The Minimal Effect of Linker Length for Fatty Acid Conjugation to a Small Protein on the Serum Half-Life Extension.

Conjugation of serum albumin or one of its ligands (such as fatty acid) has been an effective strategy to prolong the serum half-lives of drugs via neonatal Fc receptor (FcRn)-mediated recycling of albumin. So far, fatty acid (FA) has been effective in prolonging the serum half-lives for therapeutic peptides and small proteins, but not for large therapeutic proteins. Very recently, it was reported a large protein conjugated to FA competes with the binding of FcRn with serum albumin, leading to limited serum half-life extension, because primary FA binding sites in serum albumin partially overlap with FcRn binding sites. In order to prevent such competition, longer linkers between FA and the large proteins were required. Herein, we hypothesized that small proteins do not cause substantial competition for FcRn binding to albumin, resulting in the extended serum half-life. Using a small protein (28 kDa), we investigated whether the intramolecular distance in FA-protein conjugate affects the FcRn binding with albumin and serum half-life using linkers with varying lengths. Unlike with the FA-conjugated large protein, all FA-conjugated small proteins with different linkers exhibited comparable the FcRn binding to albumin and extended serum half-life.

Albumin affibody-outfitted injectable gel enabling extended release of urate oxidase-albumin conjugates for hyperuricemia treatment.

Therapeutic proteins are attractive candidates for the treatment of human diseases. However, their short half-life often limits their clinical application. To overcome this problem, injectable hydrogels have been developed as depots for controlled release of therapeutic proteins, but these systems have not yet achieved the desired extended, sustained drug release profile. Our strategy herein was to implement selective and strong interactions between the hydrogels and therapeutic proteins. Specifically, we investigated whether strong and specific interactions between human serum albumin (HSA) and albumin-binding peptide (ABP) can be used to achieve extended release of urate oxidase (Uox), a therapeutic protein for hyperuricemia treatment, from pH- and temperature-sensitive injectable hydrogels consisting of poly(ethylene glycol)-poly(ß-amino ester urethane) (PEG-PAEU) copolymer. Thus, HSA was conjugated to Uox (Uox-HSA) and ABP was introduced in PEG-PAEU (PEG-PAEU-ABP). Polymers, conjugates, and hydrogels were extensively characterized for their physicochemical characteristics and in vivo efficacy in a hyperuricemia mouse model. Briefly, the hydrogels exhibited good injectability, in vitro biocompatibility and extended drug release, and in vivo gel formation and degradability. The serum half-life of the Uox-HSA loaded in PEG-PAEU-ABP hydrogels was ~96 h in mice, which was ~88, ~5.5, and ~2 times longer than that of free native Uox, free Uox-HSA, and Uox-HSA loaded in PEG-PAEU hydrogels, respectively. In the hyperuricemia mouse model, Uox-HSA loaded in PEG-PAEU-ABP hydrogels exhibited a substantially extended period of uric acid-lowering efficacy. These results clearly show that by applying ABP-HSA strong interaction to injectable hydrogels and therapeutic protein, the concentration of the therapeutic protein can be maintained for a long period in vivo, prolonging its therapeutic effect. Further, our approach can be tailored to accommodate other therapeutic proteins, which potentially expands the clinical applicability range of these systems.

Recombinant Peptide Production Platform Coupled with Site-Specific Albumin Conjugation Enables a Convenient Production of Long-Acting Therapeutic Peptide.

The number of therapeutic peptides for human treatment is growing rapidly. However, their development faces two major issues: the poor yield of large peptides from conventional solid-phase synthesis, and the intrinsically short serum half-life of peptides. To address these issues, we investigated a platform for the production of a recombinant therapeutic peptide with an extended serum half-life involving the site-specific conjugation of human serum albumin (HSA). HSA has an exceptionally long serum half-life and can be used to extend the serum half-lives of therapeutic proteins and peptides. We used glucagon-like-peptide 1 (GLP-1) as a model peptide in the present study. A "clickable" non-natural amino acid-p-azido-l-phenylalanine (AzF)-was incorporated into three specific sites (V16, Y19, and F28) of a GLP-1 variant, followed by conjugation with HSA through strain-promoted azide-alkyne cycloaddition. All three HSA-conjugated GLP-1 variants (GLP1_16HSA, GLP1_19HSA, and GLP1_28HSA) exhibited comparable serum half-lives in vivo. However, the three GLP1_HSA variants had different in vitro biological activities and in vivo glucose-lowering effects, demonstrating the importance of site-specific HSA conjugation. The platform described herein could be used to develop other therapeutic peptides with extended serum half-lives.

