Pediatric hypereosinophilic syndrome associated with liver damage, portal vein, splenic vein and superior mesenteric vein thromboses: a case report.

BACKGROUND: The hypereosinophilic syndrome (HES) is a group of rare blood disorders characterized by persistent eosinophilia and damage to multiple organs. HES can be either primary, secondary or idiopathic. Secondary HES are commonly caused by parasitic infections, allergic reactions or cancer. We described a pediatric case of HES associated with liver damage and multiple thrombi. A 12-year-old boy with eosinophilia was complicated with severe thrombocytopenia, liver damage, portal vein, splenic vein, and superior mesenteric vein thromboses. The thrombi recanalized after treatment with methylprednisolone succinate and low molecular weight heparin. No side effects appeared after 1-month. CONCLUSIONS: Corticosteroids should be used at an early stage of HES to prevent further damage to vital organs. Anticoagulants should be recommended only in cases with thrombosis which should be actively screened as a part of evaluation of end organ damage.

[Clinical features of children with coronavirus disease 2019 caused by Delta variant infection in different age groups]. / 不同年龄段儿童Deltaå˜å¼‚æ ªæ„ŸæŸ“æ‰€è‡´æ–°åž‹å† çŠ¶ç— æ¯’æ„ŸæŸ“çš„ä¸´åºŠç‰¹å¾åˆ†æž.

OBJECTIVES: To study the clinical features of children with coronavirus disease 2019 (COVID-19) caused by Delta variant infection in different ages groups. METHODS: A total of 45 children with COVID-19 caused by Delta variant infection who were hospitalized in the designated hospital in Henan Province, China, from November 17 to December 17, 2021, were included. They were divided into three groups: <6 years group (n=16), 6-13 years group (n=16), and >13 years group (n=13). The three groups were compared in clinical features and laboratory examination data. RESULTS: COVID-19 in all age groups was mainly mild. Main manifestations included cough and expectoration in the three groups, and fever was only observed in the 6-13 years group. The <6 years group had significantly higher serum levels of aspartate aminotransferase, lactate dehydrogenase, and creatine kinase isoenzymes than the other two groups (P<0.05). The 6-13 years group had the highest proportion of children with elevated serum creatinine levels (50%). Among the three groups, only 4 children in the >13 years group had an increase in serum C-reactive protein levels. The 6-13 years group had the lowest counts of CD3+CD4+ lymphocytes, CD3+CD8+ lymphocytes, and natural killer cells in the peripheral blood among the three groups. The >13 years group had a significantly higher positive rate of SARS-CoV-2 IgG on admission than the other two groups (P<0.05). There was no significant difference in the imaging findings on chest CT among the three groups (P>0.05). CONCLUSIONS: The clinical features of COVID-19 caused by Delta variant infection in children of different age groups may be different: children aged <6 years tend to develop myocardial injury, and those aged 6-13 years have fever except cough and expectoration and tend to develop renal and immune dysfunction.

Fam96b recruits brain-type creatine kinase to fuel mitotic spindle formation.

Mitosis is a complicated and ordered process with high energy demands and metabolite fluxes. Cytosolic creatine kinase (CK), an enzyme involved in ATP homeostasis, has been shown to be essential to chromosome movement during mitotic anaphase in sea urchin. However, it remains elusive for the molecular mechanism underlying the recruitment of cytosolic CK by the mitotic apparatus. In this study, Fam96b/MIP18, a component of the MMXD complex with a function in Fe/S cluster supply, was identified as a brain-type CK (CKB)-binding protein. The binding of Fam96b with CKB was independent of the presence of CKB substrates and did not interfere with CKB activity. Fam96b was prone to oligomerize via the formation of intermolecular disulfide bonds, while the binding of enzymatically active CKB could modulate Fam96b oligomerization. Oligomerized Fam96b recruited CKB and the MMXD complex to associate with the mitotic spindle. Depletion of Fam96b or CKB by siRNA in the HeLa cells led to mitotic defects, which further resulted in retarded cell proliferation, increased cell death and aberrant cell cycle progression. Rescue experiments indicated that both Fam96b oligomerization and CKB activity were essential to the proper formation of mitotic spindle. These findings suggest that Fam96b may act as a scaffold protein to coordinate the supply and homeostasis of ATP and Fe/S clusters during mitosis.

