Phosphatase Substrate Research Articles

Enzyme-Instructed Assembly and Disassembly Processes for Targeting Downregulation in Cancer Cells.

Cancer cells differ from normal cells in both gain of functions (i.e., upregulation) and loss of functions (i.e., downregulation). While it is common to suppress gain of function for chemotherapy, it remains challenging to target downregulation in cancer cells. Here we show the combination of enzyme-instructed assembly and disassembly to target downregulation in cancer cells by designing peptidic precursors as the substrates of both carboxylesterases (CESs) and alkaline phosphatases (ALPs). The precursors turn into self-assembling molecules to form nanofibrils upon dephosphorylation by ALP, but CES-catalyzed cleavage of the ester bond on the molecules results in disassembly of the nanofibrils. The precursors selectively inhibit the cancer cells that downregulate CES (e.g., OVSAHO) but are innocuous to a hepatocyte that overexpresses CES (HepG2), while the two cell lines exhibit comparable ALP activities. This work illustrates a potential approach for the development of chemotherapy via targeting downregulation (or loss of functions) in cancer cells.

Both Intrinsic Substrate Preference and Network Context Contribute to Substrate Selection of Classical Tyrosine Phosphatases

Reversible tyrosine phosphorylation is a widespread post-translational modification mechanism underlying cell physiology. Thus, understanding the mechanisms responsible for substrate selection by kinases and phosphatases is central to our ability to model signal transduction at a system level. Classical protein-tyrosine phosphatases can exhibit substrate specificity in vivo by combining intrinsic enzymatic specificity with the network of protein-protein interactions, which positions the enzymes in close proximity to their substrates. Here we use a high throughput approach, based on high density phosphopeptide chips, to determine the in vitro substrate preference of 16 members of the protein-tyrosine phosphatase family. This approach helped identify one residue in the substrate binding pocket of the phosphatase domain that confers specificity for phosphopeptides in a specific sequence context. We also present a Bayesian model that combines intrinsic enzymatic specificity and interaction information in the context of the human protein interaction network to infer new phosphatase substrates at the proteome level.

The Temporal Ordering of Cell-Cycle Phosphorylation

Cell-cycle phosphorylation is temporally ordered, at least in part, through the sequential expression of different cyclins. Recent studies by Swaffer etal. (2016) and Godfrey etal. (2017) show that intrinsic properties of the substrate proteins contribute as well: good kinase substrates tend to be phosphorylated early, and good phosphatase substrates tend to be phosphorylated late.

Enzyme-Instructed Self-Assembly of Peptides Containing Phosphoserine to Form Supramolecular Hydrogels as Potential Soft Biomaterials.

Enzyme-instructed self-assembly (EISA) offers a facile approach to explore the supramolecular assemblies of small molecules in cellular milieu for a variety of biomedical applications. One of the commonly used enzymes is phosphatase, but the study of the substrates of phosphatases mainly focuses on the phosphotyrosine containing peptides. In this work, we examine the EISA of phosphoserine containing small peptides for the first time by designing and synthesizing a series of precursors containing only phosphoserine or both phosphoserine and phosphotyrosine. Conjugating a phosphoserine to the C-terminal of a well-established self-assembling peptide backbone, (naphthalene-2-ly)-acetyl-diphenylalanine (NapFF), affords a novel hydrogelation precursor for EISA. The incorporation of phosphotyrosine, another substrate of phosphatase, into the resulting precursor, provides one more enzymatic trigger on a single molecule, and meanwhile increases the precursors' propensity to aggregate after being fully dephosphorylated. Exchanging the positions of phosphorylated serine and tyrosine in the peptide backbone provides insights on how the specific molecular structures influence self-assembling behaviors of small peptides and the subsequent cellular responses. Moreover, the utilization of D-amino acids largely enhances the biostability of the peptides, thus providing a unique soft material for potential biomedical applications.

