The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration SMURF1-UbcH7 K29 ubiquitination of p27 and cell migration.

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.

Characterization of MYBL1 Gene in Triple-Negative Breast Cancers and the Genes' Relationship to Alterations Identified at the Chromosome 8q Loci.

The MYBL1 gene is a strong transcriptional activator involved in events associated with cancer progression. Previous data show MYBL1 overexpressed in triple-negative breast cancer (TNBC). There are two parts to this study related to further characterizing the MYBL1 gene. We start by characterizing MYBL1 reference sequence variants and isoforms. The results of this study will help in future experiments in the event there is a need to characterize functional variants and isoforms of the gene. In part two, we identify and validate expression and gene-related alterations of MYBL1, VCIP1, MYC and BOP1 genes in TNBC cell lines and patient samples selected from the Breast Invasive Carcinoma TCGA 2015 dataset available at cBioPortal.org. The four genes are located at chromosomal regions 8q13.1 to 8q.24.3 loci, regions previously identified as demonstrating a high percentage of alterations in breast cancer. We identify alterations, including changes in expression, deletions, amplifications and fusions in MYBL1, VCPIP1, BOP1 and MYC genes in many of the same patients, suggesting the panel of genes is involved in coordinated activity in patients. We propose that MYBL1, VCPIP1, MYC and BOP1 collectively be considered as genes associated with the chromosome 8q loci that potentially play a role in TNBC pathogenesis.

Inside out: Relations between the microbiome, nutrition, and eye health.

Age-related macular degeneration (AMD) is a complex disease with increasing numbers of individuals being afflicted and treatment modalities limited. There are strong interactions between diet, age, the metabolome, and gut microbiota, and all of these have roles in the pathogenesis of AMD. Communication axes exist between the gut microbiota and the eye, therefore, knowing how the microbiota influences the host metabolism during aging could guide a better understanding of AMD pathogenesis. While considerable experimental evidence exists for a diet-gut-eye axis from murine models of human ocular diseases, human diet-microbiome-metabolome studies are needed to elucidate changes in the gut microbiome at the taxonomic and functional levels that are functionally related to ocular pathology. Such studies will reveal new ways to diminish risk for progression of- or incidence of- AMD. Current data suggest that consuming diets rich in dark fish, fruits, vegetables, and low in glycemic index are most retina-healthful during aging.

Protecting Vulnerable Patients from Influenza During the COVID-19 Pandemic: An Urgent Call to Action for Health Care Professionals.

The 2020-2021 US influenza season, although mild, initially raised concerns about an unprecedented dual threat of SARS-CoV-2, the virus that causes COVID-19, circulating alongside seasonal influenza viruses. Although everyone is susceptible to influenza infection, adults with chronic health conditions (including heart disease, lung disease, and diabetes) are particularly vulnerable to influenza-related complications including hospitalization, disability, and death-as are older adults (65+ years) and adults in underserved communities, in which rates of chronic health conditions are higher. Many of the chronic health conditions associated with an increased risk of influenza-related hospitalization and mortality are the same conditions that increase the risk of severe COVID-19 outcomes. Given the impact of the COVID-19 pandemic, health care professionals must prioritize influenza vaccination for all patients, especially those with chronic health conditions.

Stabilization of p27Kip1/CDKN1B by UBCH7/UBE2L3 catalyzed ubiquitinylation: a new paradigm in cell-cycle control.

Ubiquitinylation drives many cellular processes by targeting proteins for proteasomal degradation. Ubiquitin conjugation enzymes promote ubiquitinylation and, thus, degradation of protein substrates. Ubiquitinylation is a well-known posttranslational modification controlling cell-cycle transitions and levels or/and activation levels of ubiquitin-conjugating enzymes change during development and cell cycle. Progression through the cell cycle is tightly controlled by CDK inhibitors such as p27Kip1. Here we show that, in contrast to promoting its degradation, the ubiquitin-conjugating enzyme UBCH7/UBE2L3 specifically protects p27Kip1 from degradation. Overexpression of UBCH7/UBE2L3 stabilizes p27Kip1 and delays the G1-to-S transition, while depletion of UBCH7/UBE2L3 increases turnover of p27Kip1. Levels of p21Cip1/Waf1, p57Kip2, cyclin A and cyclin E, all of which are also involved in regulating the G1/S transition are not affected by UBCH7/UBE2L3 depletion. The effect of UBCH7/UBE2L3 on p27Kip1 is not due to alteration of the levels of any of the ubiquitin ligases known to ubiquitinylate p27Kip1. Rather, UBCH7/UBE2L3 catalyzes the conjugation of heterotypic ubiquitin chains on p27Kip1 that are proteolytically incompetent. These data reveal new controls and concepts about the ubiquitin proteasome system in which a ubiquitin-conjugating enzyme selectively inhibits and may even protect, rather than promote degradation of a crucial cell-cycle regulatory molecule.-Whitcomb, E. A., Tsai, Y. C., Basappa, J., Liu, K., Le Feuvre, A. K., Weissman, A. M., Taylor, A. Stabilization of p27Kip1/CDKN1B by UBCH7/UBE2L3 catalyzed ubiquitinylation: a new paradigm in cell-cycle control.

