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PDF file - 856K, Supplementary Table 1, presenting Conditional logistic regression model relating the three biomarkers OGG1, MPG and APE1 to lung cancer. Supplementary Figure 1, presenting Association of DNA repair activity with lung cancer risk, including relative frequency plots for APE1 activity in 100 case patients and 99 matched controls subjects, relative frequency plots for the integrated DNA repair OMA score in 100 case patients and 99 matched controls subjects, and odds ratio calculated as a function of the percentiles of the distribution of the OMA score among the control subjects.

Severe congenital neutropenia (SCN) is a life-threatening marrow failure disorder, usually caused by heterozygous mutations in

The analysis of cells and tissue by bottom-up proteomics starts with lysis, followed by in-solution digestion. Lysis buffers commonly used include detergents and other reagents for achieving efficient protein solubility. However, these reagents are, for the most part, incompatible with downstream analytical instrumentation. One method for in-solution digestion and cleanup, termed suspension trapping (S-Trap), has been recently introduced. We present an evaluation of the compatibility of commonly used lysis buffers with S-Trap: SDS, urea, NP-40, RIPA, and SDS with DTT (SDT). We show that S-Trap is compatible with all of the tested buffers, with SDS and SDT performing the best. On the basis of these data, we anticipate that the method will transform experimental planning for mass-spectrometry-based proteomics, making it far more flexible and tolerable of various lysis buffers. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD011665.

Summary We developed radioactivity-based and fluorescence-based assays for the DNA repair enzyme APE1 and showed that its decreased activity is associated with increased lung cancer risk. This suggests that ‘bad DNA repair’ rather than ‘bad luck’ is involved in cancer etiology., The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case–control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655–727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751–834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3–3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that ‘bad DNA repair’, rather than ‘bad luck’, is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT.

Presented is a data set for benchmarking MS1-based label-free quantitative proteomics using a quadrupole orbitrap mass spectrometer. Escherichia coli digest was spiked into a HeLa digest in four different concentrations, simulating protein expression differences in a background of an unchanged complex proteome. The data set provides a unique opportunity to evaluate the proteomic platform (instrumentation and software) in its ability to perform MS1-intensity-based label-free quantification. We show that the presented combination of informatics and instrumentation produces high precision and quantification accuracy. The data were also used to compare different quantitative protein inference methods such as iBAQ and Hi-N. The data can also be used as a resource for development and optimization of proteomics informatics tools, thus the raw data have been deposited to ProteomeXchange with identifier PXD001385.

Summary Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.

The Nucleosome Remodeling and Deacytelase (NuRD) complex is a co-repressive complex involved in many pathological and physiological processes in the cell. Previous studies have identified one of its components, Mbd3, as a potent inhibitor for reprogramming of somatic cells to pluripotency. Following OSKM induction, early and partial depletion of Mbd3 protein followed by applying naïve ground-state pluripotency conditions, results in a highly efficient and near-deterministic generation of mouse iPS cells. Increasing evidence indicates that the NuRD complex assumes multiple mutually exclusive protein complexes, and it remains unclear whether the deterministic iPSC phenotype is the result of a specific NuRD sub complex. Since complete ablation of Mbd3 blocks somatic cell proliferation, here we aimed to identify alternative ways to block Mbd3-dependent NuRD activity by identifying additional functionally relevant components of the Mbd3/NuRD complex during early stages of reprogramming. We identified Gatad2a (also known as P66α), a relatively uncharacterized NuRD-specific subunit, whose complete deletion does not impact somatic cell proliferation, yet specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuit during both stem cell differentiation and reprogramming to pluripotency. Complete ablation of Gatad2a in somatic cells, but not Gatad2b, results in a deterministic naïve iPSC reprogramming where up to 100% of donor somatic cells successfully complete the process within 8 days. Genetic and biochemical analysis established a distinct sub-complex within the NuRD complex (Gatad2a-Chd4-Mbd3) as the functional and biochemical axis blocking reestablishment of murine naïve pluripotency. Disassembly of this axis by depletion of Gatad2a, results in resistance to conditions promoting exit of naïve pluripotency and delays differentiation. We further highlight context- and posttranslational dependent modifications of the NuRD complex affecting its interactions and assembly in different cell states. Collectively, our work unveils the distinct functionality, composition and interactions of Gatad2a-Chd4-Mbd3/NuRD subcomplex during the resolution and establishment of mouse naïve pluripotency.

