Rural-urban and racial/ethnic trends and disparities in early-onset and average-onset colorectal cancer.

BACKGROUND: Incidence rates (IRs) of early-onset colorectal cancer (EOCRC) are increasing, whereas average-onset colorectal cancer (AOCRC) rates are decreasing. However, rural-urban and racial/ethnic differences in trends by age have not been explored. The objective of this study was to examine joint rural-urban and racial/ethnic trends and disparities in EOCRC and AOCRC IRs. METHODS: Surveillance, Epidemiology, and End Results data on the incidence of EOCRC (age, 20-49 years) and AOCRC (age, ≥50 years) were analyzed. Annual percent changes (APCs) in trends between 2000 and 2016 were calculated jointly by rurality and race/ethnicity. IRs and rate ratios were calculated for 2012-2016 by rurality, race/ethnicity, sex, and subsite. RESULTS: EOCRC IRs increased 35% from 10.44 to 14.09 per 100,000 in rural populations (APC, 2.09; P < .05) and nearly 20% from 9.37 to 11.20 per 100,000 in urban populations (APC, 1.26; P < .05). AOCRC rates decreased among both rural and urban populations, but the magnitude of improvement was greater in urban populations. EOCRC increased among non-Hispanic White (NHW) populations, although rural non-Hispanic Black (NHB) trends were stable. Between 2012 and 2016, EOCRC IRs were higher among all rural populations in comparison with urban populations, including NHW, NHB, and American Indian/Alaska Native populations. By sex, rural NHB women had the highest EOCRC IRs across subgroup comparisons, and this was driven primarily by colon cancer IRs 62% higher than those of their urban peers. CONCLUSIONS: EOCRC IRs increased in rural and urban populations, but the increase was greater in rural populations. NHB and American Indian/Alaska Native populations had particularly notable rural-urban disparities. Future research should examine the etiology of these trends.

Drug screening assay based on the interaction of intact Keap1 and Nrf2 proteins in cancer cells.

BACKGROUND: The Nrf2-Keap1 interaction is the major regulatory pathway for cytoprotective responses against oxidative and electrophilic stresses. Keap1, a substrate protein of a Cul3-dependent E3 ubiquitin ligase complex, is a negative regulator of Nrf2. The use of chemicals to regulate the interaction between Keap1 and Nrf2 has been proposed as a strategy for the chemoprevention of degenerative diseases and cancers. RESULTS: The interactions between Keap1 and Nrf2 in vitro and in vivo were investigated using fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) strategies in our study. Nrf2 with its N-terminal fused to eGFP and Keap1 with its C-terminal fused to mCherry were expressed and purified in vitro. When purified eGFP-Nrf2 and Keap1-mChrry proteins were mixed together, a strong FRET signal could be detected, indicating an efficient energy transfer from eGFP to mCherry. Moreover, the FRET was detected in vivo using confocal microscopy in colon cancer HCT-116 cells that were co-transfected with eGFP-Nrf2 and Keap1-mCherry. Finally, using an eGFP BiFC approach, the Keap1-Nrf2 interaction was also detected in MCF7 cells by transfecting eGFP N-terminal fused to Nrf2 (eN158-Nrf2) and eGFP C-terminal fused to Keap1 (eC159-Keap1). Using the BiFC and FRET systems, we demonstrated that the prototypical Nrf2-activiting compound tBHQ and the antitumor drug F-dUrd might interfere with the intracellular interaction between Keap1 and Nrf2 whereas the 5-Fu have little role in activating the protective response of Nrf2 pathway in cancer cells. CONCLUSIONS: By analyzing the perturbation of the energy transfer between the donor and acceptor fluorophores and the bimolecular fluorescence complementation of eGFP, we can screen potential inhibitors for the interaction between Keap1 and Nrf2.

The ATM kinase induces microRNA biogenesis in the DNA damage response.

The DNA damage response involves a complex network of processes that detect and repair DNA damage. Here we show that miRNA biogenesis is globally induced upon DNA damage in an ATM-dependent manner. About one-fourth of miRNAs are significantly upregulated after DNA damage, while loss of ATM abolishes their induction. KH-type splicing regulatory protein (KSRP) is a key player that translates DNA damage signaling to miRNA biogenesis. The ATM kinase directly binds to and phosphorylates KSRP, leading to enhanced interaction between KSRP and pri-miRNAs and increased KSRP activity in miRNA processing. Mutations of the ATM phosphorylation sites of KSRP impaired its activity in regulating miRNAs. These findings reveal a mechanism by which DNA damage signaling is linked to miRNA biogenesis.