Intramolecular distance in the conjugate of urate oxidase and fatty acid governs FcRn binding and serum half-life in vivo.

Therapeutic proteins are indispensable for treatment of various human diseases. However, intrinsic short serum half-lives of proteins are still big hurdles for developing new therapeutic proteins or expanding applications of existing ones. Urate oxidase (Uox) is a therapeutic protein clinically used for treatment of hyperuricemia. Due to its short half-life, its application for gout treatment requires prolonging the half-life in vivo. Conjugation of a fatty acid (FA), a serum albumin (SA) ligand, to therapeutic proteins/peptides is an emerging strategy to prolong serum half-life presumably via neonatal Fc receptor (FcRn)-mediated recycling. FA conjugation was proven effective for peptides and small proteins (less than 28 kDa), but not for Uox (140 kDa). We hypothesized that the intramolecular distance in the conjugate of FA and Uox is a critical factor for effective FcRn-mediated recycling. In order to control the intramolecular distance in the conjugate, we varied linker lengths between Uox and palmitic acid (PA). There was a linear correlation between the linker length and serum half-life of PA-conjugated Uox (Uox-PA) conjugates. The longer linker led to about 7-fold greater extension of serum half-life of Uox in mice than the unmodified Uox. The trend in serum half-life extension matched well with that in the tertiary structure formation of FcRn/SA/Uox-PA in vitro. These results demonstrate that the intramolecular distance in the conjugate of Uox and FA governs the stable formation of FcRn/SA/FA-conjugated protein and serum half-life extension in vivo. These findings would also contribute to development of effective FAconjugated therapeutic proteins.

Proteomic Analysis of Hippocampus in a Mouse Model of Depression Reveals Neuroprotective Function of Ubiquitin C-terminal Hydrolase L1 (UCH-L1) via Stress-induced Cysteine Oxidative Modifications.

Chronic physical restraint stress increases oxidative stress in the brain, and dysregulation of oxidative stress can be one of the causes of major depressive disorder. To understand the underlying mechanisms, we undertook a systematic proteomic analysis of hippocampus in a chronic restraint stress mouse model of depression. Combining two-dimensional gel electrophoresis (2D-PAGE) for protein separation with nanoUPLC-ESI-q-TOF tandem mass spectrometry, we identified sixty-three protein spots that changed in the hippocampus of mice subjected to chronic restraint stress. We identified and classified the proteins that changed after chronic stress, into three groups respectively functioning in neural plasticity, metabolic processes and protein aggregation. Of these, 5 proteins including ubiquitin C-terminal hydrolase L1 (UCH-L1), dihydropyrimidinase-related protein 2 (DPYL2), haloacid dehalogenase-like hydrolase domain-containing protein 2 (HDHD2), actin-related protein 2/3 complex subunit 5 (ARPC5) and peroxiredoxin-2 (PRDX2), showed pI shifts attributable to post-translational modifications. Further analysis indicated that UCH-L1 underwent differential oxidations of 2 cysteine residues following chronic stress. We investigated whether the oxidized form of UCH-L1 plays a role in stressed hippocampus, by comparing the effects of UCH-L1 and its Cys mutants on hippocampal cell line HT-22 in response to oxidative stress. This study demonstrated that UCH-L1 wild-type and cysteine to aspartic acid mutants, but not its cysteine to serine mutants, afforded neuroprotective effects against oxidative stress; there were no discernible differences between wild-type UCH-L1 and its mutants in the absence of oxidative stress. These findings suggest that cysteine oxidative modifications of UCH-L1 in the hippocampus play key roles in neuroprotection against oxidative stress caused in major depressive disorder.

Generation of therapeutic protein variants with the human serum albumin binding capacity via site-specific fatty acid conjugation.

