Celastrol regulates the oligomeric state and chaperone activity of αB-crystallin linked with protein homeostasis in the lens.

Protein misfolding and aggregation are crucial pathogenic factors for cataracts, which are the leading cause of visual impairment worldwide. α-crystallin, as a small molecular chaperone, is involved in preventing protein misfolding and maintaining lens transparency. The chaperone activity of α-crystallin depends on its oligomeric state. Our previous work identified a natural compound, celastrol, which could regulate the oligomeric state of αB-crystallin. In this work, based on the UNcle and SEC analysis, we found that celastrol induced αB-crystallin to form large oligomers. Large oligomer formation enhanced the chaperone activity of αB-crystallin and prevented aggregation of the cataract-causing mutant ßA3-G91del. The interactions between αB-crystallin and celastrol were detected by the FRET (Fluorescence Resonance Energy Transfer) technique, and verified by molecular docking. At least 9 binding patterns were recognized, and some binding sites covered the groove structure of αB-crystallin. Interestingly, αB-R120G, a cataract-causing mutation located at the groove structure, and celastrol can decrease the aggregates of αB-R120G. Overall, our results suggested celastrol not only promoted the formation of large αB-crystallin oligomers, which enhanced its chaperone activity, but also bound to the groove structure of its α-crystallin domain to maintain its structural stability. Celastrol might serve as a chemical and pharmacological chaperone for cataract treatment.

Oral 8-aminoguanine against age-related retinal degeneration.

Visual decline in the elderly is often attributed to retinal aging, which predisposes the tissue to pathologies such as age-related macular degeneration. Currently, effective oral pharmacological interventions for retinal degeneration are limited. We present a novel oral intervention, 8-aminoguanine (8-AG), targeting age-related retinal degeneration, utilizing the aged Fischer 344 rat model. A low-dose 8-AG regimen (5 mg/kg body weight) via drinking water, beginning at 22 months for 8 weeks, demonstrated significant retinal preservation. This was evidenced by increased retinal thickness, improved photoreceptor integrity, and enhanced electroretinogram responses. 8-AG effectively reduced apoptosis, oxidative damage, and microglial/macrophage activation associated with aging retinae. Age-induced alterations in the retinal purine metabolome, characterized by elevated levels of inosine, hypoxanthine, and xanthine, were partially mitigated by 8-AG. Transcriptomics highlighted 8-AG's anti-inflammatory effects on innate and adaptive immune responses. Extended treatment to 17 weeks further amplified the retinal protective effects. Moreover, 8-AG showed temporary protective effects in the RhoP23H/+ mouse model of retinitis pigmentosa, reducing active microglia/macrophages. Our study positions 8-AG as a promising oral agent against retinal aging. Coupled with previous findings in diverse disease models, 8-AG emerges as a promising anti-aging compound with the capability to reverse common aging hallmarks.

The 18th amino acid glycine plays an essential role in maintaining the structural stabilities of γS-crystallin linking with congenital cataract.

γS-crystallin is particularly rich in the embryonic nuclear region and is crucial to the maintenance of lens transparency and optical properties. Gene mutations in crystallin are the main factors leading to congenital hereditary cataracts, which are a major cause of visual impairment in children. Some mutations located in the 18th amino acid glycine of γS-crystallin were reported to be linking with congenital cataracts. However, the pathogenic mechanism has not been elucidated. Interestingly, we previously identified a novel variant of γS-crystallin (c.53G > A; p. G18D) with progressive cortical and sutural congenital cataracts in one Chinese family. In this study, we purified the γS-crystallin wildtype and mutant proteins to investigate the effects of the G18D mutation on the structural stability of γS-crystallin. The results showed that there were tertiary structural differences between the wild-type γS-crystallin and the G18D variant. The mutation significantly impaired the stability of γS-crystallin under environmental stress and promoted aggregation. Furthermore, molecular dynamics (MD) simulations showed that the mutation altered H-bonding and surface electrostatic potential. Significantly decreased stability along with an increased tendency to aggregate under environmental stress may be the major pathogenic factors for cataracts induced by the G18D mutation.

Epitranscriptic regulation of HRAS by N6-methyladenosine drives tumor progression.