Traditional "bottom-up" proteomic approaches use proteolytic digestion, LC-MS/MS, and database searching to elucidate peptide identities and their parent proteins. Protein sequences absent from the database cannot be identified, and even if present in the database, complete sequence coverage is rarely achieved even for the most abundant proteins in the sample. Thus, sequencing of unknown proteins such as antibodies or constituents of metaproteomes remains a challenging problem. To date, there is no available method for full-length protein sequencing, independent of a reference database, in high throughput. Here, we present Database-independent Protein Sequencing, a method for unambiguous, rapid, database-independent, full-length protein sequencing. The method is a novel combination of non-enzymatic, semi-random cleavage of the protein, LC-MS/MS analysis, peptide de novo sequencing, extraction of peptide tags, and their assembly into a consensus sequence using an algorithm named "Peptide Tag Assembler." As proof-of-concept, the method was applied to samples of three known proteins representing three size classes and to a previously un-sequenced, clinically relevant monoclonal antibody. Excluding leucine/isoleucine and glutamic acid/deamidated glutamine ambiguities, end-to-end full-length de novo sequencing was achieved with 99-100% accuracy for all benchmarking proteins and the antibody light chain. Accuracy of the sequenced antibody heavy chain, including the entire variable region, was also 100%, but there was a 23-residue gap in the constant region sequence.

DNA repair is a major mechanism for minimizing mutations and reducing cancer risk. Here, we present the development of reproducible and specific enzymatic assays for methylpurine DNA glycosylase (MPG) repairing the oxidative lesions 1,N6-ethenoadenine (εA) and hypoxanthine (Hx) in peripheral blood mononuclear cells protein extracts. Association of these DNA repair activities with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean MPG-εA in case patients was 15.8 units/μg protein (95% CI 15.3-16.3), significantly higher than in control subjects-15.1 (14.6-15.5), *P = 0.011. The adjusted odds ratio for lung cancer associated with a one SD increase in MPG-εA activity (2.48 units) was significantly bigger than 1 (OR = 1.6, 95% CI = 1.1-2.4; *P = 0.013). When activity of OGG1, a different DNA repair enzyme for oxidative damage, was included in the model, the estimated odds ratio/SD for a combined MPG-εA-OGG1 score was 2.6 (95% CI 1.6-4.2) *P = 0.0001, higher than the odds ratio for each single assay. The MPG enzyme activity assays described provide robust functional risk biomarkers, with increased MPG-εA activity being associated with increased lung cancer risk, similar to the behavior of MPG-Hx. This underscores the notion that imbalances in DNA repair, including high DNA repair, usually perceived as beneficial, can cause cancer risk. Such DNA repair risk biomarkers may be useful for risk assessment of lung cancer and perhaps other cancer types, and for early detection techniques such as low-dose CT.

Only a minority of smokers develop lung cancer, possibly due to genetic predisposition, including DNA repair deficiencies. To examine whether inter-individual variations in DNA repair activity of N-methylpurine DNA glycosylase (MPG) are associated with lung cancer, we conducted a blinded, population-based, case–control study with 100 lung cancer case patients and 100 matched control subjects and analyzed the data with conditional logistic regression. All statistical tests were two-sided. MPG enzyme activity in peripheral blood mononuclear cells from case patients was higher than in control subjects, results opposite that of 8-oxoguanine DNA glycosylase (OGG1) DNA repair enzyme activity. For lung cancer associated with one standard deviation increase in MPG activity, the adjusted odds ratio was 1.8 (95% confidence interval [CI] = 1.2 to 2.6; P = .006). A combined MPG and OGG1 activities score was more strongly associated with lung cancer risk than either activity alone, with an odds ratio of 2.3 (95% CI = 1.4 to 3.6; P < .001). These results form a basis for a future panel of risk biomarkers for lung cancer risk assessment and prevention.

While the role of reduced DNA repair in susceptibility to hereditary cancers is well established, its role in sporadic cancer is less understood. One of the reasons is the lack of specific DNA repair assays that are suitable for epidemiology studies. Here we describe the development of the OGG test, an epidemiology-grade enzymatic assay for the activity of the base excision repair enzyme 8-oxoguanine DNA glycosylase, in protein extracts prepared from human blood cells. The assay is robust and reproducible, with a coefficient of variation of 10%. Using the OGG test we determined OGG activity in 120 healthy individuals. Our results show an inter-individual variation of 2.8-fold in OGG activity, from 3.6 up to 10.1 units/μg protein, with a mean value of 7.2 units/μg protein. There was no significant difference in OGG activity between males and females, or between smokers and non-smokers. Interestingly, there was a gender-specific effect of age: OGG activity was slightly but significantly lower in males older than the age of 55 years compared to younger males, but not in females at the same age groups. Analysis of OGG1 mRNA by quantitative real-time RT-PCR showed a group trend of an increase in OGG enzymatic activity with increasing mRNA expression, but the correlation between activity and mRNA in individuals was poor, indicating the importance of factors other than mRNA expression. The OGG test described is expected to be useful in studying the role of 8-oxoguanine repair in cancer, as recently demonstrated for non-small cell lung cancer [T. Paz-Elizur, M. Krupsky, S. Blumenstein, D. Elinger, E. Schechtman, Z. Livneh, J. Natl. Cancer Inst. 95 (2003) 1312–1319]. In addition, it may serve as a paradigm for the development of additional functional DNA repair tests, which are needed in order to gain further insight into the role of DNA repair in cancer risk and pathology.

