新常态下商业银行和企业两部门杠杆联动的微观机制和宏观效应

本文通过构建一个具备微观基础的局部均衡模型，研究了企业和商业银行两部门杠杆联动与资本市场信贷风险溢价之间的动态关系。研究结果发现上述两者之间存在正相关性。这一结果丰富了当前新凯恩斯主义货币经济对信贷风险溢价的内涵，将金融杠杆纳入微观动态研究框架内。在宏观层面上，企业和商业银行两部门杠杆联动一方面会减弱企业投资动力，另一方面又会导致银行“惜贷”，导致“债务-通缩”加速。由此本文建议宏观政策制定者应着重从企业和金融两部门关注当前“去杠杆”进程，在保持相对宽松的货币政策前提下，辅以预期管理，为经济转型造成良好的宏观环境

新常态下商业银行和企业两部门杠杆联动的微观机制和宏观效应

本文通过构建一个具备微观基础的局部均衡模型，研究了企业和商业银行两部门杠杆联动与资本市场信贷风险溢价之间的动态关系。研究结果发现上述两者之间存在正相关性。这一结果丰富了当前新凯恩斯主义货币经济对信贷风险溢价的内涵，将金融杠杆纳入微观动态研究框架内。在宏观层面上，企业和商业银行两部门杠杆联动一方面会减弱企业投资动力，另一方面又会导致银行“惜贷”，导致“债务-通缩”加速。由此本文建议宏观政策制定者应着重从企业和金融两部门关注当前“去杠杆”进程，在保持相对宽松的货币政策前提下，辅以预期管理，为经济转型造成良好的宏观环境

Characterization of Myosin Phosphatase Targeting Subunit 2 Knockout Mice

UNIVERSITY OF CALIFORNIA SAN DIEGOCharacterization of Myosin Phosphatase Targeting Subunit 2 Knockout Mice A Thesis submitted in partial satisfaction of the requirements for the degree Master of ScienceinBiologybyTingfei HuCommittee in charge:Professor Renate B. Pilz, Chair Professor Julian Schroeder, Co-Chair Professor Katherine Petrie2021CopyrightTingfei Hu, 2021 All rights reserved.The Thesis of Tingfei Hu is approved, and it is acceptable in quality and form for publication on microfilm and electronically:____________________________________________________________________________________________________________________________________________________________ Co-Chair______________________________________________________________________________ ChairUniversity of California San Diego2021DEDICATIONI would like to dedicate this to my family for always supporting me and enabling me to pursue my interests.TABLE OF CONTENTS Signature Page ................................................................................................... iii Dedication .......................................................................................................... iv Table of Contents ............................................................................................... v List of Figures .................................................................................................... vi Acknowledgements ............................................................................................ vii Abstract of the Thesis ......................................................................................... viii Introduction ........................................................................................................ 1 Materials and Methods ....................................................................................... 7 Results ................................................................................................................ 11 Discussion .......................................................................................................... 23 References .......................................................................................................... 25LIST OF FIGURES Figure 1. Quaternary Structure of Muscle Myosin ............................................... 2 Figure 2. Schematic Representation of Myosin Phosphatase Complex................ 6 Figure 3. Identification of MYPT2 Knockout Mice ............................................. 12 Figure 4. Antibody Test ........................................................................................ 14 Figure 5. Morphometric Measurements ............................................................... 16 Figure 6. Echocardiogram Measurements ............................................................ 18 Figure 7. Alterations in Hypertrophic Molecular Markers.................................... 22ACKNOWLEDGEMENTS I would like to first thank Dr. Renate B. Pilz and Dr. Gerry R. Boss for the opportunity to join the lab and be part of their team. Under Dr. Renate B. Pilz’s guidance the past few years, I have gained valuable insights and advice ranging from laboratory related subject matter to plausible career paths. I would also like to thank Dr. Darren Casteel for mentoring me the past few years and guiding me to become a better researcher. I would not have been able to grow in my research without his dedication to teaching and mentoring. I would also like to thank Dr. Julian Schroeder and Dr. Katherine Petrie for being my committee members. Finally, I am grateful to Dr. Renate B. Pilz and Darren Casteel for their editorial advice of my written thesis and PowerPoint presentation; additionally, I would like to thank members of the Boss-Pilz Laboratory for their helpful