Ebbing de Jong

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Purification of human β- and γ-actin from budding yeast

Journal of Cell Science, May 1, 2023

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily p... more Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified -actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins and there is an underlying concern that these proteins perform differently with actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human β-or γ-actin (i.e., cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin. Both β-or γ-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 (formin), fascin, and thymosin-β4 (Tβ4). Notably, Tβ4 and profilin bind to β-or γ-actin with higher affinity than to -actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies of actin regulation. INTRODUCTION The actin cytoskeleton is an essential, highly conserved, and abundant component of cells. Simple eukaryotes tend to express a single actin isoform, while humans display tissue and cell specific expression patterns. Although closely related, actin isoforms can subtly differ in biochemical

format_quoteBoth β-and γ-actin polymerize similarly into filaments in vitro, indicating potential functional redundancy in cytoplasmic actins during assembly.format_quote

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Loss of Myosin-1e biases MMTV-PyMT induced breast cancer towards a differentiated and secretory state

bioRxiv (Cold Spring Harbor Laboratory), Apr 27, 2022

Expression of the unconventional myosin, Myosin-1e (Myo1e), has been shown to contribute to tumor... more Expression of the unconventional myosin, Myosin-1e (Myo1e), has been shown to contribute to tumor progression in the MMTV-PyMT mouse model of mammary tumorigenesis and is associated with poor outcome in breast cancer patients. However, the specific effects of Myo1e expression on the mammary tumor cells remain unidentified. Here, we used Myo1e-KO and wild-type (WT) MMTV-PyMT mice on a pure genetic background to further investigate the molecular and cellular effects of Myo1e expression. Myo1e-WT tumors were characterized by an increased abundance of intra-epithelial macrophages and lower amounts of the extracellular matrix. Transcriptomic profiling of WT and Myo1e-KO tumors identified a pattern of differential expression of tumor suppressor and tumorpromoting genes that was consistent with the observed differences in tumor progression and morphology between the genotypes, and also revealed differential expression of genes associated with secretion and cell-cell adhesion. In agreement with the RNA-seq findings, Myo1e-expressing tumor cells exhibited increased proliferation and elevated nuclear enrichment of YAP1 transcriptional activator compared to Myo1e-KO tumor cells. To investigate tumor cellautonomous effects of Myo1e expression, we used the epithelial cell line PY-230 derived from the MMTV-PyMT-induced mouse tumor to create Myo1e-depleted cells by Crispr-mediated genome editing. Cells deficient in Myo1e had increased expression of genes encoding milk components compared to the wild-type cells. Electric cell-substrate impedance sensing (ECIS) measurements showed that depletion of Myo1e in PY-230 cells resulted in increased resistance to electrical current indicating enhanced epithelial barrier function. Overall, we find that Myo1e expression biases tumors towards a less-differentiated, pro-tumorigenic state, and that depletion of Myo1e is associated with a pro-secretory, more differentiated state.

format_quoteMyo1e depletion in Py230 cells enhances epithelial barrier function and alters tumor differentiation towards a pro-secretory state.format_quote

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Salivary proteomics in bisphosphonate‐related osteonecrosis of the jaw

Oral Diseases, Nov 29, 2013

Objective-To identify differentially expressed salivary proteins in bisphosphonate-related osteon... more Objective-To identify differentially expressed salivary proteins in bisphosphonate-related osteonecrosis of the jaw (BRONJ) patients that could serve as biomarkers for BRONJ diagnosis. Subjects and Methods-Whole saliva obtained from 20 BRONJ patients and 20 controls were pooled within groups. The samples were analyzed using iTRAQ-labeled two-dimensional liquid chromatography-tandem mass spectrometry. Results-Overall, 1340 proteins were identified. Of these, biomarker candidates were selected based on p-value (<0.001), changes in protein expression (≥1.5-fold increase or decrease), and unique peptides identified (≥2). Three comparisons made between BRONJ and controls identified 200 proteins to be differentially expressed in BRONJ patients. A majority of these proteins were predicted to have a role in drug metabolism, immunological and dermatological diseases. Of all the differentially expressed proteins, we selected metalloproteinase-9 and desmoplakin for further validation. Immunoassays confirmed increased expression of metalloproteinase-9 in individual saliva (p=0.048) and serum samples (p=0.05) of BRONJ patients. Desmoplakin was undetectable in saliva. However, desmoplakin levels tended to be lower in BRONJ serum than controls (p=0.157). Conclusions-Multiple pathological reactions are involved in BRONJ development. One or more proteins identified by this study may prove to be useful biomarkers for BRONJ diagnosis. The role of metalloproteinase-9 and desmoplakin in BRONJ requires further investigation.

