Proteome-wide mapping of short-lived proteins in human cells.

Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.

PKCλ/ι inhibition activates an ULK2-mediated interferon response to repress tumorigenesis.

The interferon (IFN) pathway is critical for cytotoxic T cell activation, which is central to tumor immunosurveillance and successful immunotherapy. We demonstrate here that PKCλ/ι inactivation results in the hyper-stimulation of the IFN cascade and the enhanced recruitment of CD8+ T cells that impaired the growth of intestinal tumors. PKCλ/ι directly phosphorylates and represses the activity of ULK2, promoting its degradation through an endosomal microautophagy-driven ubiquitin-dependent mechanism. Loss of PKCλ/ι results in increased levels of enzymatically active ULK2, which, by direct phosphorylation, activates TBK1 to foster the activation of the STING-mediated IFN response. PKCλ/ι inactivation also triggers autophagy, which prevents STING degradation by chaperone-mediated autophagy. Thus, PKCλ/ι is a hub regulating the IFN pathway and three autophagic mechanisms that serve to maintain its homeostatic control. Importantly, single-cell multiplex imaging and bioinformatics analysis demonstrated that low PKCλ/ι levels correlate with enhanced IFN signaling and good prognosis in colorectal cancer patients.

Natural products targeting the elongation phase of eukaryotic protein biosynthesis.

Covering: 2000 to 2020 The translation of mRNA into proteins is a precisely regulated, complex process that can be divided into three main stages, i.e. initiation, elongation, termination, and recycling. This contribution is intended to highlight how natural products interfere with the elongation phase of eukaryotic protein biosynthesis. Cycloheximide, isolated from Streptomyces griseus, has long been the prototype inhibitor of eukaryotic translation elongation. In the last three decades, a variety of natural products from different origins were discovered to also address the elongation step in different manners, including interference with the elongation factors eEF1 and eEF2 as well as binding to A-, P- or E-sites of the ribosome itself. Recent advances in the crystallization of the ribosomal machinery together with natural product inhibitors allowed characterizing similarities as well as differences in their mode of action. Since aberrations in protein synthesis are commonly observed in tumors, and malfunction or overexpression of translation factors can cause cellular transformation, the protein synthesis machinery has been realized as an attractive target for anticancer drugs. The therapeutic use of the first natural products that reached market approval, plitidepsin (Aplidin®) and homoharringtonine (Synribo®), will be introduced. In addition, we will highlight two other potential indications for translation elongation inhibitors, i.e. viral infections and genetic disorders caused by premature termination of translation.

Versatile Synthetic Route to Cycloheximide and Analogues That Potently Inhibit Translation Elongation.

Cycloheximide (CHX) is an inhibitor of eukaryotic translation elongation that has played an essential role in the study of protein synthesis. Despite its ubiquity, few studies have been directed towards accessing synthetic CHX derivatives, even though such efforts may lead to protein synthesis inhibitors with improved or alternate properties. Described here is the total synthesis of CHX and analogues, and the establishment of structure-activity relationships (SAR) responsible for translation inhibition. The SAR studies aided the design of more potent compounds, one of which irreversibly blocks ribosomal elongation, preserves polysome profiles, and may be a broadly useful tool for investigating protein synthesis.

A Nonpump Function of Sodium Iodide Symporter in Thyroid Cancer via Cross-talk with PTEN Signaling.