advice and discussion points. Results are currently being prepared for submission for publication of the material. Pilz, Renate; Hu, Tingfei; and Casteel, Darren. The thesis author will be co-author of this material.ABSTRACT OF THE THESIS Characterization of Myosin Phosphatase Targeting Subunit 2 Knockout Mice ByTingfei Hu Master of Science in Biology University of California San Diego, 2021Professor Renate B. Pilz, Chair Professor Julian Schroeder, Co- Chair Proper cardiac contraction is vital to maintain cardiovascular health and is modulated by the phosphorylation state of myosin regulatory light chain (MLC). The extent of MLC phosphorylation is dependent on the activities of both MLC kinase and MLC phosphatase. One prominent MLC phosphatase subunit found in cardiac muscle is Myosin Phosphatase Targeting Subunit 2 (MYPT2). It was previously shown that an overexpression of MYPT2 resulted in a decreased level of MLC phosphorylation, resulting in reduction of left ventricular contraction and induction of hypertrophy. However, no studies have examined the effect of knocking out MYPT2 in mice. Therefore, we obtained heterozygous mice, containing a MYPT2 null allele from the Mutant Mouse Resource Center, to examine their phenotype and signaling pathways involved in cardiac cell growth. Phenotypically, we observed that MYPT2 knockout mice had decreased heart size and weight, with increased fractional shortening, as assessed by cardiac echogram. For the signaling pathways, we found no alteration in AKT or ERK signaling using western blot analysis; however, qPCR analysis showed that MYPT2 knockout showed an upregulation of atrial natriuretic peptide and downregulation β-myosin heavy chain transcripts. These studies provide evidence for the critical role that MYPT2 plays in normal cardiac function.Introduction1.1 Striated Muscle Contraction Overview The sarcomere is the basic contractile unit in myocytes (1). An individual sarcomere is composed of both thin and thick filaments that slide past each other during muscle contraction and relaxation. The thin filament consists of two intertwined chains of actin with regulatory proteins troponin and tropomyosin, whereas the thick filament consists of organized bundles of myosin. When an electrochemical signal propagates down a myocyte, Ca2+ diffuses out of the sarcoplasmic reticulum and is bound to troponin. The transient attachment of Ca2+ to troponin exposes the myosin-binding sites on the actin filament, positioning myosin to its on conformation. This orientation enables the attachment of myosin head to the myosin-binding site, forming the actin-myosin crossbridge. The formation of the crossbridge permits muscle contraction as myosin pulls actin towards the M-line of the sarcomere. Following muscle contraction is muscle relaxation. When the electrochemical signal stops, Ca2+ is actively pumped back into the sarcoplasmic reticulum, detaching actin from myosin, and enabling another cycle of muscle contraction. 1.2 Structure of Myosin One functional unit of myosin contains both the myosin light chains (MLC) and myosin heavy chains (MHC) (2). The two major types of MLCs are the essential and the regulatory MLC. The three different subdomains of MHC are the coiled coil domain which mediates dimerization of two heavy chains, a lever arm with binding sites for MLC, and a head domain which forms actin-myosin cross bridges (Figure 1). The phosphorylation state of the regulatory MLC modulates the binding of the MHC head to actin, permitting muscle contraction and relaxation (3,4).Figure 1. Quaternary Structure of Muscle Myosin: Myosin is a motor protein composed of two heavy chains, two essential light chains, and two regulatory light chains. The heavy chain heads are shown in blue; the heavy chain tail is shown in green. The essential light chains are shown in pink, and the regulatory light chains are shown in orange. 1.3 Posttranslational Modification of Myosin Cardiac muscle contraction is influenced by varying posttranslational modifications of both thick and thin filaments. In this study, we focus on the phosphorylation of regulatory MLC, which is part of the thick filament. When the MLC of cardiac striated muscle is phosphorylated, contraction is not directly initiated but rather modulated. Contraction is directly initiated by Ca2+, which exposes the myosin binding site on actin and enables the attachment of MHC head, leading to actin-myosin power stroke. The phosphorylation state of MLC modulates cardiac contraction by altering the efficiency of MHC head and actin interaction (3). Phosphorylated MLC increases the efficiency of actin-myosin binding by orienting the MHC head perpendicular to actin; de-phosphorylated MLC decreases the efficiency of actin-myosin binding by reorienting the MLC head parallel to actin. 