Dopamine modification of glycolytic enzymes impairs glycolysis: possible implications for Parkinson's disease.

BACKGROUND: Parkinson's disease (PD), a chronic and severe neurodegenerative disease, is pathologically characterized by the selective loss of nigrostriatal dopaminergic neurons. Dopamine (DA), the neurotransmitter produced by dopaminergic neurons, and its metabolites can covalently modify proteins, and dysregulation of this process has been implicated in neuronal loss in PD. However, much remains unknown about the protein targets. METHODS: In the present work, we designed and synthesized a dopamine probe (DA-P) to screen and identify the potential protein targets of DA using activity-based protein profiling (ABPP) technology in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In situ pull-down assays, cellular thermal shift assays (CETSAs) and immunofluorescence were performed to confirm the DA modifications on these hits. To investigate the effects of DA modifications, we measured the enzymatic activities of these target proteins, evaluated glycolytic stress and mitochondrial respiration by Seahorse tests, and systematically analyzed the changes in metabolites with unbiased LC-MS/MS-based non-targeted metabolomics profiling. RESULTS: We successfully identified three glycolytic proteins, aldolase A, α-enolase and pyruvate kinase M2 (PKM2), as the binding partners of DA. DA bound to Glu166 of α-enolase, Cys49 and Cys424 of PKM2, and Lys230 of aldolase A, inhibiting the enzymatic activities of α-enolase and PKM2 and thereby impairing ATP synthesis, resulting in mitochondrial dysfunction. CONCLUSIONS: Recent research has revealed that enhancing glycolysis can offer protection against PD. The present study identified that the glycolytic pathway is vulnerable to disruption by DA, suggesting a promising avenue for potential therapeutic interventions. Safeguarding glycolysis against DA-related disruption could be a potential therapeutic intervention for PD.

Perfluorooctanoic acid induces Leydig cell injury via inhibition of autophagosomes formation and activation of endoplasmic reticulum stress.

Perfluorooctanoic acid (PFOA) is a man-made chemical broadly distributed in various ecological environment and human bodies, which poses potential health risks. Its toxicity, especially the male reproduction toxicity has drawn increasing attention due to declining birth rates in recent years. However, how PFOA induces male reproductive toxicity remains unclear. Here, we characterize PFOA-induced cell injury and reveal the underlying mechanism in mouse Leydig cells, which are critical to spermatogenesis in the testes. We show that PFOA induces cell injury as evidenced by reduced cell viability, cell morphology changes and apoptosis induction. RNA-sequencing analysis reveals that PFOA-induced cell injury is correlated with compromised autophagy and activated endoplasmic reticulum (ER) stress, two conserved biological processes required for regulating cellular homeostasis. Mechanistic analysis shows that PFOA inhibits autophagosomes formation, and activation of autophagy rescues PFOA-induced apoptosis. Additionally, PFOA activates ER stress, and pharmacological inhibition of ER stress attenuates PFOA-induced cell injury. Taken together, these results demonstrate that PFOA induces cell injury through inhibition of autophagosomes formation and induction of ER stress in Leydig cells. Thus, our study sheds light on the cellular mechanisms of PFOA-induced Leydig cell injury, which may be suggestive to human male reproductive health risk assessment and prevention from PFOA exposure-induced reproductive toxicity.

Reprogramming tumor microenvironment with precise photothermal therapy by calreticulin nanobody-engineered probiotics.

Targeted therapies have revolutionized traditional cancer treatments by precisely targeting tumor cells, enhancing efficacy and safety. Despite this advancement, the proportion of cancer patients eligible for such therapies remains low due to the absence of suitable targets. Here, we investigate whether the translocation of the immunogenic cell death (ICD) marker calreticulin (CALR) from the endoplasmic reticulum (ER) to the cell surface following ICD induction can serve as a target for targeted therapies. To target CALR, a nanobody Nb215 identified from a naïve VHH phage library with high binding affinity to both human and mouse CALR was employed to engineer probiotic EcN 1917. Our results demonstrated that CALR nanobody-modified EcN-215 coupled with the photothermal dye indocyanine green (ICG) was able to exert NIR-II imaging-guide photothermal therapy (PTT). Moreover, PTT with EcN-215/ICG can reshape the tumor microenvironment by enhancing the infiltration of CD45+CD3+ T cells and CD11b+F4/80+ macrophages. Furthermore, the antitumor activity of CALR-targeted EcN-215/ICG is synergistically enhanced by blocking CD47-SIRPα axis. Collectively, our study provides a proof of concept for CALR-targeted therapy. Given that CALR translocation can be induced by various anticancer therapies across numerous tumor cell lines, CALR-targeted therapies hold promise as a novel approach for treating multiple types of cancers.

Triptolide induces apoptosis and cytoprotective autophagy by ROS accumulation via directly targeting peroxiredoxin 2 in gastric cancer cells.