The sodium iodide symporter (NIS) is a classical iodide pump typically localized within the cell plasma membrane in thyroid cells, where NIS expression is believed to ensure success of mainstay radioiodide therapy in thyroid cancers. Although radioiodide uptake is generally reduced in thyroid cancer tissue, intracellular nonmembranous NIS has been reported to increase, suggesting that NIS serves a pump-independent function. Thyroid cancer is one of the major component cancers of Cowden syndrome, a subset of which is caused by germline mutations in PTEN In this study, we explored the noncanonical tumorigenic role of NIS in thyroid cancer cells in relation to PTEN signaling. PTEN knockdown in thyroid cancer cell lines stabilized intracellular NIS protein by promoting an interaction with NIS-LARG (leukemia-associated RhoA guanine exchange factor). Increased protein levels of cytoplasmic NIS enhanced RhoA activation and resulted in a promigration tumorigenic phenotype. Inhibition of NIS glycosylation through activation of the PI3K/AKT/mTOR signaling pathway contributed to mislocalization of NIS in the cytoplasm, facilitating its nonpump tumorigenic function through an interaction with LARG, which predominantly localized in the cytoplasm. Moreover, PTEN or PI3K/AKT/mTOR signaling could affect DPAGT1, a glycosylating enzyme involved in the initial step of N-linked glycosylation, to inhibit glycosylation of NIS. In summary, our results elucidate a pump-independent, protumorigenic role for NIS in thyroid cancer via its cross-talk with PTEN signaling.Significance: A novel pump-independent protumorigenic role of nonmembranous NIS challenges the presumption that radioiodine treatment of thyroid cancer is ineffective when transmembrane NIS is not expressed. Cancer Res; 78(21); 6121-33. ©2018 AACR.

USP15-dependent lysosomal pathway controls p53-R175H turnover in ovarian cancer cells.

Gain-of-function p53 mutants such as p53-R175H form stable aggregates that accumulate in cells and play important roles in cancer progression. Selective degradation of gain-of-function p53 mutants has emerged as a highly attractive therapeutic strategy to target cancer cells harboring specific p53 mutations. We identified a small molecule called MCB-613 to cause rapid ubiquitination, nuclear export, and degradation of p53-R175H through a lysosome-mediated pathway, leading to catastrophic cancer cell death. In contrast to its effect on the p53-R175H mutant, MCB-613 causes slight stabilization of p53-WT and has weaker effects on other p53 gain-of-function mutants. Using state-of-the-art genetic and chemical approaches, we identified the deubiquitinase USP15 as the mediator of MCB-613's effect on p53-R175H, and established USP15 as a selective upstream regulator of p53-R175H in ovarian cancer cells. These results confirm that distinct pathways regulate the turnover of p53-WT and the different p53 mutants and open new opportunities to selectively target them.

Signaling pathway for phagocyte priming upon encounter with apoptotic cells.

The phagocytic elimination of cells undergoing apoptosis is an evolutionarily conserved innate immune mechanism for eliminating unnecessary cells. Previous studies showed an increase in the level of engulfment receptors in phagocytes after the phagocytosis of apoptotic cells, which leads to the enhancement of their phagocytic activity. However, precise mechanisms underlying this phenomenon require further clarification. We found that the pre-incubation of a Drosophila phagocyte cell line with the fragments of apoptotic cells enhanced the subsequent phagocytosis of apoptotic cells, accompanied by an augmented expression of the engulfment receptors Draper and integrin αPS3. The DNA-binding activity of the transcription repressor Tailless was transiently raised in those phagocytes, depending on two partially overlapping signal-transduction pathways for the induction of phagocytosis as well as the occurrence of engulfment. The RNAi knockdown of tailless in phagocytes abrogated the enhancement of both phagocytosis and engulfment receptor expression. Furthermore, the hemocyte-specific RNAi of tailless reduced apoptotic cell clearance in Drosophila embryos. Taken together, we propose the following mechanism for the activation of Drosophila phagocytes after an encounter with apoptotic cells: two partially overlapping signal-transduction pathways for phagocytosis are initiated; transcription repressor Tailless is activated; expression of engulfment receptors is stimulated; and phagocytic activity is enhanced. This phenomenon most likely ensures the phagocytic elimination of apoptotic cells by stimulated phagocytes and is thus considered as a mechanism to prime phagocytes in innate immunity.

Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G0/G1 Arrest by Inhibiting Deubiquitinase USP2a.

