Elizabeth A. Whitcomb

Harmful somatic mutations: Lessons from the dark side

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.

Novel Control of S Phase of the Cell Cycle by Ubiquitin-conjugating Enzyme H7

Timely degradation of regulatory proteins by the ubiquitin proteolytic pathway (UPP) is an established paradigm of cell cycle regulation during the G2/M and G1/S transitions. Less is known about roles for the UPP during S phase. Here we present evidence that dynamic cell cycle-dependent changes in levels of UbcH7 regulate entrance into and progression through S phase. In diverse cell lines, UbcH7 protein levels are dramatically reduced in S phase but are fully restored by G2. Knockdown of UbcH7 increases the proportion of cells in S phase and doubles the time to traverse S phase, whereas UbcH7 overexpression reduces the proportion of cells in S phase. These data suggest a role for UbcH7 targets in the completion of S phase and entry into G2. Notably, UbcH7 knockdown was coincident with elevated levels of the checkpoint kinase Chk1 but not Chk2. These results argue that UbcH7 promotes S phase progression to G2 by modulating the intra-S phase checkpoint mediated by Chk1. Furthermore, UbcH7 levels appear to be regulated by a UPP. Together the data identify novel roles for the UPP, specifically UbcH7 in the regulation of S phase transit time as well as in cell proliferation.

Ubiquitin control of S phase: a new role for the ubiquitin conjugating enzyme, UbcH7.

Events within and transitions between the phases of the eukaryotic cell cycle are tightly controlled by transcriptional and post-translational processes. Prominent among them is a profound role for the ubiquitin proteasome proteolytic pathway. The timely degradation of proteins balances the increases in gene products dictated by changes in transcription. Of the dozens of ubiquitin conjugating enzymes, or E2s, functions in control of the cell cycle have been defined for only UbcH10 and Ubc3/Cdc34. Each of these E2s works primarily with one ubiquitin ligase or E3. Here we show that another E2, UbcH7 is a regulator of S phase of the cell cycle. Over-expression of UbcH7 delays entry into S phase whereas depletion of UbcH7 increases the length of S phase and decreases cell proliferation. Additionally, the level of the checkpoint kinase Chk1 increases upon UbcH7 depletion while the level of phosphorylated PTEN decreases. Taken together, these data indicate that the length of S phase is controlled in part by UbcH7 through a PTEN/Akt/Chk1 pathway. Potential mechanisms by which UbcH7 controls Chk1 levels both directly and indirectly, as well as the length of S phase are discussed and additional functions for UbcH7 are reviewed.

Modulation of Human Peripheral Blood Eosinophil Function by Tumor Necrosis Factor-Alpha

Eosinophils are important effectors in helminthic parasitic infection. Tumor necrosis factor (TNF-alpha) has been implicated as a mediator in the host response to parasitic infection and enhances eosinophil-mediated helminthotoxicity. We have examined the direct effects of recombinant human (rh) TNF on eosinophil functions of degranulation and oxidative metabolism. This report describes the minimal effects of rhTNF-alpha on eosinophil superoxide anion generation and enzyme secretion, which do not satisfactorily explain the observed increases in helminthotoxicity. In contrast to other cell types, eosinophils are unique in their differential responses to interleukin-1 beta and TNF.

Unfolded-protein response–associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract

Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens‐specific mutations that elicit an unfolded‐protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin‐dependent kinase (Cdk)‐1 inhibition, retain their LFC nuclei, and are cata‐ractous. Although a UPR was not detected in lenses expressing K6W‐Ub, they also accumulated p27 and showed failed LFCD. Induction of a UPR in human lens epithelial cells (HLECs) also induced accumulation of p27 associated with decreased levels of S‐phase kinase‐associated protein (Skp)‐2, a ubiquitin ligase that regulates mitosis. These cells also showed decreased lamin A/C phosphorylation and metaphase arrest The suppression of lamin A/C phosphorylation and metaphase transition induced by the UPR was rescued by knockdown of p27. Taken together, these data indicate that accumulation of p27, whether related to the UPR or not, prevents the phosphorylation of lamin A/C and LFCD in maturing LFCs in vivo, as well as in dividing HLECs. The former leads to cataract and the latter to metaphase arrest. These results suggest that accumulation of p27 is a common mechanism underlying retention of LFC nuclei.—Lei, L., Whitcomb, E. A., Jiang, S., Chang, M.‐L., Gu, Y., Duncan, M. K., Cvekl, A., Wang, W.‐L., Limi, S., Reneker, L. W., Shang, F., Du, L., Taylor, A., Unfolded protein response‐associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract. FASEB J. 30, 1087–1095 (2016). www.fasebj.org