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Author response: Mitochondrial protein import clogging as a mechanism of disease

Purification of Human β and γ Actin from Budding Yeast

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily p... more Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified α-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins and there is an underlying concern that these proteins perform differently with actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human β- or γ-actin (i.e., cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin. Both β- or γ-actin purified in this system polymerize and interact with various binding partners, including profilin, cofilin, mDia1 (formin), fascin, and thymosin-β4 (Tβ4). Notably, Tβ4 binds to β- or γ-actin with higher affinity than to muscle α-actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies of actin...

Mitochondrial protein import clogging as a mechanism of disease

Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cyto... more Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria is disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in adenine nucleotide translocase 1 (Ant1), and its yeast ortholog Aac2, cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis and cell viability independent of Ant1’s nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/Ant1 cause severe clogging primarily at the Translocase of the Outer Membrane (TOM) complex. This confers extreme toxicity in yeast...

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Developing Photoaffinity Probes for Dopamine Receptor D2 to Determine Targets of Parkinson’s Disease Drugs

Dopaminergic pathways control highly consequential aspects of physiology and behavior. One of the... more Dopaminergic pathways control highly consequential aspects of physiology and behavior. One of the most therapeutically important and best-studied receptors in these pathways is dopamine receptor D2 (DRD2). Unfortunately, DRD2 is challenging to study with traditional molecular biological techniques, and most drugs designed to target DRD2 are ligands for many other receptors. Here, we developed probes able to both covalently bind to DRD2 using photoaffinity labeling as well as provide a chemical handle for detection or affinity purification. These probes behaved like good DRD2 agonists in traditional biochemical assays and were able to perform in chemical biological assays of cell and receptor labeling. Rat whole brain labeling and affinity enrichment using the probe permitted proteomic analysis of the probes’ interacting proteins. Bioinformatic study of the hits revealed that the probes bound non-canonically targeted proteins in the Parkinson’s disease network as well as the retrogra...

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Mutant Preproteins Clog Global Mitochondrial Protein Import to Cause Degenerative Disease

Social Science Research Network, 2021

Pan-SHIP1/2 inhibitors promote microglia effector functions essential for CNS homeostasis

Journal of Cell Science

We show here that both SHIP1 and its paralog SHIP2 are expressed at the protein level in microgli... more We show here that both SHIP1 and its paralog SHIP2 are expressed at the protein level in microglia. To examine whether targeting of SHIP paralogs might influence microglial physiology and function we tested the capacity of SHIP1-selective, SHIP2-selective and pan-SHIP1/2 inhibitors for their ability to impact microglia proliferation, lysosomal compartment size and phagocytic function. We find that highly potent pan-SHIP1/2 inhibitors can significantly increase lysosomal compartment size and phagocytosis of dead neurons and Aβ1–42 by microglia in vitro. We show that one of the more potent and water-soluble pan-SHIP1/2 inhibitors, K161, can penetrate the blood-brain barrier and consistent with this K161 increases the capacity of CNS-resident microglia to phagocytose beta-amyloid and apoptotic neurons following systemic administration. These findings provide the first demonstration that small molecule modulation of microglia function in vivo is feasible and suggest the possibility that...

