Method for detecting acetylated PD-L1 in cell lysates by immunoprecipitation and western blot analysis.

Lysine acetylation is an important regulatory post-translational modification (PTM) that occurs sub-stoichiometrically, often representing less than 1% of the target protein. This makes studying endogenous protein acetylation extremely challenging. Recent reports suggest that several post-translational modifications (PTMs), including lysine acetylation, play a major role in the regulation the programmed cell death-ligand 1 (PD-L1), a clinically important protein target. An enrichment step is necessary to enable identification of the acetylated species by either antibody or mass spectrometry-based detection methods. This report describes a robust lab protocol for the enrichment and detection of endogenous acetylated PD-L1 protein. A recently developed acetyl lysine affinity matrix was utilized to enrich >90% of acetylated PD-L1 species, from a variety of cell lines, spanning a fourteen-fold range of target protein levels. Western blot analysis, using a highly sensitive PD-L1 antibody and optimized transfer times, was used to determine that the endogenous level of acetylated PD-L1 is in the range of 0.02-0.07% of total PD-L1. As validation, we demonstrate that acetylation levels increase to 0.11-0.17% of total PD-L1 after a 4h treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). The method described here is simple to perform in any lab equipped with tissue culture and western blot equipment.

Utilizing Optimized Tools to Investigate PTM Crosstalk: Identifying Potential PTM Crosstalk of Acetylated Mitochondrial Proteins.

Post-translational modification (PTM) crosstalk is recognized as a major cell-regulatory mechanism, and studies of several proteins have validated the premise that PTMs work in concert. Previous work by our group investigated the potential PTM crosstalk on proteins in the EGFR-Ras-c-Fos axis by utilizing a comprehensive set of PTM reagents termed Signal-Seeker toolkits. In this study, these tools were used to investigate the potential PTM crosstalk that occurs in acetylated mitochondrial proteins in response to a mitochondrial stress-inducing agent hydrogen peroxide (H2O2). Mitochondrial protein acetylation has been shown to participate in PTM crosstalk as exemplified by the regulation of the pyruvate dehydrogenase complex via kinase, phosphatase, acetyltransferase, and deacetylase activities. Changes in the acetylated state of mitochondrial proteins were investigated, in response to H2O2, using a novel anti acetyl lysine (Ac-K) antibody. Signal-Seeker PTM detection tools were used to validate the acetylation state of ten mitochondrial targets, as well as their endogenous acetylation state in response to H2O2. Importantly, the endogenous acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation state of four target mitochondrial proteins were also investigated with the toolkit. Each of the four proteins had unique PTM profiles, but diverging acetylation and ubiquitin or SUMO 2/3 signals appeared to be a common theme. This proof-of-concept study identifies the Signal-Seeker toolkits as a useful tool to investigate potential PTM crosstalk.

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins.

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein's structure and function, which may be essential for a given protein's role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Identifying Regulatory Posttranslational Modifications of PD-L1: A Focus on Monoubiquitinaton.

A set of high-affinity, high-specificity posttranslational modification (PTM) enrichment tools was developed to generate an unbiased snapshot of four key PTM profiles (tyrosine phosphorylation, acetylation, ubiquitination, and SUMOylation 2/3) for the clinically important protein programmed cell death ligand 1 (PD-L1). The results showed that epidermal growth factor (EGF) treatment induced tyrosine phosphorylation, acetylation, and ubiquitination of PD-L1. Further characterization of EGF-induced PD-L1 ubiquitination revealed a significant increase in mono- and multiubiquitination of PD-L1 that occurred on glycosylated PD-L1. EGF induced mono- and multiubiquitination of PD-L1 preceded EGF-induced increases in PD-L1 protein levels. Chemical inhibitors of the EGFR pathway, gefitnib and SCH772984, suppressed PD-L1 mono- and multiubiquitination, and inhibition of the ubiquitin E1 activating enzyme, with the chemical inhibitor PYR41, was sufficient to block EGF-stimulated increases in PD-L1 protein levels. This study highlights the significance of identifying novel PTMs for PD-L1 and reveals potentially critical regulatory mechanisms that may be valuable therapeutic targets. In a broader context, this report validates an approach whereby one can gain insight into novel mechanisms of action by a simple and unbiased analysis of a PTM profile of potentially any endogenous protein of interest.

A simple toolset to identify endogenous post-translational modifications for a target protein: a snapshot of the EGFR signaling pathway.