USP2a is a deubiquitinase responsible for stabilization of cyclin D1, a crucial regulator of cell-cycle progression and a proto-oncoprotein overexpressed in numerous cancer types. Here we report that lithocholic acid (LCA) derivatives are inhibitors of USP proteins, including USP2a. The most potent LCA derivative, LCA hydroxyamide (LCAHA), inhibits USP2a, leading to a significant Akt/GSK3ß-independent destabilization of cyclin D1, but does not change the expression of p27. This leads to the defects in cell-cycle progression. As a result, LCAHA inhibits the growth of cyclin D1-expressing, but not cyclin D1-negative cells, independently of the p53 status. We show that LCA derivatives may be considered as future therapeutics for the treatment of cyclin D1-addicted p53-expressing and p53-defective cancer types.

Vitamin D receptor is a novel transcriptional regulator for Axin1.

BACKGROUND: Axin1 is a scaffold protein in the ß-catenin destruction complex, which, if disrupted, contributes to pathogenesis of various human diseases, including colorectal carcinogenesis and inflammatory bowel diseases (IBD). We have previously demonstrated that Salmonella infection promotes the degradation and plasma sequestration of Axin1, leading to bacterial invasiveness and inflammatory responses. Vitamin D and the vitamin D receptor (VDR) appear to be important regulators of IBD and colon cancer. Although VDR and Axin1 are all involved in intestinal inflammation, it remains unclear whether these processes are related or function independently. In the current study, we hypothesize that VDR is an important regulator for the maintenance of physiological level of Axin1. METHODS: Using the intestinal epithelial conditional VDR knockout mouse model (VDRΔIEC) and cultured cell lines, influences of VDR status on the expression of Axin1 was evaluated by Western blots and real-time PCR. Loss- and gain-of-function assays were used to investigate the regulation of VDR on Axin1 at the transcriptional and translational levels. Cells were treated with cycloheximide or actinomycin for molecular mechanistic studies. Candidate genomic VDR binding sites for Axin1 were tested by chromatin immunoprecipitation (ChIP) assay. Physical interactions among VDR, Axin1, and ß-catenin were tested by immunoprecipitation. Cellular localization of Axin1 with different VDR status was determined by fractionation and immunohistochemistry. RESULTS: We found that VDR deletion led to lower protein and mRNA levels of Axin1, whereas knockdown of Axin1 did not change the expression level of VDR protein. Immunoprecipitation data did not support physical interaction between VDR and Axin1. The VDR regulation of Axin1 was through a VDR genomic binding site for Axin1 gene on the regulatory region. Fractionation data showed that cytosolic Axin1 was significantly reduced due to VDR deletion, leaving the nuclear fraction unchanged. In ileum, Axin1 was distributed in the cytosol of apical epithelium and crypts. CONCLUSION: VDR is important for the maintenance of physiological level of Axin1. The discovery of Axin1 as a VDR target gene provides novel and fundamental insights into the interactions between the VDR and ß-catenin signaling pathways.

Autophagy regulates spermatid differentiation via degradation of PDLIM1.

Spermiogenesis is a complex and highly ordered spermatid differentiation process that requires reorganization of cellular structures. We have previously found that Atg7 is required for acrosome biogenesis. Here, we show that autophagy regulates the round and elongating spermatids. Specifically, we found that Atg7 is required for spermatozoa flagella biogenesis and cytoplasm removal during spermiogenesis. Spermatozoa motility of atg7-null mice dropped significantly with some extra-cytoplasm retained on the mature sperm head. These defects are associated with an impairment of the cytoskeleton organization. Functional screening revealed that the negative cytoskeleton organization regulator, PDLIM1 (PDZ and LIM domain 1 [elfin]), needs to be degraded by the autophagy-lysosome-dependent pathway to facilitate the proper organization of the cytoskeleton. Our results thus provide a novel mechanism showing that autophagy regulates cytoskeleton organization mainly via degradation of PDLIM1 to facilitate the differentiation of spermatids.

Dexamethasone rapidly inhibits glucose uptake via non-genomic mechanisms in contracting myotubes.