Restoration of Ig Secretion: Mutation of Germline-Encoded Residues in T15L Chains Leads to Secretion of Free Light Chains and Assembled Antibody Complexes Bearing Secretion-Impaired Heavy Chains

We previously described T15H chain mutants that were impaired in assembly with L chain and in ability to be secreted from the cell. The unmutated T15L chain is unusual in that it is secretion-impaired in the absence of assembly with H chain. The T15L chain preferentially pairs with T15H in vivo, suggesting that if we introduced mutations that would allow secretion of free T15L chain, they might also lead to the secretion of the complex with the defective H chain. We mutated four positions in the germline T15L that had amino acids infrequently found in other κ-chains. Mutation to the most frequently occurring amino acid at three of the four positions allowed secretion of free L chain, while the combination of two secretion-restoring mutations was synergistic. Coexpression of secretion-restored mutant L chains with the secretion-defective mutant H chains rescued secretion of the assembled H2L2 complex, suggesting that during somatic hypermutation in vivo, deleterious mutations at the H chain may be compensated by mutations on the L chain. To our knowledge, this is the first example of mutations in IgL chains that are able to restore secretion-defective H chains to secretion competence in mammalian cells.

Dietary glycemia as a determinant of health and longevity.

The role of diet in extending lifespan and healthspan has been the subject of much research and debate. Our recent epidemiological and in vivo data suggest that carbohydrate quality can be a major determinant in prolonging eye health. Additionally, excessive carbohydrate intake can contribute to the exacerbation of many different diseases. The metabolic diversity of the tissues that are affected by excessive carbohydrate intake suggests that dietary carbohydrate quality may affect cellular homeostasis.

Common cell biologic and biochemical changes in aging and age-related diseases of the eye: toward new therapeutic approaches to age-related ocular diseases.

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.

Optimizing Nutrition to Delay Age Related Macular Degeneration

Age related macular degeneration (AMD) is the leading cause of visual loss in older adults, affecting 30–50 million individuals worldwide and over two million individuals in the USA (Ann Med 38(7):450–71, 2006; Facts about age-related macular degeneration, NIoH; 2010]. Over 15 % of the elderly are affected. While AMD does not result in complete blindness on its own, the loss of central vision can impair the ability to perform everyday activities, decreasing quality of life (Mol Aspects Med 33(4):318–75, 2012). Costs associated with AMD are in excess of

Cutting Edge: Proteasome Involvement in the Degradation of Unassembled Ig Light Chains

340 billion in the USA, and for the majority of AMD patients in the USA, there is no clinical treatment (Ann Med 38(7):450–71, 2006). Given the aging population, and changing diets toward diets that may enhance risk for AMD (see below), the prevalence of AMD is projected to grow, with over five million individuals being affected by 2050 (Facts about age-related macular degeneration, NIoH; 2010). Treatment options for AMD are limited to treating only the most advanced stages of AMD, and for only the 15 % of AMD sufferers with “wet” AMD, in which some vision is already lost (Nutrients 5(7):2405–56, 2013). Given the limited therapeutic options available for AMD, preventative interventions through dietary modification are attractive strategies. Here we review studies that suggest benefits of certain nutrients with respect to AMD, with few, if any, adverse side effects. For an exhaustive review of the literature available for these nutrients please see review by Weikel et al. (Mol Aspects Med. 33(4):318–75, 2012).