Detailed analysis of the rearrangement and expression of two mouse Vkappa genes has been used to examine B cell repertoire development. The Vkappa1-A gene is used by a large proportion (9.6%) of splenic B cells in the adult primary repertoire, whereas the Vkappa22 gene is used at a much lower frequency (0.16%). Consistent with these results, quantitative PCR (Q-PCR) assays revealed that the number of splenic B cells with rearranged Vkappa1-A genes is much greater than the number with rearranged Vkappa22 genes. Q-PCR was also performed on both normal bone marrow pre-B cells and transformed pre-B cells induced to rearrange their kappa loci at high frequency. In contrast to splenic B cell rearrangements, the numbers of Vkappa1-A and Vkappa22 rearrangements in pre-B cells differ by only two- or threefold, suggesting that the intrinsic rearrangement frequencies of these two Vkappa genes are not significantly different. Further evidence of disproportionate selection was obtained by comparing the percentages of productive rearrangements amplified from genomic splenic DNA. Sequence analysis showed 84% (37 of 44) of the Vkappa1-A rearrangements but only 57% (29 of 51) of the Vkappa22 rearrangements to be in-frame. Together these results suggest that B cells expressing Vkappa1-A-encoded light chains are preferentially selected either in the periphery or in the transition from pre-B to B cell. Sequence data also reveal a surprisingly restricted diversity of VJ junctions, apparently due to biases introduced by the rearrangement mechanism.
Summary: The ability of somatic mutation to modify the course of an immune response is well documented. However, emphasis has been placed almost exclusively on the ability of somatic mutation to improve the functional characteristics of representative antibodies. The harmful effects of somatic mutation, its dark side, have been far less well characterized. Yet evidence suggests that the number of B cells directed to wastage pathways as a result of harmful somatic mutation probably far exceeds the number of cells whose antibodies have been improved. Here we review our recent findings in understanding the structural and functional consequences of V‐region mutation.
The lens proteome undergoes dramatic composition changes during development and maturation. A defective developmental process leads to congenital cataracts that account for about 30% of cases of childhood blindness. Gene mutations are associated with approximately 50% of early-onset forms of lens opacity, with the remainder being of unknown etiology. To gain a better understanding of cataractogenesis, we utilized a transgenic mouse model expressing a mutant ubiquitin protein in the lens (K6W-Ub) that recapitulates most of the early pathological changes seen in human congenital cataracts. We performed mass spectrometry-based tandem-mass-tag quantitative proteomics in E15, P1, and P30 control or K6W-Ub lenses. Our analysis identified targets that are required for early normal differentiation steps and altered in cataractous lenses, particularly metabolic pathways involving glutathione and amino acids. Computational molecular phenotyping revealed that glutathione and taurine were spatially altered in the K6W-Ub cataractous lens. High-performance liquid chromatography revealed that both taurine and the ratio of reduced glutathione to oxidized glutathione, two indicators of redox status, were differentially compromised in lens biology. In sum, our research documents that dynamic proteome changes in a mouse model of congenital cataracts impact redox biology in lens. Our findings shed light on the molecular mechanisms associated with congenital cataracts and point out that unbalanced redox status due to reduced levels of taurine and glutathione, metabolites already linked to age-related cataract, could be a major underlying mechanism behind lens opacities that appear early in life.
The tumor co-promotor TPA is believed to enhance a wide variety of cellular processes by interacting with protein kinase C. Interleukin (IL 1) is a family of highly active molecules which augments the host response to infection. We have explored the interactions of these activators of cell function on the modulation of selected eosinophil functions. The effects of purified monocyte-derived IL 1 on the eosinophil functions of oxidative metabolism (as measured by superoxide anion production) and degranulation (as measured by release of the granular enzymes arylsulfatase and beta-glucuronidase) have been examined. Superoxide anion production by eosinophils stimulated with standard doses of the stimulant phorbol myristic acetate (TPA) (1 microgram/ml) was augmented approximately 20% by preincubation with IL 1. However, IL 1 alone had no effect on superoxide anion production. At suboptimal doses of TPA, there was a dose-dependent inhibition of superoxide anion production in the presence of IL 1. Calcium ionophore (2 X 10(-7) M) markedly enhanced superoxide anion production elicited by 0.1 ng/ml of TPA, but had only modest effects in the absence of TPA. When IL 1 was added to eosinophils stimulated by TPA in the presence of calcium ionophore, there was a dose-dependent increase in superoxide anion production. In contrast to other cell types, degranulation as measured by the release of arylsulfatase and beta-glucuronidase was not elicited by the addition of TPA (1 microgram/ml). Although calcium ionophore (2 X 10(-6) M) caused enzyme release (24.2% release of beta-glucuronidase, 29.4% release of arylsulfatase), this release was inhibited by the addition of TPA. The addition of IL 1 alone caused an approximate twofold increase in enzyme release, but pretreatment with IL 1 (1 U) reduced ionophore-mediated degranulation (p less than or equal to 0.05). Studies employing purified monocyte IL 1 were confirmed by recombinant IL 1-beta. These studies demonstrate for the first time that eosinophil function is modulated by IL 1. IL 1 may also modify the response of eosinophils to other stimuli such as ionophore and TPA. Because TPA is known to act by direct binding to protein kinase C, these studies also demonstrate that, in eosinophils, activation of protein kinase C by phorbol esters may augment one cellular function (oxidative metabolism) while inhibiting another cellular function (degranulation). Similarly, phorbol esters may act synergistically with calcium ionophore in regulation of one function (oxidative metabolism) and act antagonistically with another function (degranulation). The concept that IL 1 uniformly enhances cell function may need to be re-evaluated.