Glucocorticoids (GCs) are a class of steroid hormones that regulate multiple aspects of glucose homeostasis. In skeletal muscle, it is well established that prolonged GC excess inhibits glucose uptake and utilization through glucocorticoid receptor (GR)-mediated transcriptional changes. However, it remains obscure that whether the rapid non-genomic effects of GC on glucose uptake are involved in acute exercise stress. Therefore, we used electric pulse stimulation (EPS)-evoked contracting myotubes to determine whether the non-genomic actions of GC were involved and its underlying mechanism(s). Pretreatment with dexamethasone (Dex, 10 µM) significantly prevented contraction-stimulated glucose uptake and glucose transporter 4 (Glut4) translocation within 20 min in C2C12 myotubes. Neither GC nuclear receptor antagonist (RU486) nor protein synthesis inhibitor (cycloheximide, Chx) affected the rapid inhibition effects of Dex. AMPK and CaMKII-dependent signaling pathways were associated with the non-genomic effects of Dex. These results provide evidence that GC rapidly suppresses glucose uptake in contracting myotubes via GR-independent non-genomic mechanisms. AMPK and CaMKII-mediated Glut4 translocation may play a critical role in GC-induced rapid inhibition of glucose uptake.

Information Transfer in Gonadotropin-releasing Hormone (GnRH) Signaling: EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK)-MEDIATED FEEDBACK LOOPS CONTROL HORMONE SENSING.

Cell signaling pathways are noisy communication channels, and statistical measures derived from information theory can be used to quantify the information they transfer. Here we use single cell signaling measures to calculate mutual information as a measure of information transfer via gonadotropin-releasing hormone (GnRH) receptors (GnRHR) to extracellular signal-regulated kinase (ERK) or nuclear factor of activated T-cells (NFAT). This revealed mutual information values <1 bit, implying that individual GnRH-responsive cells cannot unambiguously differentiate even two equally probable input concentrations. Addressing possible mechanisms for mitigation of information loss, we focused on the ERK pathway and developed a stochastic activation model incorporating negative feedback and constitutive activity. Model simulations revealed interplay between fast (min) and slow (min-h) negative feedback loops with maximal information transfer at intermediate feedback levels. Consistent with this, experiments revealed that reducing negative feedback (by expressing catalytically inactive ERK2) and increasing negative feedback (by Egr1-driven expression of dual-specificity phosphatase 5 (DUSP5)) both reduced information transfer from GnRHR to ERK. It was also reduced by blocking protein synthesis (to prevent GnRH from increasing DUSP expression) but did not differ for different GnRHRs that do or do not undergo rapid homologous desensitization. Thus, the first statistical measures of information transfer via these receptors reveals that individual cells are unreliable sensors of GnRH concentration and that this reliability is maximal at intermediate levels of ERK-mediated negative feedback but is not influenced by receptor desensitization.

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbß Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor.

Rev-erbα and Rev-erbß are heme-binding nuclear receptors (NR) that repress the transcription of genes involved in regulating metabolism, inflammation, and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erbα recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases the affinity of the ligand-binding domain of Rev-erb NRs for NCoR1 peptides. One explanation for this discrepancy is that the ligand-binding domain and NCoR1 peptides used for in vitro studies cannot replicate the key features of the full-length proteins used in cellular studies. However, the combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erbß (FLRev-erbß) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo- and heme-replete FLRev-erbß·DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erbß·NCoR1 complex. The interaction between FLRev-erbß and NCoR1 as well as Rev-erbß repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating the degradation of Rev-erbß in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10(-6) s(-1)) of heme dissociation from Rev-erbß rules out a prior proposal that Rev-erbß acts as an intracellular heme sensor.

Heme oxygenase-1 accelerates erastin-induced ferroptotic cell death.

The oncogenic RAS-selective lethal small molecule Erastin triggers a unique iron-dependent form of nonapoptotic cell death termed ferroptosis. Ferroptosis is dependent upon the production of intracellular iron-dependent reactive oxygen species (ROS), but not other metals. However, key regulators remain unknown. The heme oxygenase (HO) is a major intracellular source of iron. In this study, the role of heme oxygenase in Erastin-triggered ferroptotic cancer cell death has been investigated. Zinc protoporphyrin IX (ZnPP), a HO-1 inhibitor, prevented Erastin-triggered ferroptotic cancer cell death. Furthermore, Erastin induced the protein and mRNA levels of HO-1 in HT-1080 fibrosarcoma cells. HO-1+/+ and HO-1-/- fibroblast, HO-1 overexpression, and chycloheximide-treated experiments revealed that the expression of HO-1 has a decisive effects in Erastin-triggered cell death. Hemin and CO-releasing molecules (CORM) promote Erastin-induced ferroptotic cell death, not by biliverdin and bilirubin. In addition, hemin and CORM accelerate the HO-1 expression in the presence of Erastin and increase membranous lipid peroxidation. Thus, HO-1 is an essential enzyme for iron-dependent lipid peroxidation during ferroptotic cell death.

