Masaru Miyagi

Case Western Reserve University | CWRU · Department of Pharmacology

Malaria, a devastating parasitic infection, is the leading cause of death in many developing countries. Unfortunately, the most deadliest causative agent of malaria, Plasmodium falciparum , has developed resistance to nearly all currently available antimalarial drugs. The P. falciparum Niemann-Pick type C1–related (PfNCR1) transporter has been iden...

Platelets fulfill their essential physiological roles sensing the extracellular environment through their membrane proteins. The native membrane environment provides essential regulatory cues that impact the protein structure and mechanism of action. Single-particle cryogenic electron microscopy (cryo-EM) has transformed structural biology by allow...

Background: α2-macroglobulin (α2M) is a broad-spectrum protease inhibitor that regulates both pro- and anti-coagulant processes. Additionally, it captures and neutralizes cytokines that influent hemostasis. These multifaceted roles have made it challenging to truly evaluate the contribution of α2M on hemostasis and thrombosis. The presence of α2M i...

Malaria is an extremely devastating parasitic infection that kills over half a million people each year. It is the leading cause of death in many developing countries, in part, due to a lack of resources and readily available therapeutics. Unfortunately, the most prevalent and deadliest causative agent of malaria, Plasmodium falciparum, has develop...

Functional blockade of the transforming growth factor-beta (TGFβ) signalling pathway improves the efficacy of cytotoxic and immunotherapies. Here, we conducted a phase 1b study (ClinicalTrials.gov., NCT03143985) to determine the primary endpoints of safety, tolerability, and maximal tolerated dose (200 mg twice daily) for the orally-available TGFβ...

Isoaspartic acid (isoAsp) is a common protein modification that spontaneously arises from asparagine or aspartic acid and has been linked to various diseases and health conditions. However, current methods for identifying isoAsp sites in proteins often suffer from ambiguity and have not gained widespread adoption. We developed a novel method that e...

Isoaspartic acid (isoAsp) is a common protein modification that spontaneously arises from asparagine or aspartic acid, and it has been linked to various diseases and health conditions. However, current methods for identifying isoAsp sites in proteins often suffer from ambiguity and have not gained widespread adoption. We have developed a novel meth...

Histidine residues play crucial roles in shaping the function and structure of proteins due to their unique ability to act as both acids and bases. In other words, they can serve as proton donors and acceptors at physiological pH. This exceptional property is attributed to the side-chain imidazole ring of histidine residues. Consequently, determini...

Background Dementia with Lewy bodies (DLB) and Alzheimer’s disease (AD) are the among the most common neuropathologies. Amyloid and tau biomarker changes are often incorporated in the clinical diagnosis of AD. The current DLB criteria does not include robust biofluid biomarkers. However differences in amyloid plaque and CSF neurogranin in DLB has b...

The prognosis for Multiple Myeloma (MM) patients (pts) has improved substantially over the past two decades with the success of immunomodulatory drugs (IMiDs), monoclonal antibodies, chimeric antigen receptor (CAR) T-cells and bispecific T-cell engagers (BiTEs) that broke the circulus vitiosus of tumor-induced immunosuppression to reengage the immu...

Isoaspartic acid (isoAsp) is a common protein modification that spontaneously arises from asparagine or aspartic acid, and it has been linked to various diseases and health conditions. However, current methods for identifying isoAsp sites in proteins often suffer from ambiguity and have not gained widespread adoption. We have developed a novel meth...

HER3 signaling pathway plays a major role in promoting the development of metastatic colorectal cancer (mCRC). Here, we demonstrated that endothelial cells, a key component of the liver microenvironment, secrete leucine-rich alpha-2-glycoprotein 1 (LRG1) that activates HER3 as a ligand, distinct from the canonical HER3 ligand neuregulins. Blocking...

The application of integrated systems biology to the field of structural biology is a promising new direction, although it is still in the infant stages of development. Here we report the use of single particle cryo-EM to identify multiple proteins from three enriched heterogeneous fractions prepared from human liver mitochondrial lysate. We simult...