Overexpression of Ras, in addition to the oncogenic mutations, occurs in various human cancers. However, the mechanisms for epitranscriptic regulation of RAS in tumorigenesis remain unclear. Here, we report that the widespread N6-methyladenosine (m6A) modification of HRAS, but not KRAS and NRAS, is higher in cancer tissues compared with the adjacent tissues, which results in the increased expression of H-Ras protein, thus promoting cancer cell proliferation and metastasis. Mechanistically, three m6A modification sites of HRAS 3' UTR, which is regulated by FTO and bound by YTHDF1, but not YTHDF2 nor YTHDF3, promote its protein expression by the enhanced translational elongation. In addition, targeting HRAS m6A modification decreases cancer proliferation and metastasis. Clinically, up-regulated H-Ras expression correlates with down-regulated FTO and up-regulated YTHDF1 expression in various cancers. Collectively, our study reveals a linking between specific m6A modification sites of HRAS and tumor progression, which provides a new strategy to target oncogenic Ras signaling.

Tumor Cell-Intrinsic BTLA Receptor Inhibits the Proliferation of Tumor Cells via ERK1/2.

B and T lymphocyte attenuator (BTLA) is an immune checkpoint molecule that mediates the escape of tumor cells from immunosurveillance. Consequently, BTLA and its ligand herpesvirus entry mediator (HVEM) are potentially immunotherapeutic targets. However, the potential effects of BTLA on tumor cells remain incompletely unknown. Here, we show that BTLA is expressed across a broad range of tumor cells. The depletion of BTLA or HVEM promotes cell proliferation and colony formation, which is reversed by the overexpression of BTLA in BTLA knockout cells. In contrast, overexpression of BTLA or HVEM inhibits tumor cell proliferation and colony formation. Furthermore, the proliferation of a subpopulation with high BTLA was also significantly slower than that of the low BTLA subpopulation. Mechanistically, the coordination of BTLA and HVEM inhibits its major downstream extracellular regulated protein kinase (ERK1/2) signaling pathway, thus preventing tumor cell growth. This study demonstrates that tumor cell-intrinsic BTLA/HVEM is a potential tumor suppressor and is likely to have a potential antagonist for immunotherapy, thus representing a potential biomarker for the optimal cancer immunotherapeutic treatment.

Emerging Trends in the Pathological Research of Human Papillomavirus-positive Oropharyngeal Squamous Cell Carcinoma.

Oropharyngeal squamous cell carcinomas (OPSCCs) have shown an alarming rate of increase in incidence over the past several decades, markedly in men. In the United States, transcriptionally-active human papillomavirus (HPV), particularly HPV 16, has become the highest contributive agent of OPSCCs, affecting approximately 16,000 people a year. Compared to patients with HPV-negative OPSCCs, patients with HPV-positive OPSCCs exhibit better health responses to chemoradiotherapy and an overall increase in long-term survival. Despite promising treatment options, many OPSCCs are discovered at an advanced stage, and ~20% of cases will recur after definitive treatment. Therefore, extensive research is ongoing to identify new targets for precision treatment and to stratify tumor prognosis. The aim of this review is to capture the most updated research on HPV-positive OPSCCs, emphasizing their relevance as potential new targets for precision medicine and survival prognosis.

Nicotine dose-dependent epigenomic-wide DNA methylation changes in the mice with long-term electronic cigarette exposure.

Epigenomic-wide DNA methylation profiling holds the potential to reflect both electronic cigarette exposure-associated risks and individual poor health outcomes. However, a systemic study in animals or humans is still lacking. Using the Infinium Mouse Methylation BeadChip, we examined the DNA methylation status of white blood cells in male ApoE-/- mice after 14 weeks of electronic cigarette exposure with the InExpose system (2 hr/day, 5 days/week, 50% PG and 50% VG) with low (6 mg/ml) and high (36 mg/ml) nicotine concentrations. Our results indicate that electronic cigarette aerosol inhalation induces significant alteration of 8,985 CpGs in a dose-dependent manner (FDR<0.05); 7,389 (82.2%) of the CpG sites are annotated with known genes. Among the top 6 significant CpG sites (P-value<1e-8), 4 CpG sites are located in the known genes, and most (3/5) of these genes have been related to cigarette smoking. The other two CpGs are close to/associated with the Phc2 gene that was recently linked to smoking in a transcriptome-wide associations study. Furthermore, the gene set enrichment analysis highlights the activation of MAPK and 4 cardiomyocyte/cardiomyopathy-related signaling pathways (including adrenergic signaling in cardiomyocytes and arrhythmogenic right ventricular cardiomyopathy) following repeated electronic cigarette use. The MAPK pathway activation correlates well with our finding of increased cytokine mRNA expression after electronic cigarette exposure in the same batch of mice. Interestingly, two pathways related to mitochondrial activities, namely mitochondrial gene expression and mitochondrial translation, are also activated after electronic cigarette exposure. Elucidating the relationship between these pathways and the increased circulating mitochondrial DNA observed here will provide further insight into the cell-damaging effects of prolonged inhalation of e-cigarette aerosols.

