Human p-glycoprotein (P-gp) is an ATP-binding cassette transporter that actively transports a diverse set of substrates at the plasma membrane. Specifically, P-gp is expressed most highly at important blood tissue barriers on the lumenal side of endothelial cells and secretory tissues asymmetrically where it provides generalized protection against xenobiotics due to its promiscuous substrate binding pocket. Substrates typically interact with P-gp within the inner leaflet of the plasma membrane before being effluxed through large conformation changes driven by ATP binding and hydrolysis. Since many small molecule drugs are substrates of P-gp and P-gp has the ability to transport chemically and structurally diverse molecules, delivery of bioavailable small molecule therapies and treatment of diseases beyond blood-tissue barriers may be difficult. In cancer, expression of P-gp may confer a multidrug resistance phenotype due to upregulation of the MDR1 gene, which encodes P-gp, in response to treatment with chemotherapies. Treatments of diseases beyond blood-tissue barriers and some cancers may be more complex given the protective role of P-gp coupled with it promiscuous substrate binding site.<br>Many studies of P-gp have been centered around understanding the structure function relationship of how P-gp effluxes small molecules across the plasma membrane. Here we have used a transient Vaccinia virus expression system to rapidly express many mutants of P-gp in human cells for analysis. Transient expression using the Vaccinia system was optimized to produce a large amount of protein while avoiding significant cell death. Optimization of the Vaccinia expression system has also helped to show that changes in P-gp surface expression are not correlated to changes in substrate accumulation within cells expressing P-gp, a topic that has yet to be addressed within the field of P-gp study. Reduced surface expression of P-gp to 68% maintained the same level of reduced cellular accumulation of two substrates, calcein-AM and rhodamine 123, relative to a WT P-gp control. Further study of P-gp mutations revealed a Y998A mutation had a 90% reduction of surface expression but the same reduction of cellular accumulation of rhodamine 123 further supporting that changes in surface expression do not correlate to changes in substrate transport.<br>We then sought to demonstrate how flexibility in transmembrane helix (TMH) 12 of P-gp affected overall stability and transport ability in vitro. TMH 12 in inward facing conformations shows a region of decreased hydrogen bonding in the backbone of the helix leading to a “kink” present in many crystal structures of C. elegans and mouse P-gp as well as in an occluded structure of human P-gp. Outward facing crystal structures of C. elegans, mouse, and human P-gp show TMH 12 where the backbone of the helix is fully hydrogen bonded and ordered. The change in hydrogen bonding pattern and the presence of the kink in TMH 12 suggest the importance of flexibility in the function of TMH 12. Clustal Omega was used to align the primary structure of P-gp between 8 species and a conserved sequence of 996-PDYAKA-1001 was identified aligning with the kink observed in crystallographic data. The kinked nature of this region led to our development of a rigid poly-alanine mutation and a flexible poly-glycine mutation based on the propensity of these amnio acids to form helices. The more flexible poly-glycine mutation obtained no significant transport while the poly-alanine mutation maintained some ability to transport fluorescent substrate relative to a WT control. Crosslinking of the nucleotide binding domains (NBDs) revealed a decrease of NBD dimerization likely correlating to decreased transport. Thus, some degree of flexibility within the kink region is critical for substrate transport as rigid and flexible mutations of this region abrogate transport of fluorescent substrates.<br>While the substrate binding pocket it located towards the interior of P-gp within the lipid bilayer, it has been theorized that substrates may interact with P-gp at the lipid-protein interface of the inner leaflet near portals for substrate entry formed by pairs of helices either side of the protein. To test this hypothesis, aromatic residues on TMH 12 and adjacent elbow helix 2 near the interface region of the inner leaflet, that have also been observed to interact with a cyclic peptide in a crystal structure of P-gp, were mutated to alanine. Y998, on TMH 12, was shown to interact with the cyclic peptide and is ideally located at the protein-lipid interface near a surface formed by elbow helix 2 and TMH 9 and was observed to have the largest effect on substrate accumulation. Accumulation of fluorescent substrates, relative to WT P-gp, was increased though not all substrates were affected similarly. No increase of accumulation was observed with rhodamine 123 while accumulation of BD-prazosin increased 65% relative to WT P-gp. It is to be expected that the large diversity of substrates recognized by P-gp would interact preferentially with carrying residues at the protein-lipid interface similar to observations of substrate binding at the substrate binding pocket. Variability in accumulation signifies that substrates do interact with P-gp at the lipid-protein interface and substrates interact differently at this interface similarly to substrate interaction at the substrate biding pocket.<br>