[Clinical features of children with coronavirus disease 2019 Delta variant infection after vaccination with inactivated SARS-CoV-2 vaccine]. / æŽ¥ç§æ–°åž‹å† çŠ¶ç— æ¯’ç­æ´»ç–«è‹—åŽæ„ŸæŸ“Deltaå˜å¼‚æ ªçš„æ–°åž‹å† çŠ¶ç— æ¯’è‚ºç‚Žæ‚£å„¿ä¸´åºŠç‰¹å¾åˆ†æž.

OBJECTIVES: To study the clinical features of children with coronavirus disease 2019 (COVID-19) Delta variant infection vaccinated or not vaccinated with inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. METHODS: A total of 11 children with COVID-19 Delta variant infection who were vaccinated with inactivated SARS-CoV-2 vaccine and were hospitalized in the designated hospital in Henan Province, China, from November 3 to December 17, 2021 were enrolled as the vaccinated group. Thirty-one children with COVID-19 Delta variant infection who were not vaccinated and were hospitalized during the same period were enrolled as the unvaccinated group. A retrospective analysis was performed on their epidemiological data, clinical features, and laboratory examination results. RESULTS: There was no significant difference in gender composition and disease classification between the two groups (P>0.05), and there was also no significant difference in the incidence rates of the clinical symptoms such as cough, expectoration, and fever between the two groups (P>0.05). No significant difference was found between the two groups in leukocyte count, lymphocyte percentage, alanine aminotransferase, and serum creatinine (P>0.05). Compared with the unvaccinated group, the vaccinated group had significantly lower levels of aspartate aminotransferase, lactate dehydrogenase, and creatine kinase-MB (P<0.05). There was no significant difference between the two groups in the proportion of children with elevated C-reactive protein or procalcitonin and the levels of peripheral blood cytokines (P>0.05). The vaccinated group had significantly lower counts of B lymphocytes and total T lymphocytes (CD3+) than the unvaccinated group (P<0.05). Compared with the unvaccinated group, the vaccinated group had a significantly higher positive rate of IgG on admission and at week 2 of the course of disease (P<0.05), as well as a significantly higher Ct value of nucleic acid at weeks 1 and 2 of the course of disease (P<0.05). CONCLUSIONS: Vaccination with inactivated SARS-CoV-2 vaccine may reduce myocardial injury caused by SARS-CoV-2 Delta variant. For children with SARS-CoV-2 Delta variant infection after the vaccination, more attention should be paid to their immune function.

A High-Quality Haplotype-Resolved Genome of Common Bermudagrass (Cynodon dactylon L.) Provides Insights Into Polyploid Genome Stability and Prostrate Growth.

Common bermudagrass (Cynodon dactylon L.) is an important perennial warm-season turfgrass species with great economic value. However, the reference genome is still deficient in C. dactylon, which severely impedes basic studies and breeding studies. In this study, a high-quality haplotype-resolved genome of C. dactylon cultivar Yangjiang was successfully assembled using a combination of multiple sequencing strategies. The assembled genome is approximately 1.01 Gb in size and is comprised of 36 pseudo chromosomes belonging to four haplotypes. In total, 76,879 protein-coding genes and 529,092 repeat sequences were annotated in the assembled genome. Evolution analysis indicated that C. dactylon underwent two rounds of whole-genome duplication events, whereas syntenic and transcriptome analysis revealed that global subgenome dominance was absent among the four haplotypes. Genome-wide gene family analyses further indicated that homologous recombination-regulating genes and tiller-angle-regulating genes all showed an adaptive evolution in C. dactylon, providing insights into genome-scale regulation of polyploid genome stability and prostrate growth. These results not only facilitate a better understanding of the complex genome composition and unique plant architectural characteristics of common bermudagrass, but also offer a valuable resource for comparative genome analyses of turfgrasses and other plant species.