Quantitative Proteomics Reveals Myosin and Actin as Promising Saliva Biomarkers for Distinguishing Pre- Malignant and Malignant Oral Lesions

Background: Oral cancer survival rates increase significantly when it is detected and treated ear... more Background: Oral cancer survival rates increase significantly when it is detected and treated early. Unfortunately, clinicians now lack tests which easily and reliably distinguish pre-malignant oral lesions from those already transitioned to malignancy. A test for proteins, ones found in non-invasively-collected whole saliva and whose abundances distinguish these lesion types, would meet this critical need. Methodology/Principal Findings: To discover such proteins, in a first-of-its-kind study we used advanced mass spectrometry-based quantitative proteomics analysis of the pooled soluble fraction of whole saliva from four subjects with pre-malignant lesions and four with malignant lesions. We prioritized candidate biomarkers via bioinformatics and validated selected proteins by western blotting. Bioinformatic analysis of differentially abundant proteins and initial western blotting revealed increased abundance of myosin and actin in patients with malignant lesions. We validated thos...

Salivary proteomics of healthy dogs: An in depth catalog

PLOS ONE

Objective To provide an in-depth catalog of the salivary proteome and endogenous peptidome of hea... more Objective To provide an in-depth catalog of the salivary proteome and endogenous peptidome of healthy dogs, evaluate proteins and peptides with antimicrobial properties, and compare the most common salivary proteins and peptides between different breed phylogeny groups. Methods 36 healthy dogs without evidence of periodontal disease representing four breed phylogeny groups, based upon single nucleotide polymorphism haplotypes (ancient, herding/sighthound, and two miscellaneous groups). Saliva collected from dogs was pooled by phylogeny group and analyzed using nanoscale liquid chromatography-tandem mass spectrometry. Resulting tandem mass spectra were compared to databases for identification of endogenous peptides and inferred proteins. Results 2,491 proteins and endogenous peptides were found in the saliva of healthy dogs with no periodontal disease. All dog phylogeny groups' saliva was rich in proteins and peptides with antimicrobial functions. The ancient breeds group was distinct in that it contained unique proteins and was missing many proteins and peptides present in the other groups. Conclusions and clinical relevance Using a sophisticated nanoscale liquid chromatography-tandem mass spectrometry, we were able to identify 10-fold more salivary proteins than previously reported in dogs. Seven of the top 10 most abundant proteins or peptides serve immune functions and many more with various antimicrobial mechanisms were found. This is the most comprehensive

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mRNA 3'-UTR shortening is a molecular signature of mTORC1 activation

Nature communications, Jan 15, 2015

Mammalian target of rapamycin (mTOR) enhances translation from a subset of messenger RNAs contain... more

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Mass Spectrometry-Based Proteomics: Basic Principles and Emerging Technologies and Directions

Advances in Experimental Medicine and Biology, 2013

As the main catalytic and structural molecules within living systems, proteins are the most likel... more As the main catalytic and structural molecules within living systems, proteins are the most likely biomolecules to be affected by radiation exposure. Proteomics, the comprehensive characterization of proteins within complex biological samples, is therefore a research approach ideally suited to assess the effects of radiation exposure on cells and tissues. For comprehensive characterization of proteomes, an analytical platform capable of quantifying protein abundance, identifying post-translation modifications and revealing members of protein complexes on a system-wide level is necessary. Mass spectrometry (MS), coupled with technologies for sample fractionation and automated data analysis, provides such a versatile and powerful platform. In this chapter we offer a view on the current state of MS-proteomics, and focus on emerging technologies within three areas: (1) New instrumental methods; (2) New computational methods for peptide identification; and (3) Label-free quantification. These emerging technologies should be valuable for researchers seeking to better understand biological effects of radiation on living systems.

Low-picomolar limits of detection using high-power light-emitting diodes for fluorescence

The Analyst, 2006

Fluorescence detectors are ever more frequently being used with light-emitting diodes (LEDs) as t... more Fluorescence detectors are ever more frequently being used with light-emitting diodes (LEDs) as the light source. Technological advances in the solid-state lighting industry have produced LEDs which are also suitable tools in analytical measurements. LEDs are now available which deliver 700 mW of radiometric power. While this greater light power can increase the fluorescence signal, it is not trivial to make proper use of this light. This new generation of LEDs has a large emitting area and a highly divergent beam. This presents a classic problem in optics where one must choose between either a small focused light spot, or high light collection efficiency. We have selected for light collection efficiency, which yields a light spot somewhat larger than the emitting area of the LED. This light is focused onto a flow cell. Increasing the detector cell internal diameter (i.d.) produces gains in (sensitivity)3. However, since the detector cell i.d. is smaller than the LED spot size, scattering of excitation light towards the detector remains a significant source of background signal. This can be minimized through the use of spectral filters and spatial filters in the form of pinholes. The detector produced a limit of detection (LOD) of 3 pM, which is roughly three orders of magnitude lower than other reports of LED-based fluorescence detectors. Furthermore, this LOD comes within a factor of six of much more expensive laser-based fluorescence systems. This detector has been used to monitor a separation from a gel filtration column of fluorescently labeled BSA from residual labeling reagent. The LOD of fluorescently labeled BSA is 25 pM.