Prostasin is expressed at the apical surface of normal epithelial cells and suppresses in vitro invasion of cancer cells. Prostasin re-expression in the PC-3 prostate carcinoma cells down-regulated the epidermal growth factor receptor (EGFR) protein expression and EGF-induced phosphorylation of the extracellular signal-regulated kinases (Erk1/2). We report here that prostasin and its activating enzyme matriptase are capable of inducing proteolytic cleavages in the EGFR extracellular domain (ECD) when co-expressed in the FT-293 cells, generating two amino-terminally truncated fragments EGFR135 and EGFR110, at 135 and 110 kDa. Prostasin's role in EGFR cleavage is dependent on the serine active-site but not the GPI-anchor. The modifications of EGFR were confirmed to be on the primary structure by deglycosylation. EGFR135 and EGFR110 are not responsive to EGF stimulation, indicating loss of the ligand-binding domains. EGFR110 is constitutively phosphorylated and in its presence Erk1/2 phosphorylation is increased in the absence of EGF. The protease-induced EGFR cleavages are not dependent on EGFR phosphorylation. The EGFR ECD proteolytic modification by matriptase-prostasin is also observed in the BEAS-2B normal lung epithelial cells, the BPH-1 benign prostate hyperplasia and the MDA-MB-231 breast cancer cell lines; and represents a novel mechanism for epithelial cells to modulate EGF-EGFR signaling.

The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to public health. An explicit investigation of COVID-19 immune responses, particularly the host immunity in recovered subjects, will lay a foundation for the rational design of therapeutics and/or vaccines against future coronaviral outbreaks. Here, we examined virus-specific T cell responses and identified T cell epitopes using peptides spanning SARS-CoV-2 structural proteins. These peptides were used to stimulate peripheral blood mononuclear cells (PBMCs) derived from COVID-19-recovered subjects, followed by an analysis of IFN-γ-secreting T cells by enzyme-linked immunosorbent spot (ELISpot). We also evaluated virus-specific CD4 or CD8 T cell activation by flow cytometry assay. By screening 52 matrix pools (comprised of 315 peptides) of the spike (S) glycoprotein and 21 matrix pools (comprised of 102 peptides) spanning the nucleocapsid (N) protein, we identified 28 peptides from S protein and 5 peptides from N protein as immunodominant epitopes. The immunogenicity of these epitopes was confirmed by a second ELISpot using single peptide stimulation in memory T cells, and they were mapped by HLA restrictions. Notably, SARS-CoV-2 specific T cell responses positively correlated with B cell IgG and neutralizing antibody responses to the receptor-binding domain (RBD) of the S protein. Our results demonstrate that defined levels of SARS-CoV-2 specific T cell responses are generated in some, but not all, COVID-19-recovered subjects, fostering hope for the protection of a proportion of COVID-19-exposed individuals against reinfection. These results also suggest that these virus-specific T cell responses may induce protective immunity in unexposed individuals upon vaccination, using vaccines generated based on the immune epitopes identified in this study. However, SARS-CoV-2 S and N peptides are not potently immunogenic, and none of the single peptides could universally induce robust T cell responses, suggesting the necessity of using a multi-epitope strategy for COVID-19 vaccine design.

Lipid transfer inhibitor protein (LTIP) is an important regulator of cholesteryl ester transfer protein function. We report the development of an immunoassay for LTIP and its use to quantify LTIP in plasma of varying lipid contents. A rabbit antibody against bacterially produced recombinant LTIP detected two LTIP isoforms in plasma differing in carbohydrate content. This antibody was used in a competitive, enzyme-linked immunoassay that uses partially purified LTIP bound to microtiter plates. To optimize LTIP immunoreactivity, plasma samples required preincubation in 1% Tween-20 and 0.5% Nonidet P-40. In normolipidemic plasma, LTIP averaged 83.5 mg/ml. LTIP was 31% higher in males than in females. LTIP was positively associated with HDL cholesterol in normolipidemic males but not in females. In hypertriglyceridemic males, LTIP was only 56% of control values, whereas in hypertriglyceridemic females, LTIP tended to increase. Additionally, in males with normal cholesterol and triglyceride (TG) ≤ 200 mg/dl, LTIP varied inversely with plasma TG. Overall, we have confirmed the negative association between plasma TG levels and LTIP previously suggested by a small data set, but now we demonstrate that this effect is seen only in males. The mechanisms underlying this gender-specific response to TG, and why LTIP and HDL levels correlate in males but not in females, remain to be determined.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17793号 / 農博第2014号 / 新制||農||1016(附属図書館) / 学位論文||H25||N4784(農学部図書室) / 30600 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 久米 新一, 教授 松井 徹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

Measurements of relaxin and acute-phase proteins have not been validated for use in canine serum as a method of pregnancy diagnosis. This means that handling and anesthesia is still necessary to check the pregnancy status of most non-domestic canines. Therefore, the intention of this study was to determine whether relaxin and/or acute-phase proteins could be detected in the urine and/or feces of the domestic dog in order to evaluate the potential for a noninvasive pregnancy test in canines. Blood, urine and feces were collected from 18 domestic dogs and assayed for the presence of relaxin, fibrinogen, alpha-1 acid glycoprotein, and ceruloplasmin. Urinary relaxin appeared to be significant for detecting pregnancy of 30 Days or more in the domestic dog. Additionally, further research might shed light on the presence of relaxin in the feces and fibrinogen and AGP in the urine of the domestic dog and their significance for pregnancy diagnosis.

