Expression of Cataract-linked γ-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability

Wu, Shu-Yu; Zou, Ping; Fuller, Alexandra; Mishra, Sanjay; Wang, Zhen; Schey, Kevin L.; Mchaourab, Hassane S.

doi:10.1074/jbc.m116.749606

Cited by 18 publications

(28 citation statements)

References 59 publications

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“…abnormalities that were readily visible starting at 3dpf ( Figure 1B), without affecting the overall morphology of the embryos ( Figure 1A). The nature of these defects was similar to those previously described for α-crystallin deficient [32,37] or γD-crystallin mutant transgenic lines [33]. Defective lenses exhibited phenotypic features that were either spherical, shiny crystal-like droplets spread sporadically across the lens (minor defects), or frequent droplets covering a large fraction of the lens as well as occasional large irregular protuberances located in the center of the lens (major defects), all of which could lead to opacity and changes in light scattering.…”

Section: Expression Of Both αα-Crystallin Variants Led To Various Degsupporting

confidence: 80%

“…In a previous study, we demonstrated that destabilized γD-crystallin mutant proteins formed aggregates in the zebrafish lens with a frequency that strongly correlated with the penetrance of lens defects [33]. Thus, the enhanced lens defects observed in the double transgenic embryos ( Figure 3; αΑ-R49C; γD-I4F) could result from increased aggregation frequency of γD-I4F when the αΑ-R49C transgene was co-expressed.…”

Section: Expression Of αA-r49c Increases the Propensity Of Destabilizmentioning

confidence: 76%

“…We directly tested this model utilizing a similar experimental approach with the Gal4/UAS expression system to facilitate mosaic analysis ( Figure 4A) [33]. By comparing the frequency of fluorescent punctate formation (i.e.…”

Section: Expression Of αA-r49c Increases the Propensity Of Destabilizmentioning

confidence: 99%

“…A number of advantages makes zebrafish ideal for such an endeavor including ease and cost of rearing the animals and the size of the embryo clutches [28,29]. Moreover, the transparency of the embryos during the first few days of their development and their relatively large eyes, which are functional at 3 days post fertilization (dpf), enable the examination of lens gross morphology by bright field microscopy [30][31][32][33][34]. The structure and function of the zebrafish adult lens parallel those of the mature human lens with respect to symmetry, refraction, transparency and optical function [29,35,36].…”

Section: Introductionmentioning

confidence: 99%

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Transgenic Zebrafish Models Reveal Distinct Molecular Mechanisms for Cataract-linked αA-Crystallin Mutants

Zou

Mishra

et al. 2018

Preprint

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Mutations in the small heat shock proteins a-crystallins have been linked to autosomal dominant cataracts in humans. Extensive studies in vitro have revealed a spectrum of alterations to the structure and function of these proteins including shifts in the size of the oligomer, modulation of subunit exchange and modification of their affinity to client proteins. Although mouse models of these mutants were instrumental in identifying changes in cellular proliferation and lens development, a direct comparative analysis of their effects on lens proteostasis has not been performed. Here, we have transgenically expressed cataract-linked mutants of aAand aBcrystallin in the zebrafish lens to dissect the underlying molecular changes that contribute to the loss of lens optical properties. Zebrafish lines expressing these mutants displayed a range of morphological lens defects. Phenotype penetrance and severity were dependent on the mutation even in fish lines lacking endogenous a-crystallin. The mechanistic origins of these differences were investigated by the transgenic co-expression of a destabilized human gDcrystallin mutant. We found that the R49C but not the R116C mutant of aA-crystallin promoted aggregation of gD-crystallin, although both mutants have similar affinity to client proteins in vitro. Our working model attributes these differences to the propensity of R49C, located in the buried N-terminal domain of aA-crystallin, to disulfide crosslinking as previously demonstrated in vitro. Our findings complement and extend previous work in mouse models and emphasize the need of investigating chaperone/client protein interactions in appropriate cellular context.

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Section: Expression Of Both αα-Crystallin Variants Led To Various Degsupporting

confidence: 80%

Section: Expression Of αA-r49c Increases the Propensity Of Destabilizmentioning

confidence: 76%

Section: Expression Of αA-r49c Increases the Propensity Of Destabilizmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Transgenic Zebrafish Models Reveal Distinct Molecular Mechanisms for Cataract-linked αA-Crystallin Mutants

Zou

Mishra

et al. 2018

Preprint

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“…The instruments were operated in a data dependent mode with the top five most abundant ions in each MS scan selected for fragmentation in the LTQ. Dynamic exclusion (exclude after 2 spectra, release after 30 sec, and exclusion list size of 150) was enabled 21 .…”

Section: Tandem Mass Spectrometrymentioning

confidence: 99%

Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

Jiang

Palatinus

et al. 2019

Preprint

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Background: aCT1 is a 25 amino acid therapeutic peptide incorporating the Zonula Occludens-1 (ZO-1)-binding domain of connexin43 (Cx43) that is currently in Phase III clinical testing for healing chronic skin wounds. In preclinical studies in mice, we reported that aCT1 reduces arrhythmias and improves ventricular function following cardiac injury, effects that were accompanied by increases in PKCε phosphorylation of Cx43 at serine 368 (pS368). In this study, we undertake a systematic characterization of the molecular mode-of-action of aCT1 in mitigating the effects of ischemia reperfusion injury on ventricular contractile function. Methods and Results:To determine the basis of αCT1-mediated increases in pS368 we undertook tandem mass spectrometry of reactants in an in vitro assay of PKCε phosphorylation, identifying an interaction between negatively charged amino acids in the αCT1 Asp-Asp-Leu-Glu-Iso sequence and positively charged lysines (Lys345, Lys346) in a short α-helical sequence (H2) within the Cx43 CT domain. In silico modeling provided further support of the specificity of this interaction, leading us to conclude that αCT1 has potential to directly interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift and phosphorylation assays, we characterized a series of αCT1 variant peptides, identifying sequences competent to interact with either ZO-1 PDZ2 or the Cx43 CT, but with limited or no ability to bind both polypeptides. Based on this analysis, it was found that only those peptides competent to interact with Cx43, but not ZO-1 alone, resulted in increased pS368 phosphorylation in vitro and in vivo. Moreover, in a mouse model of global ischemia reperfusion injury we determined that pre-ischemic infusion only with those peptides competent to bind Cx43 preserved left ventricular (LV) contractile function following injury. Interestingly, a short 9 amino acid (MW=1110) Cx43-binding variant of the original 25 amino acid αCT1 sequence demonstrated potent LV-protecting effects when infused either before or after ischemic injury. Conclusions:Interaction of αCT1 with the Cx43 CT, but not ZO-1 PDZ2, explains cardioprotection mediated by this therapeutic peptide. Pharmacophores targeting the Cx43 carboxyl terminus could provide a novel translational approach to preservation of ventricular function following ischemic injury.

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The growing world of small heat shock proteins: from structure to functions

Carra

Alberti

Arrigo

et al. 2017

Cell Stress and Chaperones

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Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world's experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12-15, 2016).

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Expression of Cataract-linked γ-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability

Cited by 18 publications

References 59 publications

Transgenic Zebrafish Models Reveal Distinct Molecular Mechanisms for Cataract-linked αA-Crystallin Mutants

Transgenic Zebrafish Models Reveal Distinct Molecular Mechanisms for Cataract-linked αA-Crystallin Mutants

Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

The growing world of small heat shock proteins: from structure to functions

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