Involvement of a gut-retina axis in protection against dietary glycemia-induced age-related macular degeneration.

Age-related macular degeneration (AMD) is the major cause of blindness in developed nations. AMD is characterized by retinal pigmented epithelial (RPE) cell dysfunction and loss of photoreceptor cells. Epidemiologic studies indicate important contributions of dietary patterns to the risk for AMD, but the mechanisms relating diet to disease remain unclear. Here we investigate the effect on AMD of isocaloric diets that differ only in the type of dietary carbohydrate in a wild-type aged-mouse model. The consumption of a high-glycemia (HG) diet resulted in many AMD features (AMDf), including RPE hypopigmentation and atrophy, lipofuscin accumulation, and photoreceptor degeneration, whereas consumption of the lower-glycemia (LG) diet did not. Critically, switching from the HG to the LG diet late in life arrested or reversed AMDf. LG diets limited the accumulation of advanced glycation end products, long-chain polyunsaturated lipids, and their peroxidation end-products and increased C3-carnitine in retina, plasma, or urine. Untargeted metabolomics revealed microbial cometabolites, particularly serotonin, as protective against AMDf. Gut microbiota were responsive to diet, and we identified microbiota in the Clostridiales order as being associated with AMDf and the HG diet, whereas protection from AMDf was associated with the Bacteroidales order and the LG diet. Network analysis revealed a nexus of metabolites and microbiota that appear to act within a gut-retina axis to protect against diet- and age-induced AMDf. The findings indicate a functional interaction between dietary carbohydrates, the metabolome, including microbial cometabolites, and AMDf. Our studies suggest a simple dietary intervention that may be useful in patients to arrest AMD.

Astronaut Cardiovascular Health and Risk Modification (Astro-CHARM) Coronary Calcium Atherosclerotic Cardiovascular Disease Risk Calculator.

BACKGROUND: Coronary artery calcium (CAC) is a powerful novel risk indicator for atherosclerotic cardiovascular disease (ASCVD). Currently, there is no available ASCVD risk prediction tool that integrates traditional risk factors and CAC. METHODS: To develop a CAC ASCVD risk tool for younger individuals in the general population, subjects aged 40 to 65 without prior cardiovascular disease from 3 population-based cohorts were included. Cox proportional hazards models were developed incorporating age, sex, systolic blood pressure, total and high-density lipoprotein cholesterol, smoking, diabetes mellitus, hypertension treatment, family history of myocardial infarction, high-sensitivity C-reactive protein, and CAC scores (Astro-CHARM model [Astronaut Cardiovascular Health and Risk Modification]) as dependent variables and ASCVD (nonfatal/fatal myocardial infarction or stroke) as the outcome. Model performance was assessed internally, and validated externally in a fourth cohort. RESULTS: The derivation study comprised 7382 individuals with a mean age 51 years, 45% women, and 55% nonwhite. The median CAC was 0 (25th, 75th [0,9]), and 304 ASCVD events occurred in a median 10.9 years of follow-up. The c-statistic was 0.784 for the risk factor model, and 0.817 for Astro-CHARM ( P<0.0001). In comparison with the risk factor model, the Astro-CHARM model resulted in integrated discrimination improvement (0.0252), and net reclassification improvement (0.121; P<0.0001), as well. The Astro-CHARM model demonstrated good discrimination (c=0.78) and calibration (Nam-D'Agostino χ2, 13.2; P=0.16) in the validation cohort (n=2057; 55 events). A mobile application and web-based tool were developed to facilitate clinical application of this tool ( www.AstroCHARM.org ). CONCLUSION: The Astro-CHARM tool is the first integrated ASCVD risk calculator to incorporate risk factors, including high-sensitivity C-reactive protein and family history, and CAC data. It improves risk prediction in comparison with traditional risk factor equations and could be useful in risk-based decision making for cardiovascular disease prevention in the middle-aged general population.