Proteomics Analysis of Cells in Whole Saliva from Oral Cancer Patients via Value-added Three-dimensional Peptide Fractionation and Tandem Mass Spectrometry

Molecular & Cellular Proteomics, 2007

Whole human saliva possesses tremendous potential in clinical diagnostics, particularly for condi... more Whole human saliva possesses tremendous potential in clinical diagnostics, particularly for conditions within the oral cavity such as oral cancer. Although many have studied the soluble fraction of whole saliva, few have taken advantage of the diagnostic potential of the cells present in saliva, and none have taken advantage of proteomics capabilities for their study. We report on a novel proteomics method with which we characterized for the first time cells contained in whole saliva from patients diagnosed with oral squamous cell carcinoma. Our method uses three dimensions of peptide fractionation, combining the following steps: preparative IEF using free flow electrophoresis, strong cation exchange step gradient chromatography, and microcapillary reverse-phase liquid chromatography. We determined that the whole saliva samples contained enough cells, mostly exfoliated epithelial cells, providing adequate amounts of total protein for proteomics analysis. From a mixture of four oral cancer patient samples, the analysis resulted in a catalogue of over 1000 human proteins, each identified from at least two peptides, including numerous proteins with a role in oral squamous cell carcinoma signaling and tumorigenesis pathways. Additionally proteins from over 30 different bacteria were identified, some of which putatively contribute to cancer development. The combination of preparative IEF followed by strong cation exchange chromatography effectively fractionated the complex peptide mixtures despite the closely related physiochemical peptide properties of these separations (pI and solution phase charge, respectively). Furthermore compared with our two-step method combining preparative IEF and reversephase liquid chromatography, our three-step method identified significantly more cellular proteins while retaining higher confidence protein identification enabled by peptide pI information gained through IEF. Thus, for detecting salivary markers of oral cancer and possibly other conditions of the oral cavity, the results confirm both the potential of analyzing the cells in whole saliva and doing so with our proteomics method. Molecular & Cellular

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Online Nanoscale ERLIC-MS Outperforms RPLC–MS for Shotgun Proteomics in Complex Mixtures

Journal of Proteome Research, 2012

We have explored the use of electrostatic repulsion hydrophilic interaction chromatography (ERLIC... more We have explored the use of electrostatic repulsion hydrophilic interaction chromatography (ERLIC) as an alternative to the gold-standard in shotgun proteomics: reversed-phase (RP) LC for online ESI−MS/MS. Conditions for sample solubilization and initial gradient conditions were optimized to strike a balance between peptide solubility and maximum peptide retention when using mobile phase with high organic solvent concentration. Online ERLIC-MS demonstrated a 57% increase in total peptide identifications compared to RP-MS. We examined the mechanism of this improved performance and found that it stems from ERLIC's propensity to retain longer peptides, which can be identified with greater confidence. Online nanoscale ERLIC-MS provides a powerful new tool for enhancing MS-based shotgun proteomic in a broad range of applications.