LRRK2 Phosphorylation.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene were discovered in 2004 and have been found to be the most frequently mutated gene in Parkinson's disease. LRRK2 is a large multi-domain protein with a functional GTPase and kinase domain. The signal transduction pathways in which LRRK2 is dysfunctional in the disease state are only now being resolved, but we do know that LRRK2 is, itself, a substrate of multiple kinases and phosphatases and exists in variable phosphorylated states. Autophosphorylation of LRRK2 can impact GTPase and pathological outcomes. LRRK2 serines (910/935/955/973) are differentially phosphorylated in pathogenic PD mutations and after LRRK2 kinase inhibition. The phosphorylation status of LRRK2 can therefore provide key insight into the mechanisms of kinase dysfunction during disease. This chapter will describe the identification of LRRK2 phosphorylation sites and how phosphoregulation of LRRK2 reveals its own kinase activity and regulates its ubiquitination and localization in vitro, in cells, and in tissues.

Dual colorimetric and fluorogenic probes for visualizing tyrosine phosphatase activity.

Two resorufin-based substrates for protein tyrosine phosphatase (PTP) activity have been synthesized. These substrates provide sensitive fluorogenic readouts of PTP activity in vitro and in living cells at both acidic and neutral pH. In addition, the presence of the pathogenic bacteria Staphylococcus aureus was detected visually using a colorimetric readout.

Dual-color chromogen In Situ hybridization (CISH) for the determination of Her2 status in breast cancer tissue sections

Background: A factor associated with breast cancer development is the mutation of the Her2/neu gene located on chromosome 17. Chromogen in situ hybridization (CISH) in which the color is generated with an enzymatic reaction is a promising alternative detection method to fluorescence in situ hybridization (FISH) which is nowadays used in clinical practice. The resulting color is stable and therefore usable for permanent storage, the use of a normal brightfield microscope is possible and the morphology of the sample can be evaluated more easily than with a fluorescence microscope. For the visualization of Her2/neu it is however important to also detect chromosome 17 and use it as a reference chromosome. The aim of the study is: to select the best substrates considering color production, background staining, reactivity and localization. Secondly to develop a dualcolor procedure visualizing Her2/neu and chromosome 17 to examine the possibility of using CISH as a standardized method for the detection of Her2 positive tumors and finally to compare these results with FISH. Methods: Single target CISH with eight substrates for the enzyme horseradish peroxidase (PO) and six substrates for alkaline phosphatase (AP) was performed on cervical cancer cell lines with centromere 1 (C1) and 1p36 as targets. The best substrates were subjectively selected with the use of a brightfield microscope. The procedure was expanded to a dual-color detection on cells and formalin-fixed, paraffin embedded (FFPE) breast cancer tissue sections with Her2/neu and centromere 17 (C17) as targets. In this procedure the best conjugate detection systems were worked out. Results: In the single target detection procedure the substrates DAB, Vina Green and Seramun Grun for PO and Ferangi Blue and Vulcan Fast Red for AP were found to give the best color reactions. The dual-color detection on tissue sections showed strong and good distinguishable color reactions when combining the substrates Vina Green for C17 and Vulcan Fast Red for Her2. Results of CISH and FISH were comparable when using immersion oil for the detection of the fluorescent signals. Discussion and Conclusion: Vina Green for C17 and Vulcan Fast Red for Her2/neu are the best substrates considering background staining, color distinguishability and localization and give comparable results to FISH. The study gives a further step in the direction of introducing CISH as a standardized method for the detection of Her2/neu amplification.

Detection of Human Papilloma Virus by means of Chromogen in situ hybridization (CISH)

