Cytoplasmic Surface Research Articles

Single transmembrane GPCR modulating proteins: neither single nor simple.

The discovery of G-protein coupled receptor (GPCR) accessory proteins has fundamentally redefined the pharmacological concept of GPCR signaling, demonstrating a more complex molecular basis for receptor specificity on the plasma membrane and impressionable downstream intracellular cascades. GPCR accessory proteins not only contribute to the proper folding and trafficking of receptors, but also exhibit selectable receptor preferences. The melanocortin receptor accessory proteins (MRAP1 and MRAP2) as well as receptor activity-modifying proteins (RAMPs) are two well-known single transmembrane partners for the regulation of the melanocortin receptors (MC1R-MC5R) and the glucagon receptor (GCGR), respectively. Especially, MRAP family participates in the pathological control of multiple endocrine disorders and RAMPs contribute to the endogenous regulation of glucose homeostasis. However, the precise mechanisms by which the MRAP and RAMP proteins regulate receptor signaling at the atomic resolution remain unknown. Recent progress made in the determination of RAMP2-bound GCGR complexes published on Cell (Krishna Kumar et al., 2023) unraveled the importance of RAMP2 for the promotion of extracellular receptor dynamics leading to cytoplasmic surface inactivation. Moreover, the new findings on Cell Research (Luo et al., 2023) of the adrenocorticotropic hormone (ACTH)-bound MC2R-Gs-MRAP1 complex disclosed the essential role of MRAP1 for MC2R activation and specificity of ligand recognition. In this article, we reviewed a series of key findings of MRAP proteins in the last decade, and the recent structural investigation of MRAP-MC2R and RAMP-GCGR functional complex and the expanded identification of new GPCR partners of MRAP proteins. In-depth understanding of GPCR modulation by single transmembrane accessory proteins will provide valuable insights for the therapeutic drug development to treat multiple GPCR associated human disorders.

A novel force transduction pathway from a tension sensor to the gate in the mechano-gating of MscL channel.

The bacterial mechanosensitive channel of large conductance MscL is activated exclusively by increased tension in the membrane bilayer. Despite many proposed models for MscL opening, its precise mechano-gating mechanism, particularly how the received force at the tension sensor transmits to the gate remains incomplete. Previous studies have shown that along with amphipathic N-terminus located near the cytoplasmic surface of the membrane, Phe78 residue near the outer surface also acts as a "tension sensor," while Gly22 is a central constituent of the "hydrophobic gate." Present study focused on elucidating the force transmission mechanism from the sensor Phe78 in the outer transmembrane helix (TM2) to the gate in the inner transmembrane helix (TM1) of MscL by applying the patch clamp and molecular dynamics (MD) simulations to the wild type MscL channel and its single mutants at the sensor (F78N), the gate (G22N) and their combination (G22N/F78N) double mutant. F78N MscL resulted in a severe loss-of-function, while G22N MscL caused a gain-of-function channel exhibiting spontaneous openings at the resting membrane tension. We initially speculated that the spontaneous opening in G22N mutant might occur without tension acting on Phe78 residue. To test this hypothesis, we examined the (G22N/F78N) double mutant, which unexpectedly exhibited neither spontaneous activity nor activity by a relatively high membrane tension. To understand the underlying mechanism, we conducted MD simulations and analyzed the force transduction pathway. Results showed that the mutation at the tension sensor (F78N) in TM2 caused decreased interaction of this residue not only with lipids, but also with a group of amino acids (Ile32-Leu36-Ile40) in the neighboring TM1 helix, which resulted in an inefficient force transmission to the gate-constituting amino acids on TM1. This change also induced a slight tilting of TM1 towards the membrane plane and decreased the size of the channel pore at the gate, which seems to be the major mechanism for the inhibition of spontaneous opening of the double mutant channel. More importantly, the newly identified interaction between the TM2 (Phe78) and adjacent TM1 (Ile32-Leu36-Ile40) helices seems to be an essential force transmitting mechanism for the stretch-dependent activation of MscL given that substitution of any one of these four amino acids with Asn resulted in severe loss-of-function MscL as reported in our previous work.