1.4 Myosin Light Chain Phosphatase The phosphorylation status of MLC is dependent on both the MLC kinases and MLC phosphatases. However, MLC kinases and MLC phosphatases of cardiac muscles have not been well characterized. MLC phosphatases are heterotrimers, composed of three unique subunits (5). It contains PP1cδ (a catalytic subunit), a small subunit (M20) of relatively unknown function, and a myosin binding subunit (MYPT) which contains binding sites for PP1cδ, myosin, and M20 (Figure 2). The diversity of MLC phosphatases is owed to the varying MYPT subunits, which include MYPT1, MYPT2, MBS85, MYPT3, and TIMAP (6). The identity of the MYPT is dependent on the tissue type. The prominent regulatory phosphatase subunits we focus on for this study are MYPT1 (115-kDa) and MBS85 (85-kDa) for smooth muscles and MYPT2 (110-kDa) for striated cardiac muscles. These three MYPT are the product of different genes. The MYPT1 gene is located on chromosome 12q15 – q21.2; the MYPT2 gene is located on chromosome 1q32.1; and the MBS85 gene is located on 19q13.42. When amino acid sequences are compared to MYPT1, the overall sequence similarities are 61% for MYPT2 and 39% for MBS85. The binding of MYPT family proteins to PP1cδ targets MLC phosphatase to region specific myosin, such as those in smooth and striated muscles. MYPT also regulates PP1cδ catalytic activity. Binding of the MYPT N-terminus to PP1cδ increases PP1cδ phosphatase activity (7,8); however, phosphorylation of residues in the MYPT C-terminus (by ROCK or other kinases), leads to inhibition of catalytic activity (9-12). The phosphorylated MYPT residue is located in a structural loop that binds to the PP1cδ catalytic site and blocks MLC access (13). Figure 2. Schematic Representation of Myosin Phosphatase Complex.The PP1Cδ binding domain is shown in blue, the M20 is shown in yellow, and the myosin phosphatase regulatory subunit is shown in pink. 1.5 Smooth and Striated Muscle Relaxation and Contraction Both smooth and striated muscles contain MLC phosphatase that is composed of PP1cδ, MYPT, and M20. The binding of MYPT to PP1cδ enhances the hydrolysis activity of PP1cδ on the phosphate of either the serine or threonine residues of MLC, resulting in effects that depend on the tissue type and identity of the MYPT subunit. De-phosphorylation of MLC by the MYPT1-containing complex, predominantly found in smooth muscle cells, directly drives relaxation and regulates vascular tone (14); whereas de-phosphorylation of MLC by the MYPT2-containing complex in striated muscle cells, only modulates muscle relaxation. In cardiac myocytes, the cycle of contraction and relaxation is mainly driven by Ca2+ levels. 1.6 Characterization of Mice Overexpressing MYPT2 Transgenic mice (Tg) with cardiac specific MYPT2 overexpression showed a decrease in the amount of phosphorylated MLC (14). These Tg mice appeared and behaved normally when compared to wild-type mice. However, it was found that cardiac specific overexpression of MYPT2 led to hypertrophic cardiomyopathy, and that the degree of hypertrophy correlated with the extent of MYPT2 overexpression. H&E staining of heart tissue showed that overexpression of MYPT2 led to an increase in left ventricular dimensions and an increase in heart to body weight ratio, indicating an enlargement of the heart. Overexpression of MYPT2 also led to increased expression of brain natriuretic peptide and β-myosin heavy chain, which are molecular markers for hypertrophy. The mice showed an increased in the end-systolic and end-diastolic volumes due to reduced fractional shortening. Since my project is focused on MYPT2 knockout mice, we expect to see the opposite phenotypic findings when we characterize MYPT2 knockout mice. Materials and Methods 2.1 Materials MYPT2 knockout mice were produced by the Mutant Mouse Resource Center and obtained from Jackson laboratory (MMRRC Stock No: 46179-JAX). Dulbecco’s Modified Eagle Medium (DMEM) was from Cellgro. Fetal bovine serum (FBS) was from Sigma-Aldrich. Restriction enzymes were from New England Biolabs. Protease inhibitor cocktail was from EMD Millipore Corporation (Cat#: 539131). Anti-Flag antibody was from Sigma-Aldrich. Rabbit anti-MYPT1/2 was from Abcam (Cat#: ab32519). Anti-PP1cδ antibody was from Origene (Cat#: TA308937). HRP conjugated anti-β-actin antibody was from Santa Cruz Biotechnology (Cat#: sc-47778-HRP). Peroxidase-conjugated Goat anti-Rabbit IgG was from Jackson ImmunoResearch (Cat#: 111-035-003). SuperSignal West Pico Plus was from Pierce (Cat#: 34580). IRDye 680RD Goat anti-mouse and IR DYE 800CW Goat anti-Rabbit, and Intercept Blocking Buffer were from LI-COR. Other general chemicals and supplies were from Fisher Scientific or Sigma-Aldrich. 