Introduction. The human papillomavirus (HPV) can cause infections in humans, although most women will be infected with HPV at some time in their life only a few cases will progress into invasive disease. Of all the HPV types only the ‘high-risk’ viruses are associated with cervical carcinomas. In order to prevent cervical cancer, multiple countries have implemented a cervical cancer screening programme. The detection mechanism in these screening programmes are commonly based on the Papanicolaou test, colposcopic analysis and PCR. However the sensitivity of such tests have several limitations. Another detection tool is HPV in situ hybridization either with fluorescence (FISH) or with an enzyme reaction (CISH). The use of FISH ensures high sensitivity with low endogenous background. Furthermore, this method will result in high resolution and the possibility to quantify the signal intensity. However, the fluorescent signal fades after light exposure, and samples might have auto-fluorescence. Thus in a setting where routine analysis has to be carried out the CISH procedure is more convenient, as only a standard bright-field microscope is needed for the detection procedure. The aim of this thesis is to determine if there are enzymatic substrates that have stronger reaction products in comparison to the control substrate DAB. Secondly this thesis aims to determine which conjugate detection system results in the best localisation and least background. Finally, the main aim is to investigate the possibilities of developing such an CISH that the end staining is comparable or better than the detection with FISH. Materials & Methods. Seven horseradish peroxidase substrates and six alkaline phosphatase substrates have been tested on CasKi cells with the centromere 1 and 1p36 probes as targets for the in situ hybridization reaction to determine the sensitivity and specificity of the substrates. The selected substrates were further tested with the HPV16 probe as target on SiHa cells and on formalin-fixed and paraffin-embedded (FFPE) tissue sections to determine the best conjugate detection system. The DAB DAKO substrate and the FISH with FITC were used as controls. The selected substrate in combination with the selected conjugate detection system was then tested on a series of eighteen clinical samples. Results. The horseradish peroxidase substrates Vina Green and Seramun Grün resulted in superior precipitates in comparison to the DAB DAKO control. These substrates show very high sensitivity as the spots visible for 1p36 were similar in size as the spots for 1C of the control staining. The substrates Vulcan Fast Red and Ferangi Blue are the selected substrates for the alkaline phosphatase enzyme reaction. Both these substrates have good localised sensitive reaction products with, respectively, clear red and blue precipitate products. The Av-PO system in combination with the Vina Green substrate lead to the best results in comparison to the fluorescent control. All clinical samples tested with the Av-PO system tested positive for HPV infection and had the same sensitivity and specificity as the fluorescent samples. Discussion and Conclussion. The Av-PO system results in the same specificity and sensitivity on clinical samples as the FISH. However, the ease of localizing the infected area when only the integrated pattern is present is much better with FISH. In conclusion we can say that the Vina Green substrate in combination with the Av-PO systems targeting HPV in clinical samples gives similar results as FISH. For this reason we developed a CISH method which could be used in the routine setting for histopathological diagnosis.

High throughput, quantitative analysis of human osteoclast differentiation and activity

Osteoclasts are multinuclear cells that degrade bone under both physiological and pathophysiological conditions. Osteoclasts are therefore a major target of osteoporosis therapeutics aimed at preserving bone. Consequently, analytical methods for osteoclast activity are useful for the development of novel biomarkers and/or pharmacological agents for the treatment of osteoporosis.The nucleation state of an osteoclast is indicative of its maturation and activity. To date, activity is routinely measured at the population level with only approximate consideration of the nucleation state (an ‘osteoclast population’ is typically defined as cells with ≥3 nuclei). Using a fluorescent substrate for tartrate-resistant acid phosphatase (TRAP), a routinely used marker of osteoclast activity, we developed a multi-labelled imaging method for quantitative measurement of osteoclast TRAP activity at the single cell level. Automated image analysis enables interrogation of large osteoclast populations in a high throughput manner using open source software.Using this methodology, we investigated the effects of receptor activator of nuclear factor kappa-B ligand (RANK-L) on osteoclast maturation and activity and demonstrated that TRAP activity directly correlates with osteoclast maturity (i.e. nuclei number). This method can be applied to high throughput screening of osteoclast-targeting compounds to determine changes in maturation and activity.

Hypophosphatasia: Review of Bone Mineral Metabolism, Pathophysiology, Clinical Presentation, Diagnosis, and Treatment

Hypophosphatasia (HPP) is a rare, inherited form of rickets or osteomalacia due to reduced activity of tissue non-specific alkaline phosphatase (TNSALP) caused by a loss of function mutation in the TNSALP gene marked by a low serum alkaline phosphatase. The ratio of PPi to Pi is crucial in the mineralization process. This process is regulated by the interaction of three phosphatases present in matrix vesicles: the mineralization promotors TNSALP and phosphatase orphan 1 (PHOSPHO1) and the mineralization inhibitor nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1). HPP is seen worldwide affecting all races. The broad-ranging clinical severity of HPP is correlated to the pattern of inheritance and degree of TNSALP activity, with lower levels of activity seen in the more severe autosomal recessive forms. HPP is marked by compromise of phase 2 hydroxyapatite crystal formation beyond the matrix vesicle resulting in skeletal hypomineralization, spontaneous fractures, tooth loss, and chondrocalcinosis. The major forms in declining order of severity include the following: perinatal, infantile, childhood, and adult HPP in addition to OdontoHPP and benign prenatal HPP. Biochemistry typically reveals a low serum alkaline phosphatase and elevated alkaline substrates such as pyridoxal-5′-phosphate (PLP) and phosphoethanolamine (PEA). Asfotase alfa, a recent novel enzyme replacement therapy for the treatment of HPP, has transformed this once lethal disease to one that is less severe.