CCT2 is an aggrephagy receptor for clearance of solid protein aggregates.

Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.

Modeling congenital cataract in vitro using patient-specific induced pluripotent stem cells.

Congenital cataracts are the leading cause of childhood blindness. To date, surgical removal of cataracts is the only established treatment, but surgery is associated with multiple complications, which often lead to visual impairment. Therefore, mechanistic studies and drug-candidate screening have been intrigued by the aims of developing novel therapeutic strategies. However, these studies have been hampered by a lack of an appropriate human-disease model of congenital cataracts. Herein, we report the establishment of a human congenital cataract in vitro model through differentiation of patient-specific induced pluripotent stem cells (iPSCs) into regenerated lenses. The regenerated lenses derived from patient-specific iPSCs with known causative mutations of congenital cataracts (CRYBB2 [p. P24T] and CRYGD [p. Q155X]) showed obvious opacification that closely resembled that seen in patients' cataracts in terms of opacification severity and disease course accordingly, as compared with lentoid bodies (LBs) derived from healthy individuals. Increased protein aggregation and decreased protein solubility corresponding to the patients' cataract severity were observed in the patient-specific LBs and were attenuated by lanosterol treatment. Taken together, the in vitro model described herein, which recapitulates patient-specific clinical manifestations of congenital cataracts and protein aggregation in patient-specific LBs, provides a robust system for research on the pathological mechanisms of cataracts and screening of drug candidates for cataract treatment.

Human Ccr4 and Caf1 Deadenylases Regulate Proliferation and Tumorigenicity of Human Gastric Cancer Cells via Modulating Cell Cycle Progression.

Cancer cells generally have reprogrammed gene expression profiles to meet the requirements of survival, continuous division, and metastasis. An interesting question is whether the cancer cells will be affected by interfering their global RNA metabolism. In this research, we found that human Ccr4a/b (hCcr4a/b) and Caf1a/b (hCaf1a/b) deadenylases, the catalytic components of the Ccr4-Not complex, were dysregulated in several types of cancers including stomach adenocarcinoma. The impacts of the four deadenylases on cancer cell growth were studied by the establishment of four stable MKN28 cell lines with the knockdown of hCcr4a/b or hCaf1a/b or transient knockdown in several cell lines. Depletion of hCcr4a/b or hCaf1a/b significantly inhibited cell proliferation and tumorigenicity. Mechanistic studies indicated that the cells were arrested at the G2/M phase by knocking down hCaf1a, while arrested at the G0/G1 phase by depleting hCaf1b or hCcr4a/b. The four enzymes did not affect the levels of CDKs and cyclins but modulated the levels of CDK-cyclin inhibitors. We identified that hCcr4a/b, but not hCaf1a/b, targeted the p21 mRNA in the MKN28 cells. Furthermore, depletion of any one of the four deadenylases dramatically impaired processing-body formation in the MKN28 and HEK-293T cells. Our results highlight that perturbating global RNA metabolism may severely affect cancer cell proliferation, which provides a potential novel strategy for 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.

Diverse functions of deadenylases in DNA damage response and genomic integrity.