2.2 Cell culture 293T cells were grown in Dulbecco's Modified Eagle Medium DMEM (Cellgro) with 10% fetal calf serum (Sigma-Aldrich) at 37°C with 5% CO2 in a water jacketed incubator. Stock cells were grown in 10 cm tissue culture dishes and cells were split every two to three days using 0.25% trypsin/EDTA (Gibco). 2.3 MYPT2 Knockout Mice All animal experiments were approved by the Institutional Care and Use Committee of the University of California, San Diego. 12 or 25 weeks old Mice were used (C57BL/6NJ-Ppp1r12bem1(IMPC)J/Mmjax) to generate data. They were housed in a temperature-controlled environment with a 12/12 h light/dark cycle. Mice were weaned at day 18, and genotyped by polymerase chain reaction (PCR) and agarose gel electrophoresis using genomic DNA from approximately 2 mm of clipped mouse tail. The genomic DNA was obtained by incubation of the tail in 75μL of 25mM NaOH/0.2mM EDTA at 98°C for 1h. The tube was then cooled to room temperature before 75μL of 40mM tris HCl (pH 5.5) was added. Sample was next vortexed and 1 μL was aliquoted for PCR. The PCR reaction was run using HotStart Taq polymerase (BioPioneer, catalog #: MAT-4) along with the following primers: 5’-TGTTTTCCCTACTTGCTGTG-3’ and 5’-GCTTGTGTTCTGCTGTTTG-3’. The wild-type gene product was 775 bp and the mutant gene product was 195 bp. 2.4 Expression Vectors and Transfection The expression vectors of MYPT1, MYPT2, and MBS85 were engineered with N-terminal FLAG tags. They were then transfected into 293T cells using Lipofectamine2000 (ThermoFisher). For each transfected sample, DNA/Opti-MEM and Lipofectamine2000/Opti-MEM mixtures were incubated for 5-minute at room temperature. The two incubations were mixed and incubated for an additional 20 min at room temperature. The mixtures were then added to 293T cells and kept at 37°C for 24 h before harvesting. 2.5 Western blot For transfected 293T cells, lysates were prepared by aspirating the media from the cells and directly scraping in ice cold Lysis Buffer 1 (phosphate buffered saline, 0.1% NP40 and protease inhibitor cocktail). Lysates were cleared by centrifugation at 16,000g for 10 minutes at 4° C, and cleared lysates were mixed 2:1 in 3x in sodium dodecyl sulfate sample (SDS) sample buffer. Equal amounts of lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE). For transgenic MYPT2 knockout mice, frozen tissues were pulverized, resuspended in 1x SDS sample buffer, and sonicated 2x 20 seconds on ice. Proteins were quantified using a Bradford assay, and 80 μg protein from each tissue were separated by SDS-PAGE. Proteins were transferred to Polyvinylidene difluoride (PVDF) membrane and blocked in 5% milk in Tris-buffered saline (TBS). For blots using infrared dye (IR Dye) secondary antibodies, proteins were transferred to Immobilon-FL PVDF membrane and blocked in Intercept Blocking Buffer (LI-COR). Western blots were performed with antibodies directed against Flag-epitope (1:5000), MYPT1/2/MBS85 (1:2000), PP1Cδ (1:2000) and HRP-conjugated β-actin (1:10,000). HRP-conjugated secondary antibodies were used at 1:5000 and IR-dye secondary antibodies were used at 1:10,000). Antibodies were diluted in the blocking buffer used for each condition. HRP blots were visualized using SuperSignal West Pico Plus chemiluminescent western blotting substrate and IR blots were visualized using an Odyssey Imaging System (LI-COR). 2.6 Quantitative RT-PCR Within five minutes of euthanasia of MYPT2 knockout mice, hearts were snap-frozen in liquid nitrogen then pulverized and resuspended in Trizol (Molecular Res.Center, TR118). Total RNA was isolated as per the manufacturer’s instructions. Total RNA was reverse-transcribed using iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed using a MX3005P real-time PCR detection system with Brilliant II SYBR Green Mix (Agilent Technologies) as described. All primers were intron-spanning and were tested with serial cDNA dilutions. Relative changes in mRNA expression were analyzed using the comparative 2−ΔΔCt method. 