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.

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.

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.

A micro surface tension alveolus (MISTA) in a glass microchip.

We have designed a non-membrane micro surface tension alveolus (MISTA) in a glass microchip for direct gas exchange and micro gradient control. Hemoglobin (Hb) in the liquid phase indicates the rapid gas gradient changes of O2 and CO2 shifted by the difference in pressure between the liquid and the gas.

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.

Cataract-causing mutation S228P promotes ßB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain.

ß/γ-Crystallins are predominant structural proteins in the cytoplasm of lens fiber cells and share a similar fold composing of four Greek-key motifs divided into two domains. Numerous cataract-causing mutations have been identified in various ß/γ-crystallins, but the mechanisms underlying cataract caused by most mutations remains uncharacterized. The S228P mutation in ßB1-crystallin has been linked to autosomal dominant congenital nuclear cataract. Here we found that the S228P mutant was prone to aggregate and degrade in both of the human and E. coli cells. The intracellular S228P aggregates could be redissolved by lanosterol. The S228P mutation modified the refolding pathway of ßB1-crystallin by affecting the formation of the dimeric intermediate but not the monomeric intermediate. Compared with native ßB1-crystallin, the refolded S228P protein had less packed structures, unquenched Trp fluorophores and increased hydrophobic exposure. The refolded S228P protein was prone to aggregate at the physiological temperature and decreased the protective effect of ßB1-crystallin on ßA3-crystallin. Molecular dynamic simulation studies indicated that the mutation decreased the subunit binding energy and modified the distribution of surface electrostatic potentials. More importantly, the mutation separated two interacting loops in the C-terminal domain, which shielded the hydrophobic core from solvent in native ßB1-crystallin. These two interacting loops are highly conserved in both of the N- and C-terminal domains of all ß/γ-crystallins. We propose that these two interacting loops play an important role in the folding and structural stability of ß/γ-crystallin domains by protecting the hydrophobic core from solvent access.

DsCaf1 is involved in environmental stress response of Dunaliella salina.

Regulation of mRNA fates can alter the gene-expression profile promptly and specifically. Deadenylation is a process of which the poly(A) tail is degraded by deadenylases. By regulating mRNA turnover, deadenylases are involved in various vital cellular processes including stress responses. However, it is unclear whether deadenylases play a role in the adaption/tolerance of extremophiles. In this research, we cloned the deadenylase caf1 from Dunaliella salina (dscaf1), a unicellular green alga with exceptional halotolerance. In silicon analysis indicated that compared with the mesophilic alga Chlamydomonas reinhardtii caf1, dscaf1 promoter contained more elements responsive to abiotic stresses. Dscaf1 had an extremely high expression level under hypersaline conditions. When the D. salina cells were subject to stress shock, a two-stage response was observed for dscaf1 expression. The mRNA level of dscaf1 had an immediate 2-4 fold increase and followed by an ∼10 fold increase after hyperosmotic, heat or UV treatment, while had an about 3 fold increase quickly followed by an abrupt decrease after hypoosmotic or cold shock. The dissimilarity in dscaf1 expression patterns suggested that DsCaf1 is a stress-responsive deadenylase with the ability to regulate fates of a specific group of mRNAs for a certain type of stress.

Multi-frequency complex network from time series for uncovering oil-water flow structure.

Uncovering complex oil-water flow structure represents a challenge in diverse scientific disciplines. This challenge stimulates us to develop a new distributed conductance sensor for measuring local flow signals at different positions and then propose a novel approach based on multi-frequency complex network to uncover the flow structures from experimental multivariate measurements. In particular, based on the Fast Fourier transform, we demonstrate how to derive multi-frequency complex network from multivariate time series. We construct complex networks at different frequencies and then detect community structures. Our results indicate that the community structures faithfully represent the structural features of oil-water flow patterns. Furthermore, we investigate the network statistic at different frequencies for each derived network and find that the frequency clustering coefficient enables to uncover the evolution of flow patterns and yield deep insights into the formation of flow structures. Current results present a first step towards a network visualization of complex flow patterns from a community structure perspective.