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Improved Intensity-Based Label-Free Quantification via Proximity-Based Intensity Normalization (PIN)

Journal of Proteome Research, 2014

Researchers are increasingly turning to label-free MS1 intensity-based quantification strategies ... more Researchers are increasingly turning to label-free MS1 intensity-based quantification strategies within HPLC−ESI− MS/MS workflows to reveal biological variation at the molecule level. Unfortunately, HPLC−ESI−MS/MS workflows using these strategies produce results with poor repeatability and reproducibility, primarily due to systematic bias and complex variability. While current global normalization strategies can mitigate systematic bias, they fail when faced with complex variability stemming from transient stochastic events during HPLC−ESI−MS/MS analysis. To address these problems, we developed a novel local normalization method, proximity-based intensity normalization (PIN), based on the analysis of compositional data. We evaluated PIN against common normalization strategies. PIN outperforms them in dramatically reducing variance and in identifying 20% more proteins with statistically significant abundance differences that other strategies missed. Our results show the PIN enables the discovery of statistically significant biological variation that otherwise is falsely reported or missed.

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An evaluation of the Noyori system “in reverse”: Thermodynamic and kinetic parameters of secondary alcohol transfer dehydrogenation catalyzed by [(η6-1-iPr-4-Me-C6H4)Ru(HN-CR′R″-CR′R″NTs)], R′=H, Me; Ph, R″=H, Me

Journal of Molecular Catalysis A: Chemical, 2008

The 16 electron ruthenium complexes [(η 6 -1-isopropyl-4-methyl-benzene)(XN)Ru(II)], where XN is ... more

Noncovalent labeling of myoglobin for capillary electrophoresis with laser-induced fluorescence detection by reconstitution with a fluorescent porphyrin

ELECTROPHORESIS, 2004

Noncovalent labeling of myoglobin for capillary electrophoresis with laser-induced fluorescence d... more Noncovalent labeling of myoglobin for capillary electrophoresis with laser-induced fluorescence detection by reconstitution with a fluorescent porphyrin Traditional protein labeling reactions for capillary electrophoresis (CE) with laserinduced fluorescence (LIF) detection suffer from a variety of disadvantages. The reactions can be nonquantitative on a reasonable time scale, require relatively high concentrations of protein and fluorophore, and can give multiple reaction products that can not be separated. Herein, we describe a new noncovalent labeling technique that is rapid, selective for myoglobin, and gives a simple reaction product. Myoglobin is denatured with either 5.4 M urea or low pH (2.0). The denatured myoglobin releases its nonfluorescent heme group. A fluorescent porphyrin (protoporphyrin IX (PPIX) or its zinc (II) complex, Zn-PPIX), is added to the mixture and the solution conditions are altered (dilute to 0.54 M urea or adjust pH to 7.0) to allow myoglobin refolding. Upon refolding, the protein incorporates PPIX from solution, thus making the reaction product fluorescent. The experimental conditions have been optimized for both urea and low-pH denaturation of myoglobin. The latter procedure produces a detection limit of 50 nM. Alternatively, the reaction can be performed without denaturation by a simple exchange of the porphyrins. The use of Zn-PPIX yields the most efficient reaction. The low-pH reaction is unaffected by a 2000-fold excess of bovine serum albumin.

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Sample collection and handling considerations for peptidomic studies in whole saliva; implications for biomarker discovery

Clinica Chimica Acta, 2011

Background: Proteomic studies in saliva have demonstrated its potential as a diagnostic biofluid,... more Background: Proteomic studies in saliva have demonstrated its potential as a diagnostic biofluid, however the salivary peptidome is less studied. Here we study the effects of several sample collection and handling factors on salivary peptide abundance levels. Methods: Salivary peptides were isolated using an ultrafiltration device and analyzed by tandem mass spectrometry. A panel of 41 peptides common after various treatments were quantified and normalized. We evaluated the effects of freezing rate of the samples, nutritional status of the donors (fed vs. fasted), and room-temperature sample degradation on peptide abundance levels. Repeatability of our sample processing method and our instrumental analysis method were investigated. Results: Increased sample freezing rate produced higher levels of peptides. Donor nutritional status had no influence on the levels of measured peptides. No significant difference was detected in donors' saliva following 5, 10 and 15 min of room-temperature degradation. Sample processing and instrumental variability were relatively small, with median CVs of 9.6 and 6.6. Conclusions: Peptide abundance levels in saliva are rather forgiving towards variations in sample handling and donor nutritional status. Differences in freezing methods may affect peptide abundance, so consistency in freezing samples is preferred. Our results are valuable for standardizing sample collection and handling methods for peptidomic-based biomarker studies in saliva.

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