Functional blockade of the transforming growth factor-beta (TGF-β) signaling pathway improves the efficacy of cytotoxic and immunotherapies. We conducted a phase 1b study to determine the safety, efficacy, and maximal tolerated dose (200 mg po bid) of the potent, orally-available TGF-β type I receptor kinase inhibitor vactosertib in relapsed and/or...

We applied raw human liver microsome lysate to a holey carbon grid and used cryo-electron microscopy (cryo-EM) to define its composition. From this sample we identified and simultaneously determined high-resolution structural information for ten unique human liver enzymes involved in diverse cellular processes. Notably, we determined the structure...

Genomic instability can promote inflammation and tumor development. Previous research revealed an unexpected layer of regulation of genomic instability by a cytoplasmic protein MYO10; however, the underlying mechanism remained unclear. Here, we report a protein stability-mediated mitotic regulation of MYO10 in controlling genome stability. We chara...

The use of an integrated systems biology approach to investigate tissues and organs has been thought to be impracticable in the field of structural biology, where the techniques mainly focus on determining the structure of a particular biomacromolecule of interest. Here, we report the use of cryoelectron microscopy (cryo-EM) to define the compositi...

The ability to investigate tissues and organs through an integrated systems biology approach has been thought to be unobtainable in the field of structural biology, where the techniques mainly focus on a particular biomacromolecule of interest. Here we report the use of cryo-electron microscopy (cryo-EM) to define the composition of a raw human kid...

Human 53BP1 is primarily known as a key player in regulating DNA double strand break (DSB) repair choice; however, its involvement in other biological process is less well understood. Here, we report a previously uncharacterized function of 53BP1 at heterochromatin, where it undergoes liquid-liquid phase separation (LLPS) with the heterochromatin p...

The in vivo physiological function of liquid–liquid phase separation (LLPS) that governs non–membrane-bound structures remains elusive. Among LLPS-prone proteins, TAR DNA-binding protein of 43 kD (TDP-43) is under intense investigation because of its close association with neurological disorders. Here, we generated mice expressing endogenous LLPS-d...

The maintenance of telomere length supports repetitive cell division and therefore plays a central role in cancer development and progression. Telomeres are extended by either the enzyme telomerase or the alternative lengthening of telomeres (ALT) pathway. Here, we found that the telomere-associated protein SLX4IP dictates telomere proteome composi...

In this study, we test the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca2+-sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy (DR). Nondiabetic and 2-months diabetic C57Bl/6J and calpain1 knockout (Capn1-...

The anthrax toxin protective antigen (PA), the membrane binding and pore-forming component of the anthrax toxin, was studied using 19F NMR. We site-specifically labeled PA with p-fluorophenylalanine (pF-Phe) at Phe427, a critically important residue that comprises the ϕ-clamp that is required for translocation of edema factor (EF) and lethal factor...

We previously found that the elevated abundance of the fungus Candida tropicalis is positively correlated with the bacteria Escherichia coli and Serratia marcescens in Crohn’s disease patients and the three pathogens, when co-cultured, form a robust mixed-species biofilm. The finding suggests that these three pathogens communicate and promote biofi...

The gene encoding promyelocytic leukemia protein (PML) generates several spliced isoforms. Ectopic expression of PML1 promotes the proliferation of ERα-positive MCF-7 breast cancer (BC) cells, while a loss of PML by knockdown or overexpression of PML4 does the opposite. PML is an essential constituent of highly dynamic particles called PML nuclear...

Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that are activated by proteolysis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here,...

Epigenetic modifications are known to play critical roles in the expression of genes related to differentiation and dedifferentiation. Histone lysine demethylase KDM5B (PLU-1) catalyzes the demethylation of histone H3 on Lys 4 (H3K4), which results in the repression of gene expression. KDM5B is involved in regulation of luminal and basal cell speci...

Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that are activated by proteolyis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here, w...