A statewide program providing colorectal cancer screening to the uninsured of South Carolina.

BACKGROUND: Cancer screening rates are lowest in those without insurance or a regular provider. Since 2008, the Colorectal Cancer Prevention Network (CCPN) has provided open access colonoscopy to uninsured residents of South Carolina through established, statewide partnerships and patient navigation. Herein, we describe the structure, implementation, and clinical outcomes of this program. METHODS: The CCPN provides access to colonoscopy screening at no cost to uninsured, asymptomatic patients aged 50-64 years (African Americans age 45-64 years are eligible) who live at or below 150% of the poverty line and seek medical care in free medical clinics, federally qualified health centers, or hospital-based indigent practices in South Carolina. Screening is performed by board-certified gastroenterologists. Descriptive statistics and regression analysis are used to describe the population screened, and to assess compliance rates and colonoscopy quality metrics. RESULTS: Out of >4000 patients referred to the program, 1854 were deemed eligible, 1144 attended an in-person navigation visit, and 1030 completed a colonoscopy; 909 were included in the final sample. Nearly 90% of participants exhibited good-to-excellent bowel preparation. An overall cecal intubation rate of 99% was measured. The polyp detection rate and adenoma detection rate were 63% and 36%, respectively, with male sex and urban residence positively associated with adenoma detection. Over 13% of participants had an advanced polyp, and 1% had a cancer diagnosis or surgical intervention. CONCLUSION: The CCPN program is characterized by strong collaboration with clinicians statewide, low no-show rates, and high colonoscopy quality. Future work will assess the effectiveness of the navigation approach and will explore the mechanisms driving higher adenoma detection in urban participants. Cancer 2018;124:1912-20. © 2018 American Cancer Society.

Functional characterization of the mouse Serpina1 paralog DOM-7.

The generation of authentic mouse-models for human α1-antitrypsin (A1AT)-deficiency is difficult due to the high complexity of the mouse Serpina1 gene locus. Depending on the exact mouse strain, three to five paralogs are expressed, with different proteinase inhibitory properties. Nowadays with CRISPR-technology, genome editing of complex genomic loci is feasible and could be employed for the generation of A1AT-deficiency mouse models. In preparation of a CRISPR/Cas9-based genome-engineering approach we identified cDNA clones with a functional CDS for the Serpina1-paralog DOM-7. Here, we show that DOM-7 functionally inhibits neutrophil elastase (ELANE) and chymotrypsin, and therefore needs to be considered when aiming at the generation of A1AT-deficient models.

Formative Research on Knowledge and Preferences for Stool-based Tests compared to Colonoscopy: What Patients and Providers Think.

The rates of colorectal cancer (CRC) screening in the U.S. remain below national targets, so many people at risk are not being screened. The objective of this qualitative research project was to assess patient and provider knowledge and preferences about CRC screening modalities and specifically the use of the fecal immunochemical test (FIT) as a first line screening choice. Nine focus groups were conducted with a medically underserved patient population and qualitative interviews were administered to their medical providers. Thematic analysis was used to synthesize key findings. Both providers and patients thought that the FIT would be a good option for CRC screening both as an individual choice and for an overall program approach. The test is less expensive and therefore more readily available for patients compared to colonoscopy. Overall, there was consensus that the FIT offers a reasonably priced, simple approach to CRC screening which has broad appeal to both providers and patients. Concerns identified by patients and providers included the possibility of false positives with the FIT which could be caused by test contamination or failing to perform the test properly. Patients also described feelings of disgust toward performing the FIT and difficulties in following the instructions. Study findings indicate provider and patient support for using the FIT for CRC screening at both the individual and system-wide levels of implementation. While barriers to the use of the FIT were listed, benefits of using the FIT were perceived as positive motivators to engage previously unscreened and uninsured or under-insured individuals in CRC screening.

Correction to: Formative Research on Knowledge and Preferences for Stool-based Tests compared to Colonoscopy: What Patients and Providers Think.

The original version of this article unfortunately contained a mistake. There is a typo in the coauthor name, it should be Franklin G. Berger.

Race and Prevalence of Large Bowel Polyps Among the Low-Income and Uninsured in South Carolina.