Extension of the serum half-life is an important issue in developing new therapeutic proteins and expanding applications of existing therapeutic proteins. Conjugation of fatty acid, a natural human serum albumin ligand, to a therapeutic protein/peptide was developed as a technique to extend the serum half-life in vivo by taking advantages of unusually long serum half-life of human serum albumin (HSA). However, for broad applications of fatty acid-conjugation, several issues should be addressed, including a poor solubility of fatty acid and a substantial loss in the therapeutic activity. Therefore, herein we systematically investigate the conditions and components in conjugation of fatty acid to a therapeutic protein resulting in the HSA binding capacity without compromising therapeutic activities. By examining the crystal structure and performing dye conjugation assay, two sites (W160 and D112) of urate oxidase (Uox), a model therapeutic protein, were selected as sites for fatty acid-conjugation. Combination of site-specific incorporation of a clickable p-azido-L-phenylalanine to Uox and strain-promoted azide-alkyne cycloaddition allowed the conjugation of fatty acid (palmitic acid analog) to Uox with the HSA binding capacity and retained enzyme activity. Deoxycholic acid, a strong detergent, greatly enhanced the conjugation yield likely due to the enhanced solubility of palmitic acid analog.

Heterogeneous nuclear ribonucleoprotein K inhibits heat shock-induced transcriptional activity of heat shock factor 1.

When cells are exposed to heat shock and various other stresses, heat shock factor 1 (HSF1) is activated, and the heat shock response (HSR) is elicited. To better understand the molecular regulation of the HSR, we used 2D-PAGE-based proteome analysis to screen for heat shock-induced post-translationally modified cellular proteins. Our analysis revealed that two protein spots typically present on 2D-PAGE gels and containing heterogeneous nuclear ribonucleoprotein K (hnRNP K) with trioxidized Cys132 disappeared after the heat shock treatment and reappeared during recovery, but the total amount of hnRNP K protein remained unchanged. We next tested whether hnRNP K plays a role in HSR by regulating HSF1 and found that hnRNP K inhibits HSF1 activity, resulting in reduced expression of hsp70 and hsp27 mRNAs. hnRNP K also reduced binding affinity of HSF1 to the heat shock element by directly interacting with HSF1 but did not affect HSF1 phosphorylation-dependent activation or nuclear localization. hnRNP K lost its ability to induce these effects when its Cys132 was substituted with Ser, Asp, or Glu. These findings suggest that hnRNP K inhibits transcriptional activity of HSF1 by inhibiting its binding to heat shock element and that the oxidation status of Cys132 in hnRNP K is critical for this inhibition.

Bioengineered robust hybrid hydrogels enrich the stability and efficacy of biological drugs.

Biological drugs are exquisitely tailored components offering the advantages of high specificity and efficacy that are considered safe for treating diseases. Nevertheless, the effectiveness of biological drugs is limited by their inherent short biological half-life and poor stability in vivo. Herein, we engineered a novel delivery platform based on hybrid injectable hydrogels, in which pH- and temperature-responsive biodegradable copolymers were site-specifically coupled to the sulfhydryl group of human serum albumin, which effectively enhances the stability and circulation half-life of the biological drug, recombinant uricase enzyme (Uox). The albumin ligand conjugated to the Uox allowed specific-binding of the enzyme within the protein shell, and the synthetic polymers effectively shield the protein-enzyme complex. Such close confinement exhibits strong resistance towards various physical, chemical and therapeutically relevant stressors such as temperature, pH and proteases. Subcutaneous administration of Uox-loaded bioengineered hybrid hydrogel improved the pharmacokinetics by prolonging its circulation half-life. As a consequence, the bioengineered hybrid hydrogel normalized the serum uric acid level in hypoxanthine/potassium oxonate-induced hyperuricemia mice, and no obvious side effects were observed in the major organs. The characteristic of the bioengineered hydrogel networks applicable to a variety of biological drugs by simple mixing that unlock the possibility of adapting biological drugs to therapeutic applications.

Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex.

Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.

Degradation of Redox-Sensitive Proteins including Peroxiredoxins and DJ-1 is Promoted by Oxidation-induced Conformational Changes and Ubiquitination.

Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1.

Double clicking for site-specific coupling of multiple enzymes.

A method to site-specifically couple multiple enzymes is reported. The approach is based on the site-specific incorporation of a clickable non-natural amino acid into enzymes and two compatible click reactions. The multi-enzyme reaction system exhibited enhanced catalytic efficiency over the respective free enzymes.