Background: Human papillomavirus (HPV) is linked to nearly all cases of cervical cancer. Despite available vaccines, a deeper understanding of the immune response to HPV is needed. Human α-defensin 5 (HD5), an innate immune effector peptide, blocks infection of multiple sero-types of HPV, including high-risk HPV16. While a common mechanism of α-de...

The effects of diversity of the gut microbiome on inflammation have centered mainly on bacterial flora. Recent research has implicated fungal species and their interactions with other organisms in the inflammatory process. New ways to restore microbial balance in the gut are being explored. Our goal was to identify beneficial probiotic strains that...

p>Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US with a staggeringly less than 14% survival rate for patients with metastatic disease. This poor clinical outcome for CRC patients is largely attributed to the lack of understanding of the factors that drive CRC progression, leading to a dearth of available trea...

Dauer larva is an alternative developmental stage of Caenorhabditis elegans (C. elegans) that occurs when the environmental condition is unfavorable for growth. Little is known regarding how the proteome of dauer larvae respond to poor environmental growth conditions. Such knowledge is expected to help understand the survival mechanism(s) of dauer...

Protein tyrosine nitration occurs under both physiological and pathological conditions ¹ . However, enzymes that remove this protein modification have not yet been identified. Here we report that the pseudo-phosphatase domain of protein tyrosine receptor T (PTPRT) is a denitrase that removes nitro-groups from tyrosine residues in paxillin. PTPRT no...

Diabetic cardiomyopathy is preceded by mitochondrial alterations, and progresses to heart failure. We studied whether treatment with methylene blue (MB), a compound that was reported to serve as an alternate electron carrier within the mitochondrial electron transport chain (ETC), improves mitochondrial metabolism and cardiac function in type 1 dia...

Background N-terminal acetylation is one of the most common protein modifications in eukaryotes and occurs co-translationally when the N-terminus of the nascent polypeptide is still attached to the ribosome. This modification has been shown to be involved in a wide range of biological phenomena such as protein half-life regulation, protein-protein...

The major immunogenic component of the current anthrax vaccine, anthrax vaccine adsorbed (AVA) is protective antigen (PA). We have shown recently that the thermodynamic stability of PA can be significantly improved by binding to the Von-Willebrand factor A (VWA) domain of capillary morphogenesis protein 2 (CMG2), and improvements in thermodynamic s...

The controlled and selective synthesis/clearance of biomolecules is critical for most cellular processes. In most high-throughput ‘omics’ studies, we measure the static quantities of only one class of biomolecules (e.g. DNA, mRNA, proteins or metabolites). It is, however, important to recognize that biological systems are highly dynamic in which bi...

The static levels of proteins are the net results of their production and clearance regulated by the activities of proteins involved in their synthesis, degradation, and transportation. Therefore, the information on the rates of protein synthesis and clearance is needed to understand the underlying dynamic nature of a proteome. In this chapter, the...

Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput scr...

Solvent D2O and 18O kinetic isotope effects on RNA 2’-O-transphosphorylation catalyzed by Zn2+ demonstrate an altered transition state relative to specific base catalysis. A recent model from DFT calculations involving inner sphere coordination to the non-bridging and leaving group oxygens is consistent with the data.

Acetylation of e(open)-amino group of lysine is one of the most common protein posttranslational modifications. The modification is reversible and catalyzed by lysine acetyltransferases in one direction and lysine deacetylases in the other direction. Although numerous lysine acetylation sites have been identified in many proteins involved in a dive...

Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. However, the toxicity of nucleoside inhibitors of RR is often exacerbated by their off-target effects. Therefore, a new class of non-nucleoside drugs that show enhanced specificity towards RR may be less toxic chemotherapeutics. Usi...

Cardiomyopathy is a major complication of diabetes. Our study was aimed to identify the sites of mitochondrial dysfunction, and delineate its consequences on mitochondrial metabolism in a model of type 1 diabetes. Diabetes was induced by streptozotocin injection to male Lewis rats. We found a decrease in mitochondrial biogenesis pathway and electro...