The TRAF-interacting protein (TRAIP) is a novel E2F target with peak expression in mitosis.

The TRAF-interacting protein (TRAIP) is an E3 ubiquitin ligase required for cell proliferation. TRAIP mRNA is downregulated in human keratinocytes after inhibition of the PI3K/AKT/mTOR signaling. Since E2F transcription factors are downstream of PI3K/AKT/mTOR we investigated whether they regulate TRAIP expression. E2F1 expression significantly increased the TRAIP mRNA level in HeLa cells. Reporter assays with the 1400 bp 5'-upstream promoter in HeLa cells and human keratinocytes showed that E2F1-, E2F2- and E2F4-induced upregulation of TRAIP expression is mediated by 168 bp upstream of the translation start site. Mutating the E2F binding site within this fragment reduced the E2F1- and E2F2-dependent promoter activities and protein-DNA complex formation in gel shift assays. Abundance of TRAIP mRNA and protein was regulated by the cell cycle with a peak in G2/M. Expression of GFP and TRAIP-GFP demonstrated that TRAIP-GFP protein has a lower steady-state concentration than GFP despite similar mRNA levels. Cycloheximide inhibition experiments indicated that the TRAIP protein has a half-life of around four hours. Therefore, the combination of cell cycle-dependent transcription of the TRAIP gene by E2F and rapid protein degradation leads to cell cycle-dependent expression with a maximum in G2/M. These findings suggest that TRAIP has important functions in mitosis and tumorigenesis.

ORMDL orosomucoid-like proteins are degraded by free-cholesterol-loading-induced autophagy.

Eukaryotic cells have evolved robust mechanisms to counter excess cholesterol including redistribution of lipids into different compartments and compensatory up-regulation of phospholipid biosynthesis. We demonstrate here that excess cellular cholesterol increased the activity of the endoplasmic reticulum (ER) enzyme serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme in sphingomyelin synthesis. This increased SPT activity was not due to altered levels of SPTLC1 or SPTLC2, the major subunits of SPT. Instead, cholesterol loading decreased the levels of ORMDL1, a negative regulator of SPT activity, due to its increased turnover. Several lines of evidence demonstrated that free-cholesterol-induced autophagy, which led to increased turnover of ORMDL1. Cholesterol loading induced ORMDL1 redistribution from the ER to cytoplasmic p62 positive autophagosomes. Coimmunoprecipitation analysis of cholesterol-loaded cells showed increased association between ORMDL1 and p62. The lysosomal inhibitor chloroquine or siRNA knockdown of Atg7 inhibited ORMDL1 degradation by cholesterol, whereas proteasome inhibitors showed no effect. ORMDL1 degradation was specific to free-cholesterol loading as autophagy induced by serum starvation or general ER stress did not lead to ORMDL1 degradation. ORMDL proteins are thus previously unidentified responders to excess cholesterol, exiting the ER to activate SPT and increase sphingomyelin biosynthesis, which may buffer excess cellular cholesterol.

Atypical ubiquitylation in yeast targets lysine-less Asi2 for proteasomal degradation.

Proteins are typically targeted for proteasomal degradation by the attachment of a polyubiquitin chain to ϵ-amino groups of lysine residues. Non-lysine ubiquitylation of proteasomal substrates has been considered an atypical and rare event limited to complex eukaryotes. Here we report that a fully functional lysine-less mutant of an inner nuclear membrane protein in yeast, Asi2, is polyubiquitylated and targeted for proteasomal degradation. Efficient degradation of lysine-free Asi2 requires E3-ligase Doa10 and E2 enzymes Ubc6 and Ubc7, components of the endoplasmic reticulum-associated degradation pathway. Together, our data suggest that non-lysine ubiquitylation may be more prevalent than currently considered.