BACKGROUND: Compared to whites, blacks have higher colorectal cancer incidence and mortality rates and are at greater risk for early-onset disease. The reasons for this racial disparity are poorly understood, but one contributing factor could be differences in access to high-quality screening and medical care. AIMS: The present study was carried out to assess whether a racial difference in prevalence of large bowel polyps persists within a poor and uninsured population (n = 233, 124 blacks, 91 whites, 18 other) undergoing screening colonoscopy. METHODS: Eligible patients were uninsured, asymptomatic, had no personal history of colorectal neoplasia, and were between the ages 45-64 years (blacks) or 50-64 years (whites, other). We examined the prevalence of any adenoma (conventional, serrated) and then difference in adenoma/polyp type by race and age categories. RESULTS: Prevalence for ≥1 adenoma was 37 % (95 % CI 31-43 %) for all races combined and 36 % in blacks <50 years, 38 % in blacks ≥50 years, and 35 % in whites. When stratified by race, blacks had a higher prevalence of large conventional proximal neoplasia (8 %) compared to whites (2 %) (p value = 0.06) but a lower prevalence of any serrated-like (blacks 18 %, whites 32 %; p value = 0.02) and sessile serrated adenomas/polyps (blacks 2 %, whites 8 % Chi-square p value; p = 0.05). CONCLUSIONS: Within this uninsured population, the overall prevalence of adenomas was high and nearly equal by race, but the racial differences observed between serrated and conventional polyp types emphasize the importance of taking polyp type into account in future research on this topic.

Oxidative Stress and Response to Thymidylate Synthase-Targeted Antimetabolites.

Thymidylate synthase (TYMS; EC 2.1.1.15) catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) by N(5),N(10)-methyhlenetetrahydrofolate, forming dTMP for the maintenance of DNA replication and repair. Inhibitors of TYMS have been widely used in the treatment of neoplastic disease. A number of fluoropyrimidine and folate analogs have been developed that lead to inhibition of the enzyme, resulting in dTMP deficiency and cell death. In the current study, we have examined the role of oxidative stress in response to TYMS inhibitors. We observed that intracellular reactive oxygen species (ROS) concentrations are induced by these inhibitors and promote apoptosis. Activation of the enzyme NADPH oxidase (NOX), which catalyzes one-electron reduction of O2 to generate superoxide (O2 (●-)), is a significant source of increased ROS levels in drug-treated cells. However, gene expression profiling revealed a number of other redox-related genes that may contribute to ROS generation. TYMS inhibitors also induce a protective response, including activation of the transcription factor nuclear factor E2-related factor 2 (NRF2), a critical mediator of defense against oxidative and electrophilic stress. Our results show that exposure to TYMS inhibitors induces oxidative stress that leads to cell death, while simultaneously generating a protective response that may underlie resistance against such death.

USP4 inhibits p53 through deubiquitinating and stabilizing ARF-BP1.

Tumour suppressor p53 levels in the cell are tightly regulated by controlled degradation through ubiquitin ligases including Mdm2, COP1, Pirh2, and ARF-BP1. The ubiquitination process is reversible via deubiquitinating enzymes, such as ubiquitin-specific peptidases (USPs). In this study, we identified ubiquitin-specific peptidase 4 (USP4) as an important regulator of p53. USP4 interacts directly with and deubiquitinates ARF-BP1, leading to the stabilization of ARF-BP1 and subsequent reduction of p53 levels. Usp4 knockout mice are viable and developmentally normal, but showed enhanced apoptosis in thymus and spleen in response to ionizing radiation. Compared with wild-type mouse embryonic fibroblasts (MEFs), Usp4-/- MEFs exhibited retarded growth, premature cellular senescence, resistance to oncogenic transformation, and hyperactive DNA damage checkpoints, consistent with upregulated levels and activity of p53 in the absence of USP4. Finally, we showed that USP4 is overexpressed in several types of human cancer, suggesting that USP4 is a potential oncogene.

Racial disparities in advanced-stage colorectal cancer survival.

PURPOSE: African-Americans (AA) have a higher incidence of and lower survival from colorectal cancer (CRC) compared with European Americans (EA). In the present study, statewide, population-based data from South Carolina Central Cancer Registry are used to investigate the relationship between race and age on advanced-stage CRC survival. METHODS: The study population was comprised of 3,865 advanced pathologically documented colon and rectal adenocarcinoma cases diagnosed between 01 January 1996 and 31 December 2006: 2,673 (69 %) EA and 1,192 (31 %) AA. Kaplan-Meier methods were used to generate median survival time and corresponding 95 % confidence intervals (CI) by race, age, and gender. Factors associated with survival were evaluated by fitting Cox proportional hazards regression models to generate hazard ratios (HR) and 95 % CI. RESULTS: We observed a significant interaction between race and age on CRC survival (p = 0.04). Among younger patients (<50 years), AA race was associated with a 1.34 times (95 % CI 1.06-1.71) higher risk of death compared with EA. Among older patients, we observed a modest increase in risk of death among AA men compared with EA [HR 1.16 (95 % CI 1.01-1.32)] but no difference by race between women [HR 0.94 (95 % CI 0.82-1.08)]. Moreover, we observed that the disparity in survival has worsened over the past 15 years. CONCLUSIONS: Future studies that integrate clinical, molecular, and treatment-related data are needed for advancing understanding of the racial disparity in CRC survival, especially for those <50 years old.