Proteomic analysis of INS-1 rat insulinoma cells: ER stress effects and the protective role of exenatide, a GLP-1 receptor agonist.

Beta cell death caused by endoplasmic reticulum (ER) stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP)-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1) reversed by exenatide, 2) exaggerated by exenatide, and 3) unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3ß, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.

Differential protective effects of exenatide, an agonist of GLP-1 receptor and Piragliatin, a glucokinase activator in beta cell response to streptozotocin-induced and endoplasmic reticulum stresses.

BACKGROUND: Agonists of glucagon-like peptide-1 receptor (GLP-1R) and glucokinase activators (GKA) act as antidiabetic agents by their ability protect beta cells, and stimulate insulin secretion. Oxidative and endoplasmic reticulum (ER) stresses aggravate type 2 diabetes by causing beta cell loss. It was shown that GLP-1R agonists protect beta cells from oxidative and ER stresses. On the other hand, little is known regarding how GKAs protect beta cells. We hypothesized that GKAs protect beta cells by mechanisms distinct from those underlying GLP-1R agonist and tested our hypothesis by comparing the molecular effects of exenatide, a GLP-1R agonist, and piragliatin, a GKA, on INS-1 cells under oxidative and ER-induced stresses. METHODS: BETA CELLS WERE TREATED WITH STREPTOZOTOCIN (STZ) TO INDUCE OXIDATIVE STRESS AND WITH PALMITATE OR THAPSIGARGIN (TG) TO INDUCE ER STRESS RESPECTIVELY, AND THE EFFECTS OF EXENATIDE AND PIRAGLIATIN ON THESE CELLS WERE INVESTIGATED BY: a) characterizing the kinases involved employing specific kinase inhibitors, and b) by identifying the differentially regulated proteins in response to stresses with proteomic analysis. RESULTS: Exenatide protected INS-1 cells from both ER and STZ-induced death. In contrast, piragliatin rescued the cells only from STZ-induced stress. Akt activation by exenatide appeared to contribute to its protective effects of beta cells while enhanced glucose utilization was the contributing factor in the case of piragliatin. Also, exenatide, not piragliatin, blocked changes in proteins 14-3-3ß, ε and θ, and preserved the 14-3-3θ levels under the ER stress. Isoform-specific modifications of 14-3-3, and the reduction of 14-3-3θ, commonly associated with beta cell death were assessed. CONCLUSIONS: Exenatide and piragliatin exert distinct effects on beta cell survival and thus on type 2 diabetes. This study which confirmed our hypothesis is also the first to observe specific modulation of 14-3-3 isoform in stress-induced beta cell death associated with progressive deterioration of type 2 diabetes.

Improvement of plant protein solubilization and 2-DE gel resolution through optimization of the concentration of Tris in the solubilization buffer.

It is important to solubilize acetone-precipitated proteins before isoelectric focusing (IEF) to achieve high resolution 2-DE gels. To resolve the maximum possible number of plant protein spots, we developed an improved solubilization buffer for plant proteins. We demonstrated that the resolution of 2-DE gels increased dramatically as the concentration of Tris-base increased, with maximum solubilization obtained at 200 mM Tris-base (Ly200T). The Ly200T buffer was more effective than the commonly used solubilization buffer containing 40 mM Tris at solubilizing acetone-precipitated plant proteins. Use of the Ly200T buffer to solubilize proteins resulted in an increase in intensity of approximately 30% of plant protein spots in the larger-than-40 kDa region of the gel. The Ly200T buffer also improved the resolution of abundant and basic proteins. Thus, the Ly200T buffer can be used to achieve greater resolution of protein spots in plant proteomics research.

The LRRK2 G2385R variant is a risk factor for sporadic Parkinson's disease in the Korean population.

