Physiological and pathological functions of ßB2-crystallins in multiple organs: a systematic review.

Crystallins, the major constituent proteins of mammalian lenses, are significant not only for the maintenance of eye lens stability, transparency, and refraction, but also fulfill various physiopathological functions in extraocular tissues. ßB2-crystallin, for example, is a multifunctional protein expressed in the human retina, brain, testis, ovary, and multiple tumors. Mutations in the ßB2 crystallin gene or denaturation of ßB2-crystallin protein are associated with cataracts, ocular pathologies, and psychiatric disorders. A prominent role for ßB2-crystallins in axonal growth and regeneration, as well as in dendritic outgrowth, has been demonstrated after optic nerve injury. Studies in ßB2-crystallin-null mice revealed morphological and functional abnormalities in testis and ovaries, indicating ßB2-crystallin contributes to male and female fertility in mice. Interestingly, although pathogenic significance remains obscure, several studies identified a clear correlation between ßB2 crystallin expression and the prognosis of patients with breast cancer, colorectal cancer, prostate cancer, renal cell carcinoma, and glioblastoma in the African American population. This review summarizes the physiological and pathological functions of ßB2-crystallin in the eye and other organs and tissues and discusses findings related to the expression and potential role of ßB2-crystallin in tumors.

A functional role for the cancer disparity-linked genes, CRYßB2 and CRYßB2P1, in the promotion of breast cancer.

BACKGROUND: In the USA, the breast cancer mortality rate is 41% higher for African-American women than non-Hispanic White women. While numerous gene expression studies have classified biological features that vary by race and may contribute to poorer outcomes, few studies have experimentally tested these associations. CRYßB2 gene expression has drawn particular interest because of its association with overall survival and African-American ethnicity in multiple cancers. Several reports indicate that overexpression of the CRYßB2 pseudogene, CRYßB2P1, and not CRYßB2 is linked with race and poor outcome. It remains unclear whether either or both genes are linked to breast cancer outcomes. This study investigates CRYßB2 and CRYßB2P1 expression in human breast cancers and breast cancer cell line models, with the goal of elucidating the mechanistic contribution of CRYßB2 and CRYßB2P1 to racial disparities. METHODS: Custom scripts for CRYßB2 or CRYßB2P1 were generated and used to identify reads that uniquely aligned to either gene. Gene expression according to race and tumor subtype were assessed using all available TCGA breast cancer RNA sequencing alignment samples (n = 1221). In addition, triple-negative breast cancer models engineered to have each gene overexpressed or knocked out were developed and evaluated by in vitro, biochemical, and in vivo assays to identify biological functions. RESULTS: We provide evidence that CRYßB2P1 is expressed at higher levels in breast tumors compared to CRYßB2, but only CRYßB2P1 is significantly increased in African-American tumors relative to White American tumors. We show that independent of CRYßB2, CRYßB2P1 enhances tumorigenesis in vivo via promoting cell proliferation. Our data also reveal that CRYßB2P1 may function as a non-coding RNA to regulate CRYßB2 expression. A key observation is that the combined overexpression of both genes was found to suppress cell growth. CRYßB2 overexpression in triple-negative breast cancers increases invasive cellular behaviors, tumor growth, IL6 production, immune cell chemoattraction, and the expression of metastasis-associated genes. These data underscore that both CRYßB2 and CRYßB2P1 promote tumor growth, but their mechanisms for tumor promotion are likely distinct. CONCLUSIONS: Our findings provide novel data emphasizing the need to distinguish and study the biological effects of both CRYßB2 and CRYßB2P1 as both genes independently promote tumor progression. Our data demonstrate novel molecular mechanisms of two understudied, disparity-linked molecules.

Crybb2 deficiency impairs fertility in female mice.

Beta-B2-crystallin (CRYBB2), encoded by Crybb2 gene, is a major protein in the mammalian eye lens that plays an important role in maintaining the transparency of the ocular lens. However, CRYBB2 also plays important roles in many extra-lenticular tissues and organs such as the retina, brain and testis. Our previous studies demonstrated that male Crybb2 deficient (Crybb2(-/-)) mice have reduced fertility compared with wild-type (WT) mice, while female Crybb2(-/-) mice exhibited reduced ovary weights and shorter estrous cycle percentages. Here we specifically investigated the role of CRYBB2 in the female reproductive system. Our studies revealed that ovaries from female Crybb2(-/-) mice exhibited significantly reduced numbers of primordial, secondary and pre-ovulatory follicles when compared with WT mice, while the rate of atretic follicles was also increased. Additionally, fewer eggs were collected from the oviduct of Crybb2(-/-) female mice after superovulation. Estrogen levels were higher in the metestrus and diestrus cycles of female Crybb2(-/-) mice, while progesterone levels were lower in diestrus cycles. Furthermore, the expression of survival and cell cycle genes, Bcl-2, Cdk4 and Ccnd2, were significantly decreased in granulosa cells isolated from female Crybb2(-/-) mice, consistent with the predominant expression of CRYBB2 in ovarian granulosa cells. Our results reveal a critical role for CRYBB2 in female fertility and specific effects on the proliferation and survival status of ovarian granulosa cells.