Cytoplasmic mislocalization of overexpressed FOXF1 is associated with the malignancy and metastasis of colorectal adenocarcinomas.

Our previous studies have revealed that the human FOXF1 gene, encoding a transcription factor member of the forkhead box (FOX) family, functions as a tumor suppressor and its expression is frequently silenced in breast cancer via DNA hypermethylation. Moreover, we recently reported that FOXF1 expression is preferentially silenced in colorectal cancer cell lines with inactive p53 and knockdown of FOXF1 caused genomic instability in FOXF1-expressing colorectal cancer cells with a defect in the p53-p21(WAF1) checkpoint, suggesting that FOXF1 plays a key role in colorectal tumorigenesis. Given that the in vivo role of FOXF1 in colorectal cancer remains unknown, the study here was aimed at delineating the clinical relevance of FOXF1 in colorectal adenocarcinomas. To characterize FOXF1 protein expression in colorectal cancer, designed tissue microarrays, comprising 50 cases of primary colorectal adenocarcinoma paired with matched adjacent normal tissue, were utilized in the immunohistochemistry (IHC) study. The IHC results showed that for adjacent normal colorectal tissue, the FOXF1 protein was only detected in stroma, not in epithelium, with either cytoplasmic staining (70% of total cases) or a mix of cytoplasmic and nuclear staining (6%). In contrast, for colorectal adenocarcinomas, FOXF1 staining was predominately identified in the cytoplasm of tumor epithelial cells (40% of total cases) and tumor-associated stromal cells of some cases (10%) also exhibited FOXF1 positivity in their cytoplasm. Cytoplasmic FOXF1 protein expression in tumor epithelial cells positively correlated with the histologic grade, depth of invasion, stage and lymphatic metastasis of colorectal adenocarcinomas (p<0.05). Moreover, in silico meta-analysis of Oncomine's cancer microarray database indicates that FOXF1 mRNA is overexpressed in a significant subset of colorectal adenocarcinoma tumors compared with normal colorectal tissue and other types of cancers. Our findings for the first time have revealed that the FOXF1 protein is overexpressed as well as mislocalized in cancerous epithelial cells and underexpressed/lost in tumor-associated stromal fibroblasts of colorectal adenocarcinomas, and suggest that FOXF1 is a potential prognostic marker due to its association with the malignancy and metastasis of colorectal cancer.

Cooperation between an intrinsically disordered region and a helical segment is required for ubiquitin-independent degradation by the proteasome.

The 26 S proteasomal complex, which is responsible for the bulk of protein degradation within the cell, recognizes its target substrates via covalently linked polyubiquitin moieties. However, a small but growing number of proteasomal substrates are degraded without a requirement for ubiquitinylation. One such substrate is the pyrimidine biosynthetic enzyme thymidylate synthase (EC 2.1.1.45), which catalyzes the synthesis of TMP and is the sole de novo source of TTP for DNA replication and repair. Previous work showed that intracellular proteolysis of human thymidylate synthase is directed by a degron at the polypeptide's N-terminal end, composed of an intrinsically disordered region (IDR) followed by a highly conserved amphipathic α-helix (hA). In the present report, we show that the hA helix does not function simply as an extension or scaffold for the IDR; rather, it provides a specific structural component that is necessary for degradation. Furthermore, its helical conformation is required for this function. We demonstrate that small domains from heterologous proteins can substitute for the IDR and the hA helix of human thymidylate synthase, indicating that the degradation-promoting function of these regions is not sequence-specific. The results, in general, indicate that cooperation between intrinsically disordered domains and α-helical segments is required for ubiquitin-independent degradation by the proteasome. There appears to be little sequence constraint on the ability of these regions to function as degron constituents. Rather, it is the overall conformation (or lack thereof) that is critical.

Linking tumor-associated macrophages, inflammation, and intestinal tumorigenesis: role of MCP-1.