BioSpotlight / Citations

BioTechniquesVol. 61, No. 5 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114469AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit An enriching experienceAccurate detection and quantification of low-abundance proteins by immunoblotting can be difficult since the amount of sample that can be loaded onto SDS-polyacrylamide gels is limited. However, methods to enrich for specific classes of proteins prior to gel electrophoresis can help overcome this problem. In this issue of BioTechniques, Azamat Karginov and Michael Agaphonov at the Russian Academy of Sciences describe a simple enrichment technique that greatly improves the immunoblotting of low-abundance yeast membrane proteins and is also superior to the standard method of boiling yeast cell lysates in Laemmli SDS gel-electrophoresis sample buffer. In the new approach, yeast cells are lysed in a buffer containing ammonium acetate, which protects proteins from degradation and facilitates membrane precipitation. The membrane fraction is obtained by a 10-minute centrifugation in a benchtop microcentrifuge and then resuspended in an alternative sample buffer containing SDS and a high concentration of urea, eliminating the boiling step that can cause protein degradation.See “A simple enrichment procedure improves detection of membrane proteins by immunoblotting” on page 260.Double-fish for frog embryosIn situ hybridization (ISH) for the simultaneous detection of two different transcripts in Xenopus embryos has been tricky to carry out using conventional chromogenic substrates of alkaline phosphatase (AP) since the darker signal of substrates such as nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/ BCIP) obscures the lighter signal from other substrates such as Vector Red. In addition, the opaque yolk platelets in the endodermal cells of Xenopus embryos interfere with signal detection. In this month's issue, Zachary Agricola and Sang-Wook Cha at Cincinnati Children's Hospital Medical Center describe their protocol for sequential double-fluorescence ISH (FISH) in Xenopus that resolves both of these issues. As substrates for AP development, they used Vector Red and NBT/BCIP, but these were visualized fluorescently by excitation with red and far-red light, respectively, which allowed the signal from each substrate to be detected without interference from the other. Coupled with treatment of the embryos by a clearing agent to render them transparent, this new approach enabled single-cell resolution of the expression of each target transcript.See “Two-color fluorescence in situ hybridization using chromogenic substrates in Xenopus” on page 263.Deciphering cytosine methylationThe epigenetic modifications methylcytosine (mC) and hydroxymethylcytosine (hmC) play important roles in modulating gene expression. Although techniques have emerged to identify cytosine methylation patterns, they are limited in resolution, which makes distinguishing these marks experimentally challenging for researchers. Now, Kawasaki et al. present a method to simultaneously identify cytosine, mC and hmC at single-base resolution. Their approach relies on the enzyme DNMT1, which methylates the unmethylated cytosine in a hemi-methylated CpG but not in a hemi-hydroxymethylated CpG. Using DNMT1 alongside standard bisulfite treatment, the authors were able to identify mC, hmC, and cytosine sites in single assay. They validated the new EnIGMA (Enzyme-assisted Identification of Genome Modification Assay) technique by assaying a 200-bp sequence containing multiple cytosine modifications. This new approach should help researchers better catalog cytosine modifications and their effects on gene expression patterns.Kawasaki et al. 2016. “A novel method for the simultaneous identification of methylcytosine and hydroxymethylcytosine at a single base resolution.”Nucleic Acid Research. doi:10.1093/nar/gkw994Single-cell, single-tube transcriptomics and proteomicsA fundamental mystery of molecular biology is the relationship between gene expression and protein production. Attempts to understand how RNA and protein levels co-vary within cells have led to the development of new techniques for assaying these molecules simultaneously. Genshaft et al. now offer a method for profiling single-cell transcriptomes and proteomes in the same reaction. The key to their approach is the use of the reverse transcriptase activity of DNA polymerase for both complementary DNA synthesis and proximity extension assays. Proximity extension uses two antibodies attached to short DNA sequences. When the antibodies are close, the DNA sequences bind together and are extended by DNA polymerase to generate a protein-indexed DNA readout, which can be detected by sequencing. Using this method, the authors were able to profile changes in the transcriptome and proteome of a human breast cancer cell line in response to different chemical perturbations. This multiplexed profiling technique provides a new option for scientists exploring gene—protein expression relationships.Genshaft et al. 2016. “Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction.” Genome Biology. 17:188. doi:10.1186/s13059-016-1045-6FiguresReferencesRelatedDetails Vol. 61, No. 5 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print November 2016 Information© 2016 Author(s)PDF download