Prevention of aberrant protein aggregation by anchoring the molecular chaperone αB-crystallin to the endoplasmic reticulum.

The chaperone αB-crystallin (αBC) is a member of the small heat shock protein family and its point or truncated mutants cause the muscular disorder α-crystallinopathy. The illness is histologically characterized by accumulation of protein aggregates in muscle cells. Expression of the myopathy-causing R120G mutant of αBC, harboring an arginine-to-glycine mutation at position 120, results in aggregate formation. We demonstrated that tethering αBC to the endoplasmic reticulum (ER) membrane represses the protein aggregation mediated by the R120G mutant. ER-anchored αBC decreased the amount of the R120G mutant through autophagic proteolysis. In contrast, knockdown of ATG5, an E3 ligase essential for autophagy, in ER-anchored αBC-transfected cells restored the quantity of the R120G mutant. In this context, aggregate formation was still suppressed, indicating that ER-anchored αBC profoundly constrains aggregation competency of the R120G mutant separately from downregulating the abundance of the mutant. We have proposed that protein aggregation is prevented by manipulation of the ER microenvironment with αBC, and have shed light on a novel aspect of the ER as a therapeutic target.

USP11 regulates p53 stability by deubiquitinating p53.

The p53 tumor suppressor protein coordinates the cellular responses to a broad range of cellular stresses, leading to DNA repair, cell cycle arrest or apoptosis. The stability of p53 is essential for its tumor suppressor function, which is tightly controlled by ubiquitin-dependent degradation primarily through its negative regulator murine double minute 2 (Mdm2). To better understand the regulation of p53, we tested the interaction between p53 and USP11 using co-immunoprecipitation. The results show that USP11, an ubiquitin-specific protease, forms specific complexes with p53 and stabilizes p53 by deubiquitinating it. Moreover, down-regulation of USP11 dramatically attenuated p53 induction in response to DNA damage stress. These findings reveal that USP11 is a novel regulator of p53, which is required for p53 activation in response to DNA damage.

Method-dependent variability in determination of prevalence of Campylobacter jejuni and Campylobacter coli in Canadian retail poultry.

Campylobacter is the most frequent cause of bacterial gastroenteritis in Canada, and the illness is commonly associated with poultry consumption. Whereas Canadian retail poultry is often contaminated with campylobacters, studies on the prevalence of this organism are inconsistent due to variability in sampling and microbiological methodology. To determine the current microbiological status of Canadian poultry, and to evaluate two commonly used microbiological methods, 348 raw poultry samples were collected at retail across Canada over a period of 3 years (2007 to 2010) and were analyzed for the presence of thermophilic Campylobacter species. The overall prevalence of Campylobacter spp. was found to be 42.8% by a combination of the two testing methods, with 33.9% of the samples positive for C. jejuni, 3.7% of the samples positive for C. coli, and 5.2% of the samples positive for both. Variability in Campylobacter spp. prevalence was observed in samples obtained from different regions across Canada and from poultry with or without skin, but this was not statistically significant. In co-contaminated samples, C. jejuni was preferentially recovered from Preston agar compared with mCCDA and Campy-Cefex agar, with an increase in recovery of C. coli on all selective media after 48 h of enrichment. A subset of 214 of the poultry rinses were analyzed by both Health Canada's standard method, MFLP-46 (enrichment in Park and Sanders broth), and a second method requiring enrichment in Bolton broth. Significantly more positive samples were obtained with the MFLP-46 method (40.6%) than with the alternate method (35.0%). This improved recovery with MFLP-46 may be due to the omission of cycloheximide from this method. These results demonstrate that determination of prevalence of Campylobacter spp. on poultry products may be significantly impacted by the choice of microbiological methods used. Canadian poultry continues to be a source of exposure to Campylobacter spp.