Triptolide, a natural bioactive compound derived from herbal medicine Tripterygium wilfordii, has multiple biological activities including anti-cancer effect, which is being tested in clinical trials for treating cancers. However, the exact mechanism by which Triptolide exerts its cytotoxic effects, particularly its specific protein targets, remains unclear. Here, we show that Triptolide effectively induces cytotoxicity in gastric cancer cells by increasing reactive oxygen species (ROS) levels. Further investigations reveal that ROS accumulation contributes to the induction of Endoplasmic Reticulum (ER) stress, and subsequently autophagy induction in response to Triptolide. Meanwhile, this autophagy is cytoprotective. Interestingly, through activity-based protein profiling (ABPP) approach, we identify peroxiredoxins-2 (PRDX2), a component of the key enzyme systems that act in the defense against oxidative stress and protect cells against hydroperoxides, as direct binding target of Triptolide. By covalently binding to PRDX2 to inhibit its antioxidant activity, Triptolide increases ROS levels. Moreover, overexpression of PRDX2 inhibits and knockdown of the expression of PRDX2 increases Triptolide-induced apoptosis. Collectively, these results indicate PRDX2 as a direct target of Triptolides for inducing apoptosis. Our results not only provide novel insight into the underlying mechanisms of Triptolide-induced cytotoxic effects, but also indicate PRDX2 as a promising potential therapeutic target for developing anti-gastric cancer agents.

Development of EST-SSRs based on the transcriptome of Castanopsis carlesii and cross-species transferability in other Castanopsis species.

Castanopsis carlesii (Hemsl.) Hay. is a widely distributed and dominant tree species native to subtropical China with significant ecological and economic value. Due to serious human-related disturbance, its wild resources have been increasingly reduced, and whether may result in the loss of genetic diversity. However, no population genetics studies of natural C. carlesii have been reported to date. Microsatellite markers have been a useful tool in population genetics. Therefore, we developed EST-SSR markers based on the transcriptome sequencing of C. carlesii leaves. A total of 149,380,224 clean reads were obtained, and 63,012 nonredundant unigenes with a mean length of 1,034 bp were assembled and annotated based on sequence similarity searches in the Nr, Nt, KO, SwissProt, PFAM, KOG, and GO databases. The results showed that only 5,559 (8.82%) unigenes were annotated in all seven databases, but 46,338 (73.53%) could be annotated in at least one database. A total of 31,459 potential EST-SSRs were identified in 18,690 unigenes, with an average frequency of one SSR approximately 2 kb. Among the 100 EST-SSR primer pairs designed, 49 primer pairs successfully produced the expected product by amplification, with a success rate of 49%, but only 20 primer pairs showed abundant polymorphisms. Polymorphisms were verified using 25 samples from C. carlesii in Qimen, Anhui. A total of 119 alleles were detected, with a mean number of alleles (Na) of 5.95 per locus and a mean polymorphism information content (PIC) of 0.6125. All the 20 newly developed EST-SSR markers were verified in other Castanopsis species (C. sclerophylla, C. lamontii, C. fargesii, C. eyrei and C. jucunda). Sixteen primer pairs showed successful amplification in all five Castanopsis species (80%), and the transferability ratios ranged from 90% to 100%. These developed EST-SSR markers can be applied to population genetic and germplasm evaluations of C. carlesii and related species.

Characterization of 2,2',4,4'-tetrabromodiphenyl ether (BDE47)-induced testicular toxicity via single-cell RNA-sequencing.

Background: The growing male reproductive diseases have been linked to higher exposure to certain environmental compounds such as 2,2',4,4'-tetrabromodiphenyl ether (BDE47) that are widely distributed in the food chain. However, the specific underlying molecular mechanisms for BDE47-induced male reproductive toxicity are not completely understood. Methods: Here, for the first time, advanced single-cell RNA sequencing (ScRNA-seq) was employed to dissect BDE47-induced prepubertal testicular toxicity in mice from a pool of 76 859 cells. Results: Our ScRNA-seq results revealed shared and heterogeneous information of differentially expressed genes, signaling pathways, transcription factors, and ligands-receptors in major testicular cell types in mice upon BDE47 treatment. Apart from disruption of hormone homeostasis, BDE47 was discovered to downregulate multiple previously unappreciated pathways such as double-strand break repair and cytokinesis pathways, indicative of their potential roles involved in BDE47-induced testicular injury. Interestingly, transcription factors analysis of ScRNA-seq results revealed that Kdm5b (lysine-specific demethylase 5B), a key transcription factor required for spermatogenesis, was downregulated in all germ cells as well as in Sertoli and telocyte cells in BDE47-treated testes of mice, suggesting its contribution to BDE47-induced impairment of spermatogenesis. Conclusions: Overall, for the first time, we established the molecular cell atlas of mice testes to define BDE47-induced prepubertal testicular toxicity using the ScRNA-seq approach, providing novel insight into our understanding of the underlying mechanisms and pathways involved in BDE47-associated testicular injury at a single-cell resolution. Our results can serve as an important resource to further dissect the potential roles of BDE47, and other relevant endocrine-disrupting chemicals, in inducing male reproductive toxicity.