Too sweet: Problems of protein glycation in the eye.

Laboratory and epidemiological data indicate that high blood sugar levels and/or consuming high glycemia diets are linked to multiple age-related diseases, including age-related macular degeneration, cataract, Parkinson's disease, Alzheimer's disease, diabetic retinopathy, and, apparently glaucoma. High concentrations of blood sugar and perturbations of the systems that regulate blood sugar lead to the accumulation of advanced-glycation end products (AGEs). AGEs are toxic compounds that are formed from the combination of sugars and their metabolites with biomolecules in a non-enzymatic biochemical reaction called glycation. In vitro and in vivo data indicate that high sugar consumption is associated with accumulation of AGEs in a variety of human tissues. Hyperglycemia, along with an oxidative environment and limited cell proliferation in many ocular tissues, encourages formation and precludes dilution of AGEs and associated damage by cell division. These circumstances make many eye tissues vulnerable to glycation-derived damage. Here, we summarize research regarding glycation-induced ocular tissue dysfunction and its contribution to the onset and development of eye disorders. We also discuss how management of carbohydrate nutrition may provide a low-cost way to ameliorate the progression of AGEs-related diseases, including age related macular degeneration and some cataracts, as they do for cardiovascular disease and diabetes.

Low cholesterol efflux capacity and abnormal lipoprotein particles in youth with type 1 diabetes: a case control study.

BACKGROUND: Patients with type 1 diabetes (T1DM) have increased mortality from cardiovascular disease (CVD). Risk factors for CVD include an elevation of LDL (LDLp) and small HDL (sHDLp) particles, and a decrease in reverse cholesterol transport i.e. HDL-cholesterol efflux capacity (CEC). Our objective was to compare lipoprotein particles and CEC between T1DM and healthy controls (HC) and to explore the associations between NMR lipid particles and cholesterol efflux. METHODS: 78 patients with T1DM and 59 HC underwent fasting lipoprotein profile testing by NMR and measurements of CEC by cell-based method. The associations between NMR lipid particles with CEC were analyzed using multivariable linear regression models. RESULTS: Youth with T1DM had higher total LDLp 724 [(563-985) vs 622 (476-794) nmol/L (P = 0.011)] (Maahs et al. in Circulation 130(17):1532-58, 2014; Shah et al. in Pediatr Diabetes 16(5):367-74, 2015), sHDLp [11.20 (5.7-15.3) vs 7.0 (3.2-13.1) µmol/L (P = 0.021)], and lower medium HDLp [11.20 (8.5-14.5) vs 12.3 (9-19.4), (P = 0.049)] and lower CEC (0.98 ± 0.11% vs 1.05 ± 0.15%, P = 0.003) compared to HC. Moreover, CEC correlated with sHDLp (ß = - 0.28, P = 0.045) and large HDLp (ß = 0.46, P < 0.001) independent of age, sex, ethnicity, BMIz, HbA1c, hsCRP and total HDLp in the diabetic cohort. CONCLUSIONS: Youth with T1DM demonstrated a more atherogenic profile including higher sHDL and LDLp and lower CEC. Future efforts should focus on considering adding lipoprotein particles and CEC in CVD risk stratification of youth with T1DM. Trial registration Clinical Trials Registration Number NCT02275091.

The Role of Microbiota in Retinal Disease.

The ten years since the first publications on the human microbiome project have brought enormous attention and insight into the role of the human microbiome in health and disease. Connections between populations of microbiota and ocular disease are now being established, and increased accessibility to microbiome research and insights into other diseases is expected to yield enormous information in the coming years. With the characterization of the ocular microbiome, important insights have already been made regarding corneal and conjunctival tissues. Roles for non-ocular microbiomes in complex retinal diseases are now being evaluated. For example, the gut microbiome has been implicated in the pathogenesis of uveitis. This short review will summarize the few studies linking gut or oral microbiota to diabetic retinopathy (DR), glaucoma, and age-related macular degeneration (AMD). We will also conjecture where the most significant findings still remain to be elucidated. Finally, we will propose the gut-retina axis, related but distinct from the gut-brain axis.

Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract.