Tumor-associated macrophages are associated with poor prognosis in certain cancers. Monocyte chemoattractant protein 1 (MCP-1) is thought to be the most important chemokine for recruitment of macrophages to the tumor microenvironment. However, its role on tumorigenesis in a genetic mouse model of colon cancer has not been explored. We examined the role of MCP-1 on tumor-associated macrophages, inflammation, and intestinal tumorigenesis. Male Apc(Min/+), Apc(Min/+)/MCP-1(-/-) or wild-type mice were euthanized at 18 wk of age and intestines were analyzed for polyp burden, apoptosis, proliferation, ß-catenin, macrophage number and phenotype, markers for cytotoxic T lymphocytes and regulatory T cells, and inflammatory mediators. MCP-1 deficiency decreased overall polyp number by 20% and specifically large polyp number by 45% (P < 0.05). This was consistent with an increase in apoptotic cells (P < 0.05), but there was no change detected in proliferation or ß-catenin. MCP-1 deficiency decreased F4/80-positive cells in both the polyp tissue and surrounding intestinal tissue (P < 0.05) as well as expression of markers associated with M1 (IL-12 and IL-23) and M2 macrophages (IL-13, CD206, TGF-ß, and CCL17) (P < 0.05). MCP-1 knockout was also associated with increased cytotoxic T lymphocytes and decreased regulatory T cells (P < 0.05). In addition, MCP-1(-/-) offset the increased mRNA expression of IL-1ß and IL-6 in intestinal tissue and IL-1ß and TNF-α in polyp tissue (P < 0.05), and prevented the decrease in SOCS1 expression (P < 0.05). We demonstrate that MCP-1 is an important mediator of tumor growth and immune regulation that may serve as an important biomarker and/or therapeutic target in colon cancer.

Functional dissection of the N-terminal degron of human thymidylate synthase.

Human thymidylate synthase (hTS; EC 2.1.1.45) is one of a small group of proteasomal substrates whose intracellular degradation occurs in a ubiquitin-independent manner. Previous studies have shown that proteolytic breakdown of the hTS polypeptide is directed by an intrinsically disordered 27-residue domain at the N-terminal end of the molecule. This domain, in co-operation with an α-helix spanning amino acids 31-45, functions as a degron, in that it has the ability to destabilize a heterologous polypeptide to which it is attached. In the present study, we provide evidence indicating that it is the 26S isoform of the proteasome that is responsible for intracellular degradation of the hTS polypeptide. In addition, we have used targeted in vitro mutagenesis to show that an Arg-Arg motif at residues 10-11 is required for proteolysis, an observation that was confirmed by functional analysis of the TS N-terminus from other mammalian species. The effects of stabilizing mutations on hTS degradation are maintained when the enzyme is provided with an alternative means of proteasome association; thus such mutations perturb one or more post-docking steps in the degradation pathway. Surprisingly, deletion mutants missing large segments of the disordered domain still function as proteasomal substrates; however, degradation of such mutants occurs by a mechanism that is distinct from that for the wild-type protein. Taken together, our results provide information on the roles of specific subregions within the intrinsically disordered N-terminal domain of hTS in regulation of degradation, leading to a deeper understanding of mechanisms underlying the ubiquitin-independent proteasomal degradation pathway.

Hematopoietic derived cell infiltration of the intestinal tumor microenvironment in Apc Min/+ mice.

Tumors consist of a heterogeneous population of neoplastic cells infiltrated by an equally heterogeneous collection of nonneoplastic cells that comprise the tumor microenvironment. Tumor growth, invasion, and metastasis depend on multiple interactions between these cells. To assess their potential as therapeutic targets or vehicles for tumor specific delivery of therapeutic agents, we examined the contribution of bone marrow derived cells (BMDCs) to the intestinal tumor microenvironment. Hematopoietic stem cells expressing the enhanced green fluorescent protein (eGFP) were transplanted into lethally irradiated ApcMin/+ mice, and their engraftment was analyzed by confocal microscopy. The results showed abundant infiltration of eGFP cells into the small intestine, colon, and spleen compared to heart, muscle, liver, lung, and kidney. Within the intestine, there was a pronounced gradient of engraftment along the anterior to posterior axis, with enhanced infiltration into adenomas. Immunofluorescence analysis showed that osteopontin was expressed in tumor stromal cells but not in nontumor stromal populations, suggesting that gene expression in these cells is distinct. Tumor vasculature in ApcMin/+ mice was chaotic compared to normal intestinal regions. Our data suggest that BMDCs can be harnessed for tumor-targeted therapies to enhance antitumor efficacy.