Shaping membrane interfaces in lipid vesicles mimicking the cytoplasmic leaflet of myelin through variation of cholesterol and myelin basic protein contents

Myelin basic protein (MBP) is an intrinsically disordered protein and in the central nervous system (CNS) mainly responsible for connecting the cytoplasmic surfaces of the multilamellar, compact myelin. Increased posttranslational modification of MBP is linked to both, the natural development (from adolescent to adult brains) of myelin, and features of multiple sclerosis. Here, we study how a combination of this intrinsically disordered myelin protein with varying the natural cholesterol content may alter the characteristics of myelin-like membranes and interactions between these membranes. Large unilamellar vesicles (LUVs) with a composition mimicking the cytoplasmic leaflet of myelin were chosen as the model system, in which different parameters contributing to the interactions between the lipid membrane and MBP were investigated. While we use cryo-transmission electron microscopy (TEM) for imaging, dynamic light scattering (DLS) and electrophoretic measurements through continuously-monitored phase-analysis light scattering (cmPALS) were used for a more global overview of particle size and charge, and electron paramagnetic resonance (EPR) spectroscopy was utilized for local behavior of lipids in the vesicles' membranes in aqueous solution. The cholesterol content was varied from 060 % in these LUVs and measurements were performed in the presence and absence of MBP. We find that the composition of the lipid layers is relevant to the interaction with MBP. Not only the size, the shape and the aggregation behavior of the vesicles depend on the cholesterol content, but also within each membrane, cholesterol's freedom of movement, its environmental polarity and its distribution were found to depend on the content using the EPR-active spin-labeled cholesterol (CSOSL). In addition, DLS and EPR measurements probing the transition temperatures of the lipid phases allow a correlation of specific behavior with the human body temperature of 37 °C. Overall, our results aid in understanding the importance of the native cholesterol content in the healthy myelin membrane, which serves as the basis for stable and optimum protein-bilayer interactions. Although studied in this specific myelin-like system, from a more general and materials science-oriented point of view, we could establish how membrane and vesicle properties depend on cholesterol and/or MBP content, which might be useful generally when specific membrane and vesicle characteristics are sought for.

Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles

Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGFβ1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-α, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators.AbyDEs_V2PATMNaEJqximCVideo

Function and dynamics of the intrinsically disordered carboxyl terminus of β2 adrenergic receptor

Advances in structural biology have provided important mechanistic insights into signaling by the transmembrane core of G-protein coupled receptors (GPCRs); however, much less is known about intrinsically disordered regions such as the carboxyl terminus (CT), which is highly flexible and not visible in GPCR structures. The β2 adrenergic receptor’s (β2AR) 71 amino acid CT is a substrate for GPCR kinases and binds β-arrestins to regulate signaling. Here we show that the β2AR CT directly inhibits basal and agonist-stimulated signaling in cell lines lacking β-arrestins. Combining single-molecule fluorescence resonance energy transfer (FRET), NMR spectroscopy, and molecular dynamics simulations, we reveal that the negatively charged β2AR-CT serves as an autoinhibitory factor via interacting with the positively charged cytoplasmic surface of the receptor to limit access to G-proteins. The stability of this interaction is influenced by agonists and allosteric modulators, emphasizing that the CT plays important role in allosterically regulating GPCR activation.

Optimal Selection of IFN-α-Inducible Genes to Determine Type I Interferon Signature Improves the Diagnosis of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies specific to self-molecules in the nucleus, cytoplasm, and cell surface. The diversity of serologic and clinical manifestations observed in SLE patients challenges the development of diagnostics and tools for monitoring disease activity. Elevated type I interferon signature (IFN- I) in SLE leads to dysregulation of innate and adaptive immune function, resulting in autoantibodies production