Post translational modification by ubiquitination can target proteins for degradation, allow the interaction of proteins to form complexes or direct relocalization of proteins to different subcellular compartments. As such, ubiquitin controls a variety of essential cellular processes. Previously we demonstrated that the ubiquitin conjugating enzyme, UbcH7 controls both the entry into and progression through S phase of the cell cycle (Whitcomb et. al 2009 Mol. Biol. Cell v20:1–9). In our search for cell cycle regulatory proteins which might be involved in the UbcH7‐mediated control of the cell cycle, we discovered that UbcH7 affects the level of the cyclin dependent kinase inhibitor, p27, but does not affect the similarly regulated cyclin dependent kinase, p21. Interestingly, depletion of UbcH7 affects p27 levels primarily in the cytosol. While nuclear p27 controls the G1 to S transition of the cell cycle through inhibition of the cyclin dependent kinases, cytosolic p27 plays a role in cell migration. Thus, we asked whether manipulation of UbcH7 also affected cell migration and find that depletion of UbcH7 inhibits cell migration. These results suggest that UbcH7 might be an attractive drug target as it plays roles in controlling both the cell cycle and cell migration. This work was supported by grants NIH EY13250 and USDA 581950–5100‐060–01A to AT.
Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin-conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized, and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293, and HELA cells. However, the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1 but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated the colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in the stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.
Reviews of information about AMD, cataract, and glaucoma make it apparent that while each eye tissue has its own characteristic metabolism, structure, and function, there are common perturbations to homeostasis that are associated with age-related dysfunction. The commonalities appeared to be biochemical stresses and their sequelae. Recognition of shared etiologic factors for age-related debilities allows rationalization of comparable risk factor-disease incidence relationships—such as nutritional risk factors for AMD and cataract (as well as cardiovascular disease and diabetes)—and informs about potential new therapeutic avenues, such as stress reducers (i.e., antioxidants) and/or proteolysis enhancers. It also maximizes the return on the investment in research effort and costs. For example, drugs or nutrients that protect against AMD may also prove effective against cataract, glaucoma, or/and other age-related neurodegenerative debilities.
This article summarizes cell biologic and biochemical changes in aging and age-related diseases of the eye. Clearly, this is a larger challenge with a richer literature than can be properly treated in a short review such as this. In this short review, we focus on age-related stresses and current and anticipated means to diminish the stress. Recognizing that almost all age-related diseases such as Alzheimer and Parkinson diseases, cataract, AMD, glaucoma, diabetes, and the premature aging diseases such as progeria, have in common the accumulation of damaged proteins, we select three aspects of age-related biochemical changes that are common to most eye tissues: oxidative stresses; problems associated with and/or due to damaged proteins that accumulate in the retina, lens, and cornea; and intracellular degradative capacities that usually keep levels of damaged proteins in check in early life or when tissues are not stressed, but that may fail upon stress or aging (Figs. 1, ,2).2). We offer apologies to investigators whose work we do not cite or can acknowledge only via reviews.1
Figure 1
Scheme of proposed relationship between chronic stress, protective capacities, proteolytic editing machinery and age-related disease. When young, proteins are intact and cell and tissue functions are retained. Upon exposure to various stresses (red) including ...
Figure 2
Upon aging, proteins are damaged. This damage includes various modifications such as oxidations, reaction with other moieties such as sugar derivatives, cross linking (yellow), and lysis. These processes accelerate upon aging (follow blue line) and parallel ...
The most rapidly growing segment of many societies is the elderly. The prevalence of cataract, AMD, and glaucoma accelerates with age. Among those who are aged 75 years or older, prevalence rates of cataract, AMD, and glaucoma are approximately 60%, 15%, and 20% of the population, respectively. These estimates almost double for people aged just 10 years older. Like most tissues in general, most eye tissues suffer from the accumulation of damaged proteins. Such accumulation appears to involve post-synthetic modifications to proteins and limits on the proteolytic capacities that are normally available to degrade and remove the altered or obsolete proteins before they transform into cytotoxic aggregates. Collectively, we call the sum of synthesis, post-synthetic modification, editing and removal of proteins “proteopoise.” Compromises to proteopoise are also thought to be etiologic for many age-related neuropathies and premature aging syndromes.1–7 Herein, we work our way from the anterior of the eye, or cornea, through to the lens and on to the posterior segment or retina, recalling common themes of age-related changes and protein quality control.