Two-color fluorescence in situ hybridization using chromogenic substrates in Xenopus.

Xenopus embryo yolk proteins present a serious barrier to fluorescence microscopy. Previously, in situ assays of gene transcripts in whole embryos was limited to the use of chromogenic alkaline phosphatase substrates, which restricted researchers' ability to gauge coexpression of transcripts. Here, we describe a modified in situ hybridization (ISH) protocol that uses fluorescent substrates and a novel yolk-clearing technique for simultaneous visualization of the expression of two genes in whole Xenopus embryos with high resolution. This protocol employs two well-known fluorescent substrates, nitro blue tetrazolium/5-bromo- 4-chloro-3-indolyl-phosphate (NBT/BCIP) and Vector Red, in a sequential dual in situ hybridization procedure. Subsequent clearing of the samples with refractive-index-matching solution (RIMS) renders the samples amenable to confocal microscopy, allowing imaging at sufficiently high resolution to discern coexpression of transcripts within individual cells.

Role of myosin light chain phosphatase in cardiac physiology and pathophysiology

Maintenance of contractile performance of the heart is achieved in part by the constitutive 40% phosphorylation of myosin regulatory light chain (RLC) in sarcomeres. The importance of this extent of RLC phosphorylation for optimal cardiac performance becomes apparent when various mouse models and resultant phenotypes are compared. The absence or attenuation of RLC phosphorylation results in poor performance leading to heart failure, whereas increased RLC phosphorylation is associated with cardiac protection from stresses. Although information is limited, RLC phosphorylation appears compromised in human heart failure which is consistent with data from mouse studies. The extent of cardiac RLC phosphorylation is determined by the balanced activities of cardiac myosin light chain kinases and phosphatases, the regulatory mechanisms of which are now emerging. This review thusly focuses on kinases that may participate in phosphorylating RLC to make the substrate for cardiac myosin light chain phosphatases, in addition to providing perspectives on the family of myosin light chain phosphatases and involved signaling mechanisms. Because biochemical and physiological information about cardiac myosin light chain phosphatase is sparse, such studies represent an emerging area of investigation in health and disease.

Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca2+/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.

Development and in vitro evaluation of zeta potential changing self-emulsifying drug delivery systems for enhanced mucus permeation

The aim of this study was the development of zeta potential changing self-emulsifying drug delivery systems (SEDDS). Various cationic surfactants were incorporated into a formulation consisting of 30% Cremophor EL, 30% Capmul MCM, 30% Captex 355 and 10% propylene glycol (w/w). A substrate of intestinal alkaline phosphatase (IAP), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid sodium (PA), was thereafter incorporated into SEDDS. Size, zeta potential and polydispersity index were determined. Phosphate release studies were performed using three different models, namely, isolated IAP, Caco-2 cell monolayer and rat intestinal mucosa and the amount of released phosphate was quantified by malachite green assay. Interaction of SEDDS and mucus was investigated regarding surface charges and mucus diffusion studies were performed using rotating tube technique. SEDDS were diluted 1:100 in 100mM HEPES buffer and a negative zeta potential was obtained. By addition of isolated IAP, 15% to 20% phosphate was liberated from SEDDS within 3h and a shift of zeta potential from negative to positive was observed. On Caco-2 cell monolayer and rat intestinal mucosa, 12% and 23% phosphate were released, respectively, from SEDDS diluted 1:1000 in glucose-HEPES buffer. Positively charged droplets were bound to negatively charged mucus resulting in a decrease of zeta potential, whereas negatively charged SEDDS showed no interaction. Furthermore, negatively charged SEDDS diffused faster through mucus layer as higher extent of incorporated Lumogen was present in deeper mucus segments in comparison to positively charged ones. Accordingly, zeta potential changing SEDDS provide an effective mucus permeation combined with higher cellular uptake when droplets reach absorptive epithelium membrane.