Although the ocular lens shares many features with other tissues, it is unique in that it retains its cells throughout life, making it ideal for studies of differentiation/development. Precipitation of proteins results in lens opacification, or cataract, the major blinding disease. Lysines on ubiquitin (Ub) determine fates of Ub-protein substrates. Information regarding ubiquitin proteasome systems (UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressed in the lens a mutant Ub containing a K6W substitution (K6W-Ub). Protein profiles of lenses that express wild-type ubiquitin (WT-Ub) or K6W-Ub differ by only ∼2%. Despite these quantitatively minor differences, in K6W-Ub lenses and multiple model systems we observed a fourfold Ca(2+) elevation and hyperactivation of calpain in the core of the lens, as well as calpain-associated fragmentation of critical lens proteins including Filensin, Fodrin, Vimentin, ß-Crystallin, Caprin family member 2, and tudor domain containing 7. Truncations can be cataractogenic. Additionally, we observed accumulation of gap junction Connexin43, and diminished Connexin46 levels in vivo and in vitro. These findings suggest that mutation of Ub K6 alters UPS function, perturbs gap junction function, resulting in Ca(2+) elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between UPS and calpain-based degradative systems and advance our understanding of roles for Ub K6 in eye development. They also inform about new approaches to delay cataract and other protein precipitation diseases.

Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly.

Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIß (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation.

Disassembly of the lens fiber cell nucleus to create a clear lens: The p27 descent.

The eye lens is unique among tissues: it is transparent, does not form tumors, and the majority of its cells degrade their organelles, including their cell nuclei. A mystery for over a century, there has been considerable recent progress in elucidating mechanisms of lens fiber cell denucleation (LFCD). In contrast to the disassembly and reassembly of the cell nucleus during mitosis, LFCD is a unidirectional process that culminates in destruction of the fiber cell nucleus. Whereas p27Kip1, the cyclin-dependent kinase inhibitor, is upregulated during formation of LFC in the outermost cortex, in the inner cortex, in the nascent organelle free zone, p27Kip1 is degraded, markedly activating cyclin-dependent kinase 1 (Cdk1). This process results in phosphorylation of nuclear Lamins, dissociation of the nuclear membrane, and entry of lysosomes that liberate DNaseIIß (DLAD) to cleave chromatin. Multiple cellular pathways, including the ubiquitin proteasome system and the unfolded protein response, converge on post-translational regulation of p27Kip1. Mutations that impair these pathways are associated with congenital cataracts and loss of LFCD. These findings highlight new regulatory nodes in the lens and suggest that we are close to understanding this fascinating terminal differentiation process. Such knowledge may offer a new means to confront proliferative diseases including cancer.

Acute Kidney Injury and In-Hospital Mortality after Coronary Artery Bypass Graft versus Percutaneous Coronary Intervention: A Nationwide Study.

BACKGROUND: Post-procedural acute kidney injury (AKI) is associated with significantly increased short- and long-term mortalities, and renal loss. Few studies have compared the incidence of post-procedural AKI and in-hospital mortality between 2 major modalities of revascularization - coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) - and results have been inconsistent. METHODS: We generated a propensity score-matched cohort that includes a total of 286,670 hospitalizations with multi-vessel coronary disease undergoing CABG or PCI (2004-2012) from the National Inpatient Sample database. We compared incidence of AKI, AKI requiring renal replacement therapy (RRT), in-hospital mortality, hospital stay, and charges between CABG and PCI groups. RESULTS: The incidence of AKI after CABG was higher than PCI (8.9 vs. 4.5%, OR 2.05, 95% CI 1.99-2.12, p < 0.001). The incidence of AKI requiring RRT was also higher after CABG (1.1 vs. 0.5%, OR 2.14, 95% CI 1.96-2.34, p < 0.001). Likewise, in-hospital mortality was higher after CABG than PCI (2.0 vs. 1.4%, OR 1.44, 95% CI 1.35-1.52, p < 0.001). Among patients with pre-existing chronic kidney disease (stages I-IV), those undergoing CABG was associated with 2.0-2.3-fold higher odds of developing AKI than those undergoing PCI. The patients treated with CABG had a significantly longer hospital stay and higher hospital charges. CONCLUSIONS: Patients undergoing CABG are associated with (1) increased risk of developing post-procedural AKI, (2) higher likelihood of receiving RRT, and (3) worse short-term survival. Long-term renal outcome remains to be studied.

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 cataractous. 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.

Contribution of the Nurses' Health Study to the Epidemiology of Cataract, Age-Related Macular Degeneration, and Glaucoma.