DNA damage response (DDR) is a coordinated network of diverse cellular processes including the detection, signaling, and repair of DNA lesions, the adjustment of metabolic network and cell fate determination. To deal with the unavoidable DNA damage caused by either endogenous or exogenous stresses, the cells need to reshape the gene expression profile to allow efficient transcription and translation of DDR-responsive messenger RNAs (mRNAs) and to repress the nonessential mRNAs. A predominant method to adjust RNA fate is achieved by modulating the 3'-end oligo(A) or poly(A) length via the opposing actions of polyadenylation and deadenylation. Poly(A)-specific ribonuclease (PARN) and the carbon catabolite repressor 4 (CCR4)-Not complex, the major executors of deadenylation, are indispensable to DDR and genomic integrity in eukaryotic cells. PARN modulates cell cycle progression by regulating the stabilities of mRNAs and microRNA (miRNAs) involved in the p53 pathway and contributes to genomic stability by affecting the biogenesis of noncoding RNAs including miRNAs and telomeric RNA. The CCR4-Not complex is involved in diverse pathways of DDR including transcriptional regulation, signaling pathways, mRNA stabilities, translation regulation, and protein degradation. The RNA targets of deadenylases are tuned by the DDR signaling pathways, while in turn the deadenylases can regulate the levels of DNA damage-responsive proteins. The mutual feedback between deadenylases and the DDR signaling pathways allows the cells to precisely control DDR by dynamically adjusting the levels of sensors and effectors of the DDR signaling pathways. Here, the diverse functions of deadenylases in DDR are summarized and the underlying mechanisms are proposed according to recent findings. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Disease RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms.

Translation Efficiency and Degradation of ER-Associated mRNAs Modulated by ER-Anchored poly(A)-Specific Ribonuclease (PARN).

Translation is spatiotemporally regulated and endoplasmic reticulum (ER)-associated mRNAs are generally in efficient translation. It is unclear whether the ER-associated mRNAs are deadenylated or degraded on the ER surface in situ or in the cytosol. Here, we showed that ER possessed active deadenylases, particularly the poly(A)-specific ribonuclease (PARN), in common cell lines and mouse tissues. Consistently, purified recombinant PARN exhibited a strong ability to insert into the Langmuir monolayer and liposome. ER-anchored PARN was found to be able to reshape the poly(A) length profile of the ER-associated RNAs by suppressing long poly(A) tails without significantly influencing the cytosolic RNAs. The shortening of long poly(A) tails did not affect global translation efficiency, which suggests that the non-specific action of PARN towards long poly(A) tails was beyond the scope of translation regulation on the ER surface. Transcriptome sequencing analysis indicated that the ER-anchored PARN trigged the degradation of a small subset of ER-enriched transcripts. The ER-anchored PARN modulated the translation of its targets by redistributing ribosomes to heavy polysomes, which suggests that PARN might play a role in dynamic ribosome reallocation. During DNA damage response, MK2 phosphorylated PARN-Ser557 to modulate PARN translocation from the ER to cytosol. The ER-anchored PARN modulated DNA damage response and thereby cell viability by promoting the decay of ER-associated MDM2 transcripts with low ribosome occupancy. These findings revealed that highly regulated communication between mRNA degradation rate and translation efficiency is present on the ER surface in situ and PARN might contribute to this communication by modulating the dynamic ribosome reallocation between transcripts with low and high ribosome occupancies.

Lanosterol modulates proteostasis via dissolving cytosolic sequestosomes/aggresome-like induced structures.

Sequestration of misfolded proteins into distinct cellular compartments plays a pivotal role in proteostasis and proteopathies. Cytoplasmic ubiquitinated proteins are sequestered by p62/SQSTM1 to deposit in sequestosomes or aggresome-like induced structures (ALIS). Most aggresome or ALIS regulators identified thus far are recruiters, while little is known about the disaggregases or dissolvers. In this research, we showed that lanosterol synthase and its enzymatic product lanosterol effectively reduced the number and/or size of sequestosomes/ALIS/aggresomes formed by endogenous proteins in the HeLa and HEK-293A cells cultured under both non-stressed and stressed conditions. Supplemented lanosterol did not affect the proteasome and autophagic activities, but released the trapped proteins from the p62-positive inclusions accompanied with the activation of HSF1 and up-regulation of various heat shock proteins. Our results suggested that the coordinated actions of disaggregation by lanosterol and refolding by heat shock proteins might facilitate the cells to recycle proteins from aggregates. The disaggregation activity of lanosterol was not shared by cholesterol, indicating that lanosterol possesses additional cellular functions in proteostasis regulation. Our findings highlight that besides protein modulators, the cells also possess endogenous low-molecular-weight compounds as efficient proteostasis regulators.