Identification of a novel post-translational modification (PTM) for a target protein, defining its physiologic role, and studying its potential crosstalk with other PTMs is a challenging process. A set of highly sensitive tools termed Signal-Seeker kits was developed, which enables rapid and simple detection of post-translational modifications on any target protein.  The methodology for these tools utilizes affinity purification of modified proteins from a cell or tissue lysate and immunoblot analysis. These tools utilize a single lysis system that is effective at identifying endogenous, dynamic PTM changes, as well as the potential crosstalk between PTMs. As a proof-of-concept experiment, the acetylation, tyrosine phosphorylation, SUMOylation 2/3, and ubiquitination profiles of the EGFR - Ras - c-Fos axis were examined in response to EGF stimulation. All 10 previously identified PTMs of this signaling axis were confirmed using these tools, and it also identified acetylation as a novel modification of c-Fos. This axis in the EGF/EGFR signaling pathway was chosen because it is a well-established signaling pathway with proteins localized in the membrane, cytoplasmic, and nuclear compartments that ranged in abundance from 4.18x108 (EGFR) to 1.35x104 (c-Fos) molecules per A431 cell. These tools enabled the identification of low abundance PTMs, such as c-Fos Ac, at 17 molecules per cell. These studies highlight how pervasive PTMs are, and how stimulants like EGF induce multiple PTM changes on downstream signaling axis. Identification of endogenous changes and potential crosstalk between multiple PTMs for a target protein or signaling axis will provide regulatory mechanistic insight to investigators.

Terson syndrome: an underrecognized cause of reversible vision loss in patients with subarachnoid hemorrhage.

Terson syndrome is a known complication of subarachnoid hemorrhage (SAH) that causes potentially reversible vision loss. It develops after SAH because of vitreous hemorrhage caused by retinal capillary disruption. Case series report an incidence of Terson syndrome in approximately 8%-15% of patients with SAH. Nonetheless, the medical literature regarding this condition is primarily found within neurosurgical and ophthalmologic journals with little mention within the rehabilitation medicine literature. Physiatrists must be aware of this clinical presentation to coordinate the care of patients with SAH who develop vision loss and develop a rehabilitation plan that addresses the co-morbid motor, sensory, and cognitive impairments. Physiatrists may be the first to identify visual loss, are well equipped to emphasize compensatory strategies, and are well positioned to coordinate surgical treatment for visual recovery in appropriate cases. In this report, we describe the case of a young woman with SAH and Terson syndrome through her acute hospital admission, rehabilitation treatment, ophthalmologic management, and outcome, describing the salient epidemiology, pathophysiology, diagnostic workup, and treatment options.

A tubulin polymerization microassay used to compare ligand efficacy.

We developed tubulin purification strategies that allowed sufficient material to be produced for compound-screening projects. Tubulins were polymerized in the presence of compounds using either turbidometric or fluorescence polymerization assays. IC50 and EC50 values were calculated and used to determine ratios between host and target tubulin (TT) (e.g., IC50-neuronal tubulin/IC50-TT). This ratio can be compared between compounds to identify the ones which are most selective for a particular TT. We found ratios for different compounds ranged from 0.16 to 4.0 between neuronal and cancer cell tubulin indicating that the sequence and posttranslational heterogeneity between these tubulins are sufficient to identify selective ligands for the TT. Likewise, compounds compared between neuronal and fungal tubulin had ratios ranging from 0.03 to 0.60, and compounds compared between neuronal to plant tubulin had ratios ranging from 0.03 to 52. Considering these data, we believe cancer cell tubulin-targeted drugs could be obtained with ratios in excess of 20, herbicides with ratios in excess of 200, and fungicides in excess of 200.

Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators.

Kinesins are a group of related molecular motor proteins that have great potential as targets for antimitotic drug development. We have developed two novel assays, one end-point and one kinetic, that are useful for the discovery and optimization of kinesin modulators. Both assays measure inorganic phosphate (Pi) generated by microtubule-activated kinesin adenosine triphosphatase activity. The assays were validated using the mitotic Eg5 kinesin-specific inhibitor, monastrol. A panel of nine kinesin motor domain proteins, representing 8 of the 14 classes of kinesins, was screened. The coefficient of variation for both assays was determined to be 4-14% depending on the panel member. Using the Eg5 kinetic assay with monastrol the IC50 value was 12 microM, which agrees well with previously published results. Two other closely related mitotic kinesins (AnBimC and MKLP1) were found to have IC50 values in the millimolar range. The other panel members (kinesin heavy chain, chromokinesin KIF4A, KIF3C, CENP-E, MCAK, and KIFC3) were not significantly inhibited by millimolar levels of monastrol. It is anticipated that screening of the nine-member panel of kinesins in these assays will serve as a platform for the discovery and development of specific kinesin modulators.