OBJECTIVES: To review the contribution of the Nurses' Health Study (NHS) to understanding the genetic and lifestyle factors that influence the risk of cataract, age-related macular degeneration, and glaucoma. METHODS: We performed a narrative review of the publications of the NHS between 1976 and 2016. RESULTS: The NHS has helped to elucidate the roles of genetics, lifestyle factors (e.g., cigarette smoking associated with cataract extraction and age-related macular degeneration), medical conditions (e.g., diabetes associated with cataract extraction and glaucoma), and dietary factors (e.g., greater carotenoid intake and lower glycemic diet associated with lower risk of age-related macular degeneration) in the etiology of degree and progression of lens opacities, cataract extraction, age-related macular degeneration, primary open-angle glaucoma, and exfoliation glaucoma. CONCLUSIONS: The findings from the NHS, combined with those of other studies, have provided compelling evidence to support public health recommendations for helping to prevent age-related eye diseases: abstinence from cigarette smoking, maintenance of healthy weight and diabetes prevention, and a healthy diet rich in fruits and vegetables.

Gene-Diet Interactions in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a prevalent blinding disease, accounting for roughly 50 % of blindness in developed nations. Very significant advances have been made in terms of discovering genetic susceptibilities to AMD as well as dietary risk factors. To date, nutritional supplementation is the only available treatment option for the dry form of the disease known to slow progression of AMD. Despite an excellent understanding of genes and nutrition in AMD, there is remarkably little known about gene-diet interactions that may identify efficacious approaches to treat individuals. This review will summarize our current understanding of gene-diet interactions in AMD with a focus on animal models and human epidemiological studies.

Identification of potential glycan cancer markers with sialic acid attached to sialic acid and up-regulated fucosylated galactose structures in epidermal growth factor receptor secreted from A431 cell line.

We have used powerful HPLC-mass spectrometric approaches to characterize the secreted form of epidermal growth factor receptor (sEGFR). We demonstrated that the amino acid sequence lacked the cytoplasmic domain and was consistent with the primary sequence reported for EGFR purified from a human plasma pool. One of the sEGFR forms, attributed to the alternative RNA splicing, was also confirmed by transcriptional analysis (RNA sequencing). Two unusual types of glycan structures were observed in sEGFR as compared with membrane-bound EGFR from the A431 cell line. The unusual glycan structures were di-sialylated glycans (sialic acid attached to sialic acid) at Asn-151 and N-acetylhexosamine attached to a branched fucosylated galactose with N-acetylglucosamine moieties (HexNAc-(Fuc)Gal-GlcNAc) at Asn-420. These unusual glycans at specific sites were either present at a much lower level or were not observable in membrane-bound EGFR present in the A431 cell lysate. The observation of these di-sialylated glycan structures was consistent with the observed expression of the corresponding α-N-acetylneuraminide α-2,8-sialyltransferase 2 (ST8SiA2) and α-N-acetylneuraminide α-2,8-sialyltransferase 4 (ST8SiA4), by quantitative real time RT-PCR. The connectivity present at the branched fucosylated galactose was also confirmed by methylation of the glycans followed by analysis with sequential fragmentation in mass spectrometry. We hypothesize that the presence of such glycan structures could promote secretion via anionic or steric repulsion mechanisms and thus facilitate the observation of these glycan forms in the secreted fractions. We plan to use this model system to facilitate the search for novel glycan structures present at specific sites in sEGFR as well as other secreted oncoproteins such as Erbb2 as markers of disease progression in blood samples from cancer patients.

Newborn mouse lens proteome and its alteration by lysine 6 mutant ubiquitin.

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. To study the developmental processes that require intact ubiquitin, we executed the most extensive characterization of the lens proteome to date. We quantified lens protein expression changes in multiple replicate pools of P1 wild-type and K6W-Ub-expressing mouse lenses. Lens proteins were digested with trypsin, peptides were separated using strong cation exchange and reversed-phase liquid chromatography, and tandem mass (MS/MS) spectra were collected with a linear ion trap. Transgenic mice that expressed low levels of K6W-Ub (low expressers) had normal, clear lenses at birth, whereas the lenses that expressed high levels of K6W-Ub (higher expressers) had abnormal lenses and cataracts at birth. A total of 2052 proteins were identified, of which 996 were reliably quantified and compared between wild-type and K6W-Ub transgenic mice. Consistent with a delayed developmental program, fiber-cell-specific proteins, such as γ-crystallins (γA, γB, γC, and γE), were down-regulated in K6W-Ub higher expressers. Up-regulated proteins were involved in energy metabolism, signal transduction, and proteolysis. The K6W-Ub low expressers exhibited delayed onset and milder cataract consistent with smaller changes in protein expression. Because lens protein expression changes occurred prior to lens morphological abnormalities and cataract formation in K6W-Ub low expressers, it appears that expression of K6W-Ub sets in motion a process of altered protein expression that results in developmental defects and cataract.