2.7 Echocardiography To perform echocardiography, 24-week-old male mice had their chest hair removed using Nair hair removal lotion. Mice were anesthetized with 5% isoflurane and maintained on 0.5% isoflurane. Electrocardiograms were monitored with probes inserted into the upper and lower torso. Cardiac function was recorded with a VisualSonics, SonoSite FUJIFILM, Vevo 2100 ultrasound system with a linear transducer 32–55 MHz by a single, highly experienced operator who was blinded to the genotype of the mice. 2.8 Statistical Analysis Data are reported as mean ± standard deviation. Comparisons between groups were performed by two-tailed Student’s T-Test. A value of p < 0.05 was considered statistically significant. Results 3.1 Homozygous MYPT2 Knockout Mice are Viable It was previously shown that global homozygous MYPT1 knockout mice are not viable (15). Whether global homozygous MYPT2 knockout mice would be non-viable as well is unknown. We obtained male and female heterozygous MYPT2 knockout mice from the Jackson laboratory, generated with the CRISPR/Cas9 technology by the Mutant Mouse Resource Center, and set up breeding between them. We found that homozygous MYPT2 knockout mice were born and viable as determined by genotyping at the time of weaning (Figure 3A). The knockout mice were kept to at least 24-weeks of age, and they appeared normal and showed no obvious health or behavioral issues when compared to wild-type mice. To examine MYPT expression, we used heart lysates from 24-week-old mice and performed Western blot using MYPT1/2 antibodies. As seen in Figure 3B, MYPT2 expression was lower in heterozygous mice and absent in the homozygous knockout mice. Expression of catalytic PP1Cδ subunit also decreased when MYPT2 expression was decreased. MYPT1 expression was much lower than MYPT2 expression and remained relatively constant throughout the three genotypes, suggesting no apparent compensatory upregulation of MYPT1. There was no MBS85 expression detected in the heart lysates. AB Figure 3. Identification of MYPT2 Knockout Mice. (A) Agarose gel electrophoresis of MYPT2 genotyping PCR products in wild-type (+/+), heterozygous (+/-), and homozygous (-/-) mice. The wild-type allele produced a 775 base pair product, and the mutant allele produced a 195 base pair product. (B) Western blots of heart lysates from 24-week-old mice for myosin regulatory subunits and PP1Cδ for +/+, +/-, and -/- mice. 3.2 MYPT1/2 Antibody Test MYPT1, MYPT2, and MBS85 are homologues of each other, with MYPT1 and MYPT2 being closely related and MBS85 being more distant. Since these proteins have similar amino acid sequences, we used a commercially available antibody known to cross-recognize MYPT1 and MYPT2. However, its recognition of MBS85 and its sensitivity to each protein are unknown. To assess the antibody’s specificity and sensitivity, we transfected 293T cells with Flag-tagged expression vectors for MYPT1, MYPT2, and MBS85. The cell lysates were then analyzed by Western blot and probed with an anti-Flag antibody followed by an infra-red IR-Dye 680RD secondary antibody. Imaging was done using a LI-COR Odyssey Fc system. As seen in Figure 3B, the expression of MYPT1 and MYPT2 were similar, and the expression of MBS85 was ~5-fold higher. Probing the same membrane with the anti-MYPT1/2 antibody followed by an IR-Dye 800CW secondary, we again observe similar expression levels of MYPT1 and MYPT2 (Figure 4B). Interestingly, the apparent MBS85 signal was only ~1.4-fold higher than that of MYPT1/2, indicating that the antibody was less sensitive toward MBS85.AB Figure 4. Antibody Test. (A) Cell lysates from 293T cells expressing Flag-tagged MYPT1, MYPT2, or MBS85 were probed with mouse anti-Flag antibody followed by an IR Dye 680RD antibody Protein expression levels were quantified using a LI-COR Odyssey imaging system. (B) The same blot, as shown in A, was probed with rabbit anti-MYPT1/2 antibody followed by an IR Dye 800CW antibody. Proteins were quantified as described for A.3.3 Body and Tissue Weights Since global MYPT2 knockout mice have yet to be characterized, we assessed whether knocking out MYPT2 has a general effect on the body and tissue weights. The body weights were not significantly diff

PKGIα is activated by metal-dependent oxidation in vitro but not in intact cells.