Introduction: Bivalirudin and heparin are the two most commonly used anticoagulants used during Percutaneous Coronary Intervention (PCI). The results of Randomized Controlled Trials (RCTs) comparing bivalirudin versus heparin monotherapy in the era of radial access are controversial, questioning the positive impact of bivalirudin on bleeding. The purpose of this systematic review is to summarize the results of RCTs comparing the efficacy and safety of bivalirudin versus heparin with or without Glycoprotein IIb/IIIa Inhibitors (GPI). Methods: This systematic review was performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses PRISMA statements for reporting systematic reviews. We searched the National Library of Medicine PubMed, Clinicaltrial.gov and the Cochrane Central Register of Controlled Trials to include clinical studies comparing bivalirudin with heparin in patients undergoing PCI. Sixteen studies met inclusion criteria and were reviewed for the summary. Findings: Several RCTs and meta-analyses have demonstrated the superiority of bivalirudin over heparin plus routine GPI use in terms of preventing bleeding complications but at the expense of increased risk of ischemic complications such as stent thrombosis. The hypothesis of post-PCI bivalirudin infusion to mitigate the risk of acute stent thrombosis has been tested in various RCTs with conflicting results. In comparison, heparin offers the advantage of having a reversible agent, of lower cost and reduced incidence of ischemic complications. Conclusion: Bivalirudin demonstrates its superiority over heparin plus GPI with better clinical outcomes in terms of less bleeding complications, thus making it as anticoagulation of choice particularly in patients at high risk of bleeding. Further studies are warranted for head to head comparison of bivalirudin to heparin monotherapy to establish an optimal heparin dosing regimen and post-PCI bivalirudin infusion to affirm its beneficial effect in reducing acute stent thrombosis.

Multidrug resistance (MDR) in breast cancer treatment is the major cause leading to the failure of chemotherapy. P-glycoprotein (P-gp), the product of the human MDR1 gene, plays a key role in resistance to chemotherapy and confers cross-resistance to many structurally unrelated anticancer drugs. We have previously reported that integrin αvβ6 plays a critical role in breast cancer invasion and metastasis. However, whether and how αvβ6 is associated with P-gp and regulated by potential genetic mechanisms in breast cancer remains unclear. In the present study, we further investigated the reversal effect and underlying mechanisms of MDR in breast cancer. Two small interfering RNA constructs (pSUPER-β6shRNAs) targeting two different regions of the β6 gene have been designed to inhibit αvβ6 expression by transfecting them into adriamycin-resistant MCF-7/ADR cell lines. Suppression of αvβ6 dramatically downregulated the levels of MDR1 gene mRNA and P-gp. In particular, β6shRNA-mediated silencing of αvβ6 gene increased significantly the cellular accumulation of Rhodamine 123 and markedly decreased drug efflux ability, suggesting that β6shRNAs indeed inhibit P-gp mediated drug efflux and effectively overcome drug resistance. In addition, inhibition of integrin αvβ6 suppressed the expression of ERK1/2. Interestingly, our data demonstrate that suppression of integrin αvβ6 caused significant downregulation of Bcl-2, Bcl-xL and upregulation of caspase 3, Bad, accompanied by increasing activity of cytochrome C. A possible connection between αvβ6 and P-gp in drug resistance biology is suggested. Taken together, β6shRNA could efficiently inhibit αvβ6 and MDR1 expression in vitro and these findings may offer specifically useful means to reverse MDR in breast cancer therapy.

With the explosion of protein tools as popular platforms for discovery and therapeutics, we see greater need for regulator systems that work congruently within these frameworks, especially safe and effective tools that can be implemented in humans. To this end, we endeavor to create orthogonal, precise and flexible protein modulators that can be easily employed to control protein tools with little need to iterate design for novel contexts. Hepatitis C NS3 protease is employed as a stabilizable linker between protein domains, enabling control over protein localization with FDA approved anti-viral drugs. The power of this tool is demonstrated by controlling gene expression through the controlled tether and release of a transcription factor. Inteins have already been employed to modulate proteins in synthetic contexts, however we observe that natural systems lack the avenues of control necessary to make them indispensable. We employ existing protein tools to construct a system of modular protein association, as well as drug and light inducible schema that reveal gaps in our knowledge of how to repurpose inteins in vivo. Despite this, we use inteins in the construction of a novel cargo delivery platform based on the fusogenic properties of the viral envelope glycoprotein from Vesicular stomatitis virus (VSV-G). We confirm reduced tropism of cargo delivery based on an intein lock-and-key mechanism that has implications for both biosafety as well as targeted delivery in vivo of natively folded proteins to target cells. / 2024-05-23T00:00:00Z