S synthesizing enzymes after chronic treatment with GYY4137. Plasma levels of NO and H2S were significantly elevated after treatment with GYY4137. However, H2S production in the mesenteric bed showed a marginal elevation in diabetic tissues compared to controls. CONCLUSION: the results indicate that GYY4137 may be a novel therapeutic tool to prevent diabetes-associated vascular dysfunction.

Effects of glutamate and sulphur containing amino acids on ammonia toxicity and methionine sulphoximine convulsions in mice.

Arginine and large doses of glutamate (greater than 500 mg/kg) were found to reduce ammonia toxicity transiently in mice. Smaller doses of glutamate (greater than or equal to 200 mg/kg) were effective when administered with glucose. Cysteic acid, homocysteine and methionine, but not taurine reduced ammonia toxicity. All 4 amino acids reduced the number of convulsions induced by methionine sulphoximine. It is proposed that taurine has a general anticonvulsant action and that cysteic acid, homocysteine and methionine may have a specific effect on the action of ammonia on the central nervous system.