An increasing number of studies indicate that reduced DNA-repair capacity is associated with increased cancer risk. Using a functional assay for the removal of the oxidative DNA lesion 8-oxoguanine by the DNA-repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), we have previously shown that reduced OGG activity is a risk factor in lung cancer. Here, we report that OGG activity in peripheral blood mononuclear cells from 37 cases with squamous cell carcinoma of the head and neck (SCCHN) was significantly lower than in 93 control subjects, frequency matched for age and gender. Retesting of OGG activity 3 to 4 years after diagnosis and successful treatment of 18 individuals who recovered from the disease showed that OGG activity values were similar to those determined at diagnosis, suggesting that reduced OGG activity in case patients was not caused by the disease. Logistic regression analysis indicated that the adjusted odds ratio (OR) associated with a unit decrease in OGG activity was statistically significantly increased [OR, 2.3; 95% confidence interval (95% CI), 1.5–3.4]. Individuals in the lowest tertile of OGG activity exhibited an increased risk of SCCHN with an OR of 7.0 (95% CI, 2.0–24.5). The combination of smoking and low OGG was associated with a highly increased estimated relative risk for SCCHN. These results suggest that low OGG is associated with the risk of SCCHN, and if confirmed by additional epidemiologic studies, screening of smokers for low OGG activity might be used as a strategy for the prevention of lung cancer and SCCHN. (Cancer Res 2006; 66(24): 11683-9)

Peroxisomes are ubiquitous and dynamic organelles that house many important pathways of cellular metabolism. In recent years it has been demonstrated that mitochondria are tightly connected with peroxisomes and are defective in several peroxisomal diseases. Indeed, these two organelles share metabolic routes as well as resident proteins and, at least in mammals, are connected via a vesicular transport pathway. However the exact extent of cross-talk between peroxisomes and mitochondria remains unclear. Here we used a combination of high throughput genetic manipulations of yeast libraries alongside high content screens to systematically unravel proteins that affect the transport of peroxisomal proteins and peroxisome biogenesis. Follow up work on the effector proteins that were identified revealed that peroxisomes are not randomly distributed in cells but are rather localized to specific mitochondrial subdomains such as mitochondria–ER junctions and sites of acetyl-CoA synthesis. Our approach highlights the intricate geography of the cell and suggests an additional layer of organization as a possible way to enable efficient metabolism. Our findings pave the way for further studying the machinery aligning mitochondria and peroxisomes, the role of the juxtaposition, as well as its regulation during various metabolic conditions. More broadly, the approaches used here can be easily applied to study any organelle of choice, facilitating the discovery of new aspects in cell biology.

DNA repair is a prime mechanism for preventing DNA damage, mutation, and cancers. Adopting a functional approach, we examined the association with lung cancer risk of an integrated DNA repair score, measured by a panel of three enzymatic DNA repair activities in peripheral blood mononuclear cells. The panel included assays for AP endonuclease 1 (APE1), 8-oxoguanine DNA glycosylase (OGG1), and methylpurine DNA glycosylase (MPG), all of which repair oxidative DNA damage as part of the base excision repair pathways. A blinded population-based case–control study was conducted with 96 patients with lung cancer and 96 control subjects matched by gender, age (±1 year), place of residence, and ethnic group (Jews/non-Jews). The three DNA repair activities were measured, and an integrated DNA repair OMA (OGG1, MPG, and APE1) score was calculated for each individual. Conditional logistic regression analysis revealed that individuals in the lowest tertile of the integrated DNA repair OMA score had an increased risk of lung cancer compared with the highest tertile, with OR = 9.7; 95% confidence interval (CI), 3.1–29.8; P < 0.001, or OR = 5.6; 95% CI, 2.1–15.1; P < 0.001 after cross-validation. These results suggest that pending validation, this DNA repair panel of risk factors may be useful for lung cancer risk assessment, assisting prevention and referral to early detection by technologies such as low-dose computed tomography scanning. Cancer Prev Res; 7(4); 398–406. ©2013 AACR.