Semiholoenzyme optimizes activity and stability of a hyperthermostable iron-superoxide dismutase.

Metal ion coordination is an essential step for the maturation of metalloenzymes. Generally, the metal coordination sites are thought to be fully occupied to achieve the maximum activity and stability. In this research, we compared the structural features, activity and stability of the apo-, semiholo- and holo-forms of a hyperthermostable tetrameric Fe-superoxide dismutase (SOD). Strikingly, the three forms of enzymes had similar compact tetrameric structures. Removal of iron ions destabilized subunit-subunit interactions during guanidine hydrochloride-induced unfolding. The partially metalized semiholoenzyme possessed most of the activity and identical hyperthermostability of the holoenzyme, but weaker propensity to aggregate. Furthermore, both of the iron content and activity of the semiholoenzyme were unaffected by a 200-fold excess iron ions in solutions, suggesting that conformation of the apo-subunits were forced to the close state by the iron-containing subunits. These observations suggest that fully metalized enzyme is probably nonessential for multimeric metalloenzymes and the semiholoenzyme may be a better choice. The unique properties of semiholoenzyme also provide the organisms a compromised solution to survival under metal deficiency conditions.

The Intrinsically Disordered C-Terminal Domain Triggers Nucleolar Localization and Function Switch of PARN in Response to DNA Damage.

Poly(A)-specific ribonuclease (PARN), a multifunctional multi-domain deadenylase, is crucial to the regulation of mRNA turnover and the maturation of various non-coding RNAs. Despite extensive studies of the well-folding domains responsible for PARN catalysis, the structure and function of the C-terminal domain (CTD) remains elusive. PARN is a cytoplasm-nucleus shuttle protein with concentrated nucleolar distribution. Here, we identify the nuclear and nucleolar localization signals in the CTD of PARN. Spectroscopic studies indicated that PARN-CTD is intrinsically disordered with loosely packed local structures/tertiary structure. Phosphorylation-mimic mutation S557D disrupted the local structure and facilitated the binding of the CTD with the well-folded domains, with no impact on PARN deadenylase activity. Under normal conditions, the nucleolus-residing PARN recruited CBP80 into the nucleoli to repress its deadenylase activity, while DNA damage-induced phosphorylation of PARN-S557 expelled CBP80 from the nucleoli to discharge activity inhibition and attracted nucleoplasm-located CstF-50 into the nucleoli to activate deadenylation. The structure switch-induced function switch of PARN reshaped the profile of small nuclear non-coding RNAs to respond to DNA damage. Our findings highlight that the structure switch of the CTD induced by posttranslational modifications redefines the subset of binding partners, and thereby the RNA targets in the nucleoli.

Dissimilarity in the Contributions of the N-Terminal Domain Hydrophobic Core to the Structural Stability of Lens ß/γ-Crystallins.

Vertebrate lens ß/γ-crystallins share a conserved tertiary structure consisting of four Greek-key motifs divided into two globular domains. Numerous inherited mutations in ß/γ-crystallins have been linked to cataractogenesis. In this research, the folding mechanism underlying cataracts caused by the I21N mutation in ßB2 was investigated by comparing the effect of mutagenesis on the structural features and stability of four ß/γ-crystallins, ßB1, ßB2, γC, and γD. Our results showed that the four ß/γ-crystallins differ greatly in solubility and stability against various stresses. The I21N mutation greatly impaired ßB2 solubility and native structure as well as its stability against denaturation induced by guanidine hydrochloride, heat treatment, and ultraviolet irradiation. However, the deleterious effects were much weaker for mutations at the corresponding sites in ßB1, γC, and γD. Molecular dynamics simulations indicated that the introduction of a nonnative hydrogen bond contributed to twisting Greek-key motif I outward, which might direct the misfolding of the I21N mutant of ßB2. Meanwhile, partial hydration of the hydrophobic interior of the domain induced by the mutation destabilized ßB1, γC, and γD. Our findings highlight the importance of nonnative hydrogen bond formation and hydrophobic core hydration in crystallin misfolding caused by inherited mutations.

Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association.

Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K+ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K+, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.

Lanosterol and 25-hydroxycholesterol dissociate crystallin aggregates isolated from cataractous human lens via different mechanisms.

Cataract, a crystallin aggregation disease, is the leading cause of human blindness worldwide. Surgery is the only established treatment of cataracts and no anti-cataract drugs are available thus far. Recently lanosterol and 25-hydroxycholesterol have been reported to redissolve crystallin aggregates and partially restore lens transparency in animals. However, the efficacies of these two compounds have not been quantitatively studied ex vivo using patient tissues. In this research, we developed a quantitative assay applicable to efficacy validations and mechanistic studies by a protocol to isolate protein aggregates from the surgically removed cataractous human lens. Our results showed that both compounds were effective for human cataractous samples with EC50 values at ten micromolar level. The efficacies of both compounds strongly depended on cataract severity. Lanosterol and 25-hydroxycholesterol were two mechanistically different lead compounds of anti-cataract drug design.

Introduction of an extra tryptophan fluorophore by cataract-associating mutations destabilizes ßB2-crystallin and promotes aggregation.

ß/γ-Crystallins are predominant structural proteins in vertebrate lens with unique properties of extremely high solubility, long-term stability and resistance to UV damage. Four conserved Trp residues in ß/γ-crystallins account for UV absorbance and thereafter fluorescence quenching to avoid photodamage. Herein we found that ßB2-crystallin Trp fluorescence was greatly enhanced by the introduction of an extra unquenched Trp fluorophore by cataract-associated mutations S31W and R145W. Both mutations impaired oligomerization, decreased stability and promote thermal aggregation, while S31W was more deleterious. S31W accelerated ßB2-crystallin aggregation under UV damaging conditions, whereas R145W delayed. These observations suggested that the introduction of an extra Trp fluorophore had complicated effects on ßB2-crystallin stability and aggregation against various stresses. Our findings highlight that the number of Trp fluorophores in ß/γ-crystallin is evolutionarily optimized to exquisitely perform their structural roles in the lens.

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.

The cataract-causing mutation G75V promotes γS-crystallin aggregation by modifying and destabilizing the native structure.

Congenital cataract is one of the leading causes of childhood blindness worldwide. About half of heredity cataracts are caused by mutations in various crystallins. However, the underlying mechanisms have not been elucidated for most of crystallin mutations. In this research, we studied the effect of a cataract-causing mutation G75V on γS-crystallin structure, stability and aggregatory propensity. Spectroscopic experiments indicated that the mutation had little impact on γS-crystallin oligomeric status and secondary structure components, but led to large perturbations in tertiary structure. Compared with the WT protein, the G75V mutant had more solvent-accessible Trp fluorophores and hydrophobic exposure. The modified native state of mutant γS-crystallin was more susceptible to environmental stresses such as heat treatment, guanidine hydrochloride and acid conditions. The destabilized mutated protein was more prone to form large aggregates when denatured by high temperature or UV-irradiation. The thermal aggregation of the G75V mutant could be successfully inhibited by excess amount of αA-crystallin with a higher efficiency than the WT protein. Our results suggested that the aberrant modifications in γS-crystallin structure might contribute to the lower stability and higher aggregatory potency of the mutated protein, which subsequently resulted in cataracts in the patients.