Nonretinoid chaperones improve rhodopsin homeostasis in a mouse model of retinitis pigmentosa.

Rhodopsin-associated (RHO-associated) retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified nonretinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules toward the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Furthermore, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in RhoP23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL), indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the RhoP23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in RhoP23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in RhoP23H/+ mice. Collectively, we provide proof of principle that nonretinoid chaperones are promising drug candidates in treating RHO-associated RP.

LCDR regulates the integrity of lysosomal membrane by hnRNP K-stabilized LAPTM5 transcript and promotes cell survival.

Lysosome plays important roles in cellular homeostasis, and its dysregulation contributes to tumor growth and survival. However, the understanding of regulation and the underlying mechanism of lysosome in cancer survival is incomplete. Here, we reveal a role for a histone acetylation-regulated long noncoding RNA termed lysosome cell death regulator (LCDR) in lung cancer cell survival, in which its knockdown promotes apoptosis. Mechanistically, LCDR binds to heterogenous nuclear ribonucleoprotein K (hnRNP K) to regulate the stability of the lysosomal-associated protein transmembrane 5 (LAPTM5) transcript that maintains the integrity of the lysosomal membrane. Knockdown of LCDR, hnRNP K, or LAPTM5 promotes lysosomal membrane permeabilization and lysosomal cell death, thus consequently resulting in apoptosis. LAPTM5 overexpression or cathepsin B inhibitor partially restores the effects of this axis on lysosomal cell death in vitro and in vivo. Similarly, targeting LCDR significantly decreased tumor growth of patient-derived xenografts of lung adenocarcinoma (LUAD) and had significant cell death using nanoparticles (NPs)-mediated systematic short interfering RNA delivery. Moreover, LCDR/hnRNP K/LAPTM5 are up-regulated in LUAD tissues, and coexpression of this axis shows the increased diagnostic value for LUAD. Collectively, we identified a long noncoding RNA that regulates lysosome function at the posttranscriptional level. These findings shed light on LCDR/hnRNP K/LAPTM5 as potential therapeutic targets, and targeting lysosome is a promising strategy in cancer treatment.

A novel F30S mutation in γS-crystallin causes autosomal dominant congenital nuclear cataract by increasing susceptibility to stresses.

Despite of increasingly accumulated genetic variations of autosomal dominant congenital cataracts (ADCC), the causative genes of many ADCC patients remains unknown. In this research, we identified a novel F30S mutation in γS-crystallin from a three-generation Chinese family with ADCC. The patients possessing the F30S mutation exhibited nuclear cataract phenotype. The potential molecular mechanism underlying ADCC by the F30S mutation was investigated by comparing the structural features, stability and aggregatory potency of the mutated protein with the wild type protein. Spectroscopic experiments indicated that the F30S mutation did not affect γS-crystallin secondary structure compositions, but modified the microenvironments around aromatic side-chains. Thermal and chemical denaturation studies indicated that the mutation destabilized the protein and increased its aggregatory potency. The mutation altered the two-state unfolding of γS-crystallin to a three-state unfolding with the accumulation of an unfolding intermediate. The almost identical values in the changes of Gibbs free energies for transitions from the native state to intermediate and from the intermediate to unfolded state suggested that the mutation probably disrupted the cooperativity between the two domains during unfolding. Our results expand the genetic variation map of ADCC and provide novel insights into the molecular mechanism underlying ADCC caused by mutations in ß/γ-crystallins.

[Bioinformatics analysis of genes related to poor prognosis of human hepatocellular carcinoma and its clinical significance].

OBJECTIVE: To screen genes associated with poor prognosis of hepatocellular carcinoma (HCC) and to explore the clinical significance of these genes. METHODS: The proper expression profile data of HCC was obtained from the Gene Expression Omnibus (GEO) database, and the differentially expressed genes (DEGs) were identified by differential expression analysis. The DAVID and String database were used for function enrichment analysis and to construct the protein-protein interaction (PPI) network respectively. The Cancer Genome Atlas (TCGA) database and the Cox Proportional Hazard Model were used for prognosis analysis of the DEGs. RESULTS: A eligible human HCC data set (GSE84402) met the requirements. A total of 1141 differentially expressed genes were identified, including 720 up-regulated and 421 down-regulated genes. The results of function enrichment analysis and PPI network performed that CDK1、CDC6、CCNA2、CHEK1、CENPE 、PIK3R1、RACGAP1、BIRC5、KIF11 and CYP2B6 were prognosis key genes. And the prognosis analysis showed that the expressions of CDC6、PIK3R1、KIF11 and RACGAP1 were increased, and the expression of CENPE was decreased, which was closely related to prognosis of HCC. CONCLUSION: CDC6、CENPE、PIK3R1、KIF11 and RACGAP1 may be closely related to poor prognosis of HCC, and can be used as molecular biomarkers for future research of HCC prognosis.