Efficient DNA repair mechanisms comprise a critical component in the protection against human cancer, as indicated by the high predisposition to cancer of individuals with germ-line mutations in DNA repair genes. This includes biallelic germ-line mutations in the MUTYH gene, encoding a DNA glycosylase that is involved in the repair of oxidative DNA damage, which strongly predispose humans to a rare hereditary form of colorectal cancer. Extensive research efforts including biochemical, enzymological and genetic studies in model organisms established that the oxidative DNA lesion 8-oxoguanine is mutagenic, and that several DNA repair mechanisms operate to prevent its potentially mutagenic and carcinogenic outcome. Epidemiological studies on the association with sporadic cancers of single nucleotide polymorphisms in genes such as OGG1, involved in the repair of 8-oxoguanine yielded conflicting results, and suggest a minor effect at best. A new approach based on the functional analysis of DNA repair enzymatic activity showed that reduced activity of 8-oxoguanine DNA glycosylase (OGG) is a risk factor in lung and head and neck cancer. Moreover, the combination of smoking and low OGG activity was associated with a higher risk, suggesting a potential strategy for risk assessment and prevention of lung cancer, as well as other types of cancer.

DNA is continuously damaged by environmental agents and by intracellular byproducts of metabolism [1,2]. If left unrepaired, this DNA damage will cause mutations due to miscoding during replication, thereby increasing cancer risk [1,3]. Therefore, DNA repair is expected to be a major mechanism that protects organisms against cancer. Indeed, in several hereditary diseases that cause high predisposition to cancer, the mutated genes associated with the disorder encode defective DNA repair proteins [3].

DNA repair has a major role in suppressing the rate of accumulation of mutations. Therefore, variations in DNA repair are likely to play an important role in determining cancer risk. While there is compelling evidence that defects in DNA repair cause high predisposition to several hereditary cancers, there is a paucity of data on the role of DNA repair in sporadic cancers. We present our approach of using functional DNA repair tests, rather than gene polymorphism, to study the potential of DNA repair enzymes to serve as biomarkers for lung cancer risk. We have previously developed a functional DNA repair blood test for the enzymatic repair of the oxidative DNA lesion 8-oxoguanine, and found that reduced OGG activity is a risk factor in non-small cell lung cancer. Moreover the combination of smoking and low OGG activity was associated with a greatly increased lung cancer risk (Paz-Elizur et al, JNCI 95 (2003) 1312-1319). The use of OGG activity as a potential biomarker for lung cancer risk is validated in collaboration with the M. D. Anderson Cancer Center, under the sponsorship of the Associate Members Program of the Early Detection Research Network (EDRN, NCI, NIH).

Cellular function is critically regulated through degradation of substrates by the proteasome. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows capture, isolation and analysis of proteasome-cleaved peptides. Application of MAPP to cancer cell lines as well as primary immune cells reveals dynamic modulation of the cellular degradome in response to various stimuli, such as pro-inflammatory signals. Further, we demonstrate analysis of minute amounts of clinical samples by studying cells from peripheral blood of patients with systemic lupus erythematosus (SLE). We find increased degradation of histones in patient immune cells, which suggests a role for aberrant proteasomal degradation in the pathophysiology of SLE. Taken together, MAPP offers a broadly applicable method to facilitate the study of the cellular degradation landscape in various cellular conditions and diseases involving changes in proteasomal degradation, including protein aggregation diseases, autoimmunity and cancer.

Background: Although smoking is a major cause of lung cancer, only a proportion of smokers develop lung cancer, suggesting a genetic predisposition in some individuals. Because tobacco smoking is associated with the increased formation of DNA lesions, including those induced from oxidative damage, we investigated whether the activity of the DNA repair enzyme 8-oxoguanine DNA N-glycosylase (OGG), which repairs the oxidative DNA lesion 8-oxoguanine, is associated with lung cancer. Methods: We conducted a molecular epidemiologic case–control study that included 68 case patients with non–small-cell lung cancer and 68 healthy control subjects, frequency matched for age and sex. Enzymatic OGG activity was determined in protein extracts prepared from peripheral blood mononuclear cells or lung tissue by assaying the cleavage product of a radiolabeled synthetic DNA oligonucleotide containing an 8-oxoguanine residue. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined by conditional logistic regression. All statistical tests were two-sided. Results: OGG activity was lower in peripheral blood mononuclear cells from case patients than in those from control subjects. After adjustment for age and smoking status, individuals in the lowest tertile of OGG activity had an increased risk of non–small-cell lung cancer compared with individuals in the highest tertile (OR = 4.8, 95% CI = 1.5 to 15.9). The adjusted OR associated with a unit decrease in OGG activity was statistically significantly increased (OR = 1.9, 95% CI = 1.3 to 2.8). There was no interaction between OGG activity and smoking status. The estimated relative risk of lung cancer for smokers with low OGG activity was 34- or 124-fold higher for smokers with a low OGG activity of 6.0 or 4.0 U/µg protein, respectively, than for nonsmokers with a normal OGG activity of 7.0 U/µg protein, illustrating the cumulative effect of low OGG activity and smoking. Conclusions: Low OGG activity is associated with an increased risk of lung cancer. Although prospective studies are needed to validate the results, they suggest that smoking cessation in individuals with reduced OGG activity might be an effective strategy in lung cancer prevention. [J Natl Cancer Inst 2003;95:1312–19]