Type I cGMP-dependent protein kinases (PKGIs) are important components of various signaling pathways and are canonically activated by nitric oxide- and natriuretic peptide-induced cGMP generation. However, some reports have shown that PKGIα can also be activated in vitro by oxidizing agents. Using in vitro kinase assays, here, we found that purified PKGIα stored in PBS with Flag peptide became oxidized and activated even in the absence of oxidizing agent; furthermore, once established, this activation could not be reversed by reduction with DTT. We demonstrate that activation was enhanced by addition of Cu2+ before storage, indicating it was driven by oxidation and mediated by trace metals present during storage. Previous reports suggested that PKGIα Cys43, Cys118, and Cys196 play key roles in oxidation-induced kinase activation; we show that activation was reduced by C118A or C196V mutations, although C43S PKGIα activation was not reduced. In contrast, under the same conditions, purified PKGIβ activity only slightly increased with storage. Using PKGIα/PKGIβ chimeras, we found that residues throughout the PKGIα-specific autoinhibitory loop were responsible for this activation. To explore whether oxidants activate PKGIα in H9c2 and C2C12 cells, we monitored vasodilator-stimulated phosphoprotein phosphorylation downstream of PKGIα. While we observed PKGIα Cys43 crosslinking in response to H2O2 (indicating an oxidizing environment in the cells), we were unable to detect increased vasodilator-stimulated phosphoprotein phosphorylation under these conditions. Taken together, we conclude that while PKGIα can be readily activated by oxidation in vitro, there is currently no direct evidence of oxidation-induced PKGIα activation in vivo

A substitution in cGMP-dependent protein kinase 1 associated with aortic disease induces an active conformation in the absence of cGMP

Type 1 cGMP-dependent protein kinases (PKGs) play important roles in human cardiovascular physiology, regulating vascular tone and smooth-muscle cell phenotype. A mutation in the human PRKG1 gene encoding cGMP-dependent protein kinase 1 (PKG1) leads to thoracic aortic aneurysms and dissections. The mutation causes an arginine-to-glutamine (RQ) substitution within the first cGMP-binding pocket in PKG1. This substitution disrupts cGMP binding to the pocket, but it also unexpectedly causes PKG1 to have high activity in the absence of cGMP via an unknown mechanism. Here, we identified the molecular mechanism whereby the RQ mutation increases basal kinase activity in the human PKG1α and PKG1β isoforms. Although we found that the RQ substitution (R177Q in PKG1α and R192Q in PKG1β) increases PKG1α and PKG1β autophosphorylation in vitro, we did not detect increased autophosphorylation of the PKG1α or PKG1β RQ variant isolated from transiently transfected 293T cells, indicating that increased basal activity of the RQ variants in cells was not driven by PKG1 autophosphorylation. Replacement of Arg-177 in PKG1α with alanine or methionine also increased basal activity. PKG1 exists as a parallel homodimer linked by an N-terminal leucine zipper, and we show that the WT chain in WT-RQ heterodimers partly reduces basal activity of the RQ chain. Using hydrogen/deuterium-exchange MS, we found that the RQ substitution causes PKG1β to adopt an active conformation in the absence of cGMP, similar to that of cGMP-bound WT enzyme. We conclude that the RQ substitution in PKG1 increases its basal activity by disrupting the formation of an inactive conformation