Hydrogen–deuterium exchange (HDX) is a technique that measures the exchange of protein hydrogens for deuteriums in a D2O-containing buffer, providing readout of the structural dynamics. Histidine hydrogen–deuterium exchange mass spectrometry (His-HDX-MS) is a variation of this technique that measures the slow HDX of imidazole C2 hydrogens of histid...

Little is known regarding how the synthesis and degradation of individual proteins changes during the life of an organism. Such knowledge is vital to understanding the aging process. To fill this knowledge gap, we monitored newly synthesized proteins on a proteome scale in Caenorhabditis elegans over time during adulthood using a SILAC-based label-...

A method of extracting a polypeptide from a biological sample includes contacting the biological sample with an extraction reagent to form a solution of the biological sample and the extraction reagent. The extraction reagent includes perfluorooctanoic and can be used at a concentration effective to solubilize the polypeptides in the biological sam...

Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that have central roles in the cardiovascular system. Numerous studies have focused on the signaling pathways mediated by PARs; however, the structural rearrangements that initiate these pathways are unknown. PARs are activated when proteolytic cleavage exposes a tethered l...

Protective antigen (PA) mediates entry of edema factor (EF) and lethal factor (LF) into the cytoplasmic space of the cells through the formation of a membrane spanning pore. To do this, PA must initially bind to a host cellular receptor. Recent mass spectrometry analysis of PA using histidine hydrogen-deuterium exchange (His-HDX) has shown that bin...

Proteolytic (18)O-labeling has been widely used in quantitative proteomics since it can uniformly label all peptides from different kinds of proteins. There have been multiple algorithms and tools developed over the last few years to analyze high-resolution proteolytic (16)O/(18)O labeled mass spectra. We have developed a software package, O18Quant...

Optimized enzymatic conditions incorporate a single oxygen atom into digested peptides using a peptidase. The incorporation of a single oxygen atom is especially useful for proteolytic 18O labeling in comparative proteomics. The optimized proteolytic 18O labeling minimizes the generation of a mixture of isotopic isoforms of the peptides resulting f...

We present a mass spectrometric method for analyzing protein structure and function, based on the imidazole C-2 or histidine Cε1 hydrogen/deuterium (H/D) exchange reaction, which is intrinsically second order with respect to the concentrations of the imidazolium cation and OD- in D2O. The second-order rate constant (k2) of this reaction was calcula...

With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still cons...

The anthrax protective antigen (PA) is an 83 kDa protein that is one of three protein components of the anthrax toxin, an AB toxin secreted by Bacillus anthracis. PA is capable of undergoing several structural changes, including oligomerization to either a heptameric or octameric structure called the prepore, and at acidic pH a major conformational...

A method for comparative proteomics using a peptidase under enzymatic conditions that permits the optimal incorporation of two oxygen atoms into a digested peptide. The method employs a peptidase to incorporate two 18O atoms into a peptide set derived from a population of proteins at a conditioned state, which is compared to a second peptide set in...

The development of effective treatments for osteoarthritis (OA) has been hampered by a poor understanding of OA at the cellular and molecular levels. Emerging as a disease of the 'whole joint', the importance of the biochemical contribution of various tissues, including synovium, bone and articular cartilage, has become increasingly significant. Ba...

The ubiquitous inducible transcription factor NF-κB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-κB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation...

Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2'-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/base catalysis, although the...

In mammalian skeletal muscle, Ca²⁺ release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca²⁺-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO2) through O2-dependent production of hydroge...

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by...

Ribonuclease A (RNase A) is a widely used model system illustrating basic principles of enzyme catalysis. Yet, ambiguity regarding the transition state it stabilizes precludes a complete understanding of its mechanism. Here, we present kinetic isotope effect analyses that provide direct evidence for a late, anionic transition state for RNase A cata...

Nucleocytoplasmic shuttling of class IIa of histone deacetylases (HDACs) is a key mechanism that controls cell fate and animal development. We have identified the filamin B (FLNB) as a novel HDAC7-interacting protein that is required for temporal and spatial regulation of vascular endothelial growth factor (VEGF)-mediated HDAC7 cytoplasmic sequestr...