Synthesis, Evaluation, and Structure-Activity Relationship Study of Lanosterol Derivatives To Reverse Mutant-Crystallin-Induced Protein Aggregation.

We describe here the development of potent synthetic analogues of the naturally occurring triterpenoid lanosterol to reverse protein aggregation in cataracts. Lanosterol showed superiority to other scaffolds in terms of efficacy and generality in previous studies. Various modified lanosterol derivatives were synthesized via modification of the side chain, ring A, ring B, and ring C. Evaluation of these synthetic analogues draws a clear picture for SAR. In particular, hydroxylation of the 25-position in the side chain profoundly improved the potency, and 2-fluorination further enhanced the biological activity. This work also revealed that synthetic lanosterol analogues could reverse multiple types of mutant-crystallin aggregates in cell models with excellent potency and efficacy. Notably, lanosterol analogues have no cytotoxicity but can improve the viability of the HLE-B3 cell line. Furthermore, representative compound 6 successfully redissolved the aggregated crystallin proteins from the amyloid-like fibrils in a concentration-dependent manner.

[Prognosis-related miRNA bioinformatics screening of lung adenocarcinoma and its clinical significance].

OBJECTIVE: To investigate the prognosis-related miRNA histological features and clinical significance of lung adenocarcinoma. METHODS: Using The Cancer Genome Atlas (TCGA) data, the miRNA expression profile data of human lung adenocarcinoma were searched for differential analysis, and the prognosis-related miRNAs were screened by Cox risk regression model. The targeted miRNAs were predicted by mirwalk analysis platform, KEGG functional enrichment analysis, and finally, predict the function of prognosis-related miRNAs. RESULTS: A total of 46 differential miRNAs in lung adenocarcinoma were screened, including 19 up-regulated and 27 down-regulated. Six prognostic-related miRNAs were screened by Cox survival analysis, namely hsa-mir-21, hsa-mir-142, hsa-mir-200a high expression, hsa-mir-101, hsa-let-7c, hsa-mir-378e low expression, hsa-mir-21 and hsa-mir-378e were associated with poor prognosis in patients with lung adenocarcinoma, and the survival time was shortened significantly (P<0.05, AUC=0.618). KEGG analysis showed that the above prognosis-related miRNA targeting regulatory genes were related with immune response pathways, miRNA and cancer pathways, metabolic pathways and so on. CONCLUSIONS: Hsa-mir-21 and hsa-mir-378e are associated with poor prognosis of lung adenocarcinoma, and may be used as a molecular marker for prognosis of lung adenocarcinoma after further clinical verification.

Congenital microcornea-cataract syndrome-causing mutation X253R increases ßB1-crystallin hydrophobicity to promote aggregate formation.

The high solubility and lifelong stability of crystallins are crucial to the maintenance of lens transparency and optical properties. Numerous crystallin mutations have been linked to congenital cataract, which is one of the leading causes of newborn blindness. Besides cataract, several crystallin mutations have also been linked to syndromes such as congenital microcornea-cataract syndrome (CMCC). However, the molecular mechanism of CMCC caused by crystallin mutations remains elusive. In the present study, we investigated the mechanism of CMCC caused by the X253R mutation in ßB1-crystallin. The exogenously expressed X253R proteins were prone to form p62-negative aggregates in HeLa cells, strongly inhibited cell proliferation and induced cell apoptosis. The intracellular X253R aggregates could be successfully redissolved by lanosterol but not cholesterol. The extra 26 residues at the C-terminus of ßB1-crystallin introduced by the X253R mutation had little impact on ßB1-crystallin structure and stability, but increased ßB1-crystallin hydrophobicity and decreased its solubility. Interestingly, the X253R mutant fully abolished the aggregatory propensity of ßB1- and ßA3/ßB1-crystallins at high temperatures, suggesting that X253R was an aggregation-inhibition mutation of ß-crystallin homomers and heteromers in dilute solutions. Our results suggest that an increase in hydrophobicity and a decrease in solubility might be responsible for cataractogenesis induced by the X253R mutation, while the cytotoxic effect of X253R aggregates might contribute to the defects in ocular development. Our results also highlight that, at least in some cases, the aggregatory propensity in dilute solutions could not fully mimic the behaviours of mutated proteins in the crowded cytoplasm of the cells.