The benefits of being ß-crystallin heteromers: ßB1-crystallin protects ßA3-crystallin against aggregation during co-refolding.

ß-Crystallins are the major structural proteins in mammalian lens, and their stability is critical in maintaining the transparency and refraction index of the lens. Among the seven ß-crystallins, ßA3-crystallin and ßB1-crystallin, an acidic and a basic ß-crystallin, respectively, can form heteromers in vivo. However, the physiological roles of the heteromer have not been fully elucidated. In this research, we studied whether the basic ß-crystallin facilitates the folding of acidic ß-crystallin. Equilibrium folding studies revealed that the ßA3-crystallin and ßB1-crystallin homomers and the ßA3/ßB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. ßA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of ßA3/ßB1-crystallin was close to that of ßB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the ß-crystallins. A comparison of the Gibbs free energy of the equilibrium folding suggested that the formation of heteromer contributed to the stabilization of the dimer interface. On the other hand, ßA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by ßB1-crystallin in a dose-dependent manner during the kinetic co-refolding. However, the protection is not observed in the presence of the pre-existed well-folded ßB1-crystallin. These findings suggested that the formation of ß-crystallin heteromers not only stabilizes the unstable acidic ß-crystallin but also protects them against aggregation during refolding from the stress-denatured states.

N-terminal extension of beta B1-crystallin: identification of a critical region that modulates protein interaction with beta A3-crystallin.

The human lens proteins beta-crystallins are subdivided into acidic (betaA1-betaA4) and basic (betaB1-betaB3) subunit groups. These structural proteins exist at extremely high concentrations and associate into oligomers under physiological conditions. Crystallin acidic-basic pairs tend to form strong heteromolecular associations. The long N-terminal extensions of beta-crystallins may influence both homo- and heteromolecular interactions. However, identification of the critical regions of the extensions mediating protein associations has not been previously addressed. This was studied by comparing the self-association and heteromolecular associations of wild-type recombinant betaA3- and betaB1-crystallins and their N-terminally truncated counterparts (betaA3DeltaN30 and betaB1DeltaN56) using several biophysical techniques, including analytical ultracentrifugation and fluorescence spectroscopy. Removal of the N-terminal extension of betaA3 had no effect on dimerization or heteromolecular tetramer formation with betaB1. In contrast, the level of self-association of betaB1DeltaN56 increased, resulting in homotetramer formation, and heteromolecular association with betaA3 was blocked. Limited proteolysis of betaB1 produced betaB1DeltaN47, which is similar to intact protein formed dimers but in contrast showed enhanced heteromolecular tetramer formation with betaA3. The tryptic digestion was physiologically significant, corresponding to protease processing sites observed in vivo. Molecular modeling of the N-terminal betaB1 extension indicates structural features that position a mobile loop in the vicinity of these processing sites. The loop is derived from residues 48-56 which appear to be critical for mediating protein interactions with betaA3-crystallin.

Elongation of axons during regeneration involves retinal crystallin beta b2 (crybb2).

Adult retinal ganglion cells (RGCs) can regenerate their axons in vitro. Using proteomics, we discovered that the supernatants of cultured retinas contain isoforms of crystallins with crystallin beta b2 (crybb2) being clearly up-regulated in the regenerating retina. Immunohistochemistry revealed the expression of crybb within the retina, including in filopodial protrusions and axons of RGCs. Cloning and overexpression of crybb2 in RGCs and hippocampal neurons increased axonogenesis, which in turn could be blocked with antibodies against beta-crystallin. Conditioned medium from crybb2-transfected cell cultures also supported the growth of axons. Finally real time imaging of the uptake of green fluorescent protein-tagged crybb2 fusion protein showed that this protein becomes internalized. These data are the first to show that axonal regeneration is related to crybb2 movement. The results suggest that neuronal crystallins constitute a novel class of neurite-promoting factors that likely operate through an autocrine mechanism and that they could be used in neurodegenerative diseases.