The G2385R (SNP accession no. rs34778348) and R1628P (rs33949390) variants of leucine-rich repeat kinase 2 (LRRK2, PARK8) are emerging as an important risk factor for Parkinson's disease (PD) in the ethnic Chinese and Japanese populations. The purpose of this study was to investigate whether these variants are a genetic risk factor in sporadic PD patients in the Korean population. A total of 923 patients and 422 healthy subjects were included. The variants were screened by a SNaPshot assay. The LRRK2 G2385R variant was detected in 82 PD patients (8.9%, two homozygous and 80 heterozygous) and in 21 normal controls (5.0%, all heterozygous). The frequency of the LRRK2 G2385R variant in PD was significantly higher than in normal controls (adjusted odds ratio 1.83, p = 0.0170, 95% confidence interval 1.11-3.00). There were no differences in the mean age at onset or gender between the G2385R carriers and the non-carriers in PD patients. The LRRK2 R1628P variant was very rare (0.78% in patients versus 0.26% in controls) in the tested 384 patient-control pairs, and was not a significant risk factor. This study supports that the LRRK2 G2385R variant may be a genetic risk factor for sporadic PD in the Korean population.

Comparative proteomic analysis of blue light signaling components in the Arabidopsis cryptochrome 1 mutant.

An Arabidopsis hy4 mutant that is specifically impaired in its ability to undergo blue light dependent photomorphogenesis was used to identify cryptochrome 1 signaling-related components. Proteomic analysis revealed about 205 differentially expressed protein spots in the blue light-irradiated hy4 mutant compared to the wild-type. The proteins corresponding to 28 up-regulated and 33 down-regulated spots were identified. Obvious morphological changes in the hy4 mutant were closely related to the expression of various transcription factors. Our findings suggest that blue light signals may be involved in many cellular processes including disease resistance and stress responses.

Identification of phytochrome-interacting protein candidates in Arabidopsis thaliana by co-immunoprecipitation coupled with MALDI-TOF MS.

Phytochrome-interacting proteins have been extensively studied to elucidate light-signaling pathway in plants. However, most of these proteins have been identified by yeast two-hybrid screening using the C-terminal domain of phytochromes. We used co-immunoprecipitation followed by proteomic analysis in plant cell extracts in an attempt to screen for proteins interacting either directly or indirectly with native holophytochromes including the N-terminal domain as well as C-terminal domain. A total of 16 protein candidates were identified, and were selected from 2-DE experiments. Using MALDI-TOF MS analysis, 7 of these candidates were predicted to be putative phytochrome A-interacting proteins and the remaining ones to be phytochrome B-interacting proteins. Among these putative interacting proteins, protein phosphatase type 2C (PP2C) and a 66-kDa protein were strong candidates as novel phytochrome-interacting proteins, as knockout mutants for the genes encoding these two proteins had impaired light-signaling functions. A transgenic knockout Arabidopsis study showed that a 66-kDa protein candidate regulates hypocotyl elongation in a light-specific manner, and altered cotyledon development under white light during early developmental stages. The PP2C knockout plants also displayed light-specific changes in hypocotyl elongation. These results suggest that co-immunoprecipitation, followed by proteomic analysis, is a useful method for identifying novel interacting proteins and determining real protein-protein interactions in the cell.

Proteomic analysis of the response of Arabidopsis chloroplast proteins to high light stress.

Light is an essential environmental factor in the progression of plant growth and development but prolonged exposure to high levels of light stress can cause cellular damage and ultimately result in the death of the plant. Plants can respond defensively to this stress for a limited period and this involves changes to their gene expression profiles. Proteomic approaches were therefore applied to the study of the response to high light stress in the Arabidopsis thaliana plant species. Wild-type Arabidopsis was grown under normal light (100 micromol photons.m(-2).s(-1)) conditions and then subjected to high light (1000 micromol photons.m(-2).s(-1)) stress. Chloroplasts were then isolated from these plants and both soluble and insoluble proteins were extracted and subjected to two-dimensional (2-D) gel electrophoresis. The resolved proteins were subsequently identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and comparative database analysis. 64 protein spots, which were identified as candidate factors that responded to high light stress, were then selected for analysis and 52 of these were successfully identified using MALDI-TOF-MS analysis. 35 of the 52 identified proteins were found to decrease their expression levels during high light stress and a further 14 of the candidate proteins had upregulated expression levels under these conditions. Most of the proteins that were downregulated during high light stress are involved in photosynthesis pathways. However, many of the 14 upregulated proteins were identified as previously well-known high light stress-related proteins, such as heat shock proteins (HSPs), dehydroascorbate reductase (DHAR), and superoxide dismutase (SOD). Three novel proteins that were more highly expressed during periods of high light stress but had no clear functional relationship to these conditions, were also identified in this study.