Membrane insertion of αA-crystallin is oligomer-size dependent.

Vertebrate lens is one of the tissues with the highest soluble protein concentration. The predominant soluble proteins in lens fiber cells are crystallins, and among them, α-crystallins belong to the small heat shock protein family with chaperone-like activity. Although α-crystallins are highly soluble in waters, α-crystallins have been detected in the membrane-bound fraction of lens, which will increase in the aged or cataractous lens. In this research, we found αA-crystallin exhibited a complex thermal transition with remarkable changes in secondary and quaternary structures. Treatment of αA-crystallin at high temperatures induced larger oliogomers with higher hydrophobic exposure. Both heat-treated and untreated αA-crystallin could insert into lipid monolayer directly as revealed by monolayer surface pressure experiments. Heat-treatment facilitated the membrane insertion of αA-crystallin and increased the membrane-bound fraction in the cells. The membrane-binding ability of αA-crystallin could be altered by cataract-causing mutations R116C, R116H and Y118D. Our results suggested that the irreversible changes in oligomer size induced by various stresses might promote the membrane association of αA-crystallin and therefore might play a role in aged cataract. Alternations in the membrane binding ability of α-crystallins might be important to the understanding of both aged and congenital cataracts.

Congenital Cataract-Causing Mutation G129C in γC-Crystallin Promotes the Accumulation of Two Distinct Unfolding Intermediates That Form Highly Toxic Aggregates.

Cataract is a lens opacification disease prevalent worldwide. Cataract-causing mutations in crystallins generally lead to the formation of light-scattering particles in the lens. However, it remains unclear for the detailed structural and pathological mechanisms of most mutations. In this study, we showed that the G129C mutation in γC-crystallin, which is associated with autosomal dominant congenital nuclear cataract, perturbed the unfolding process by promoting the accumulation of two distinct aggregation-prone intermediates under mild denaturing conditions. The abnormally accumulated intermediates escaped from the chaperone-like function of αA-crystallin during refolding. Molecular dynamics simulations indicated that the mutation altered domain pairing geometry and allowed the penetration of extra solvent molecules into the domain binding interface, thereby weakening domain binding energy. Under mild denaturation conditions, the increased domain movements may facilitate the formation of non-native oligomers via domain swapping, which further assembled into amyloid-like fibrils. The intermediate that appeared at 1.6M guanidine hydrochloride was more compact and less aggregatory than the one populated at 0.9 M guanidine hydrochloride, which was caused by the increased solvation of acidic residues in the ion-pairing network via the competitive binding of guanidinium ions. More importantly, both the amyloid-like fibrils preformed in vitro and intracellular aggresomes formed by exogenously overexpressed mutant proteins significantly inhibited cell proliferation and induced cell death. The combined data from spectroscopic, structural and cellular studies strongly suggest that both the formation of light-scattering aggregates and the toxic effects of the aggregates may contribute to the onset and development of cataract.

Lanosterol reverses protein aggregation in cataracts.

The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.

Cataract-causing mutation R233H affects the stabilities of ßB1- and ßA3/ßB1-crystallins with different pH-dependence.

Disease-causing mutations can be stabilizing or destabilizing. Missense mutations of structural residues are generally destabilizing, while stabilizing mutations are usually linked to alterations in protein functions. Stabilizing mutations are rarely identified in mutations linked to congenital cataract, a disease caused by the opacification of the lens. In this research, we found that R233H mutation had little impact on ßB1-crystallin structure, solubility and thermal stability under neutral solution pH conditions. The mutation increased ßB1 stability against guanidine hydrochloride-induced denaturation, suggesting that Arg233 might be a functional residue. Further analysis indicated that the R233H mutation did not affect the formation of ßA3/ßB1 heteromer, but significantly reduced heteromer stability against heat- and guanidine hydrochloride-induced denaturation. The R233H mutation negatively affected the thermal stabilities and aggregatory propensities of ßB1 and ßA3/ßB1 with different pH-dependence, implying that the protonation of His side chains during acidification played a regulatory role in crystallin stability and aggregation. Molecular dynamic simulations indicated that Arg233 is one of the residues forming an inter-subunit ion-pairing network with intrinsically dynamic nature. Based on these observations, we proposed that the highly dynamic ion-pairing network contributed to the tradeoff among ßB1 solubility, stability, aggregatory propensity and function of protecting ßA3.