Zein as biodegradable material for effective delivery of alkaline phosphatase and substrates in biokits and biosensors

A biodegradable material, zein, is proposed as a reagent delivery platform for biokits and biosensors based on alkaline phosphatase (ALP) activity/inhibition in the presence of phosphatase substrates. The immobilization and release of both the substrate and/or the active ALP, in a biodegradable and low-cost material such as zein, a prolamin from maize, and in combination with glycerol as plasticizer have been investigated. Three zein-based devices are proposed for several applications: (1) inorganic phosphorus estimation in water of different sources (river, lake, coastal water and tap water) with a detection limit of 0.2mg/L – compared to at least 1mg/L required by legislation, (2) estimation of ALP in saliva and (3) chlorpyrifos control in commercial preparations. The single-use kits developed are low cost, easy and fast to manufacture and are stable for at least 20 days at −20°C, so the zein film can preserve and deliver both the enzyme and substrates.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization.

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Rif1 Regulates the Fate of DNA Entanglements during Mitosis.

SummaryClearance of entangled DNA from the anaphase mid-region must accurately proceed in order for chromosomes to segregate with high fidelity. Loss of Taz1 (fission yeast ortholog of human TRF1/TRF2) leads to stalled telomeric replication forks that trigger telomeric entanglements; the resolution of these entanglements fails at ≤20°C. Here, we investigate these entanglements and their promotion by the conserved replication/repair protein Rif1. Rif1 plays no role in taz1Δ fork stalling. Rather, Rif1 localizes to the anaphase mid-region and regulates the resolution of persisting DNA structures. This anaphase role for Rif1 is genetically separate from the role of Rif1 in S/G2, though both roles require binding to PP1 phosphatase, implying spatially and temporally distinct Rif1-regulated phosphatase substrates. Rif1 thus acts as a double-edged sword. Although it inhibits the resolution of taz1Δ telomere entanglements, it promotes the resolution of non-telomeric ultrafine anaphase bridges at ≤20°C. We suggest a unifying model for Rif1’s seemingly diverse roles in chromosome segregation in eukaryotes.

Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism

Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 phosphatase complex.

Development of an Avirulent Salmonella Surrogate for Modeling Pathogen Behavior in Pre- and Postharvest Environments.

Recurrent outbreaks of bacterial gastroenteritis linked to the consumption of fresh fruits and vegetables highlight the paucity of understanding of the ecology of Salmonella enterica under crop production and postharvest conditions. These gaps in knowledge are due, at least in part, to the lack of suitable surrogate organisms for studies for which biosafety level 2 is problematic. Therefore, we constructed and validated an avirulent strain of Salmonella enterica serovar Typhimurium. The strain lacks major Salmonella pathogenicity islands SPI-1, SPI-2, SPI-3, SPI-4, and SPI-5 as well as the virulence plasmid pSLT. Deletions and the absence of genomic rearrangements were confirmed by genomic sequencing, and the surrogate behaved like the parental wild-type strain on selective media. A loss-of-function (phoN) selective marker allowed the differentiation of this strain from wild-type strains on a medium containing a chromogenic substrate for alkaline phosphatase. Lack of virulence was confirmed by oral infection of female BALB/c mice. The strain persisted in tomatoes, cantaloupes, leafy greens, and soil with the same kinetics as the parental wild-type and selected outbreak strains, and it reached similar final population levels. The responses of this strain to heat treatment and disinfectants were similar to those of the wild type, supporting its potential as a surrogate for future studies on the ecology and survival of Salmonella in production and processing environments. There is significant interest in understanding the ecology of human pathogens in environments outside of their animal hosts, including the crop production environment. However, manipulative field experiments with virulent human pathogens are unlikely to receive regulatory approval due to the obvious risks. Therefore, we constructed an avirulent strain of S. enterica serovar Typhimurium and characterized it extensively.