Replacement of Val3 in human thymidylate synthase affects its kinetic properties and intracellular stability .

Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of approximately 27 amino acids that is not present in bacterial TSs. The extension, which is disordered in all reported crystal structures of TSs, has been considered to play a primary role in protein turnover but not in catalytic activity. In mammalian cells, the variant V3A has a half-life similar to that of wild-type human TS (wt hTS) while V3T is much more stable; V3L, V3F, and V3Y have half-lives approximately half of that for wt hTS. Catalytic turnover rates for most Val3 mutants are only slightly diminished, as expected. However, two mutants, V3L and V3F, have strongly compromised dUMP binding, with K(m,app) values increased by factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of the loop of residues 181-197, which prevents substrate binding. In the crystal structure of V3L, electron density corresponding to a leucine residue is present in a position that stabilizes the loop of residues 181-197 in the inactive conformation. Since this density is not observed in other mutants and all other leucine residues are ordered in this structure, it is likely that this density represents Leu3. In the crystal structure of a V3F.FdUMP binary complex, the nucleotide is bound in an alternative mode to that proposed for the catalytic complex, indicating that the high K(m,app) value is caused not by stabilization of the inactive conformer but by substrate binding in a nonproductive, inhibitory site. These observations show that the N-terminal extension affects the conformational state of the hTS catalytic region. Each of the mechanisms leading to the high K(m,app) values can be exploited to facilitate design of compounds acting as allosteric inhibitors of hTS.

Hemoglobin and its scavenger protein haptoglobin associate with apoA-1-containing particles and influence the inflammatory properties and function of high density lipoprotein.

Hemoglobin (Hb) uniquely associates with proinflammatory HDL in atherogenic mice and coronary heart disease (CHD) patients. In this paper, we report that Hb and its scavenger proteins, haptoglobin (Hp) and hemopexin (Hx) are significantly increased in apoA-1-containing particles of HDL both in mouse models of hyperlipidemia and in CHD patients, when compared with wild type mice and healthy donors, respectively. We further demonstrate that the association of Hb, Hp, and Hx proteins with HDL positively correlates with inflammatory properties of HDL and systemic inflammation in CHD patients. Interestingly, HDL from Hp(-/-) mice under atherogenic conditions does not accumulate Hb and is anti-inflammatory, suggesting that (i) Hp is required for the association of Hb with HDL and (ii) Hb x Hp complexes regulate the inflammatory properties of HDL. Moreover, treatment of apoE(-/-) mice with an apoA-1 mimetic peptide resulted in significant dissociation of Hb x Hp complexes from HDL and improvement of HDL inflammatory properties. Our data strongly suggest that HDL can become proinflammatory via the Hb x Hp pathway in mice and humans, and dissociation of Hb x Hp x Hx complexes from apoA-1-containing particles of HDL may be a novel target for the treatment of CHD.

The intrinsically disordered N-terminal domain of thymidylate synthase targets the enzyme to the ubiquitin-independent proteasomal degradation pathway.

The ubiquitin-independent proteasomal degradation pathway is increasingly being recognized as important in regulation of protein turnover in eukaryotic cells. One substrate of this pathway is the pyrimidine biosynthetic enzyme thymidylate synthase (TS; EC 2.1.1.45), which catalyzes the reductive methylation of dUMP to form dTMP and is essential for DNA replication during cell growth and proliferation. Previous work from our laboratory showed that degradation of TS is ubiquitin-independent and mediated by an intrinsically disordered 27-residue region at the N-terminal end of the molecule. In the current study we show that this region, in cooperation with an alpha-helix formed by the next 15 residues, functions as a degron, i.e. it is capable of destabilizing a heterologous protein to which it is fused. Comparative analysis of the primary sequence of TS from a number of mammalian species revealed that the N-terminal domain is hypervariable among species yet is conserved with regard to its disordered nature, its high Pro content, and the occurrence of Pro at the penultimate site. Characterization of mutant proteins showed that Pro-2 protects the N terminus against N(alpha)-acetylation, a post-translational process that inhibits TS degradation. However, although a free amino group at the N terminus is necessary, it is not sufficient for degradation of the polypeptide. The implications of these findings to the proteasome-targeting function of the N-terminal domain, particularly with regard to its intrinsic flexibility, are discussed.

Publisher Correction: Early-onset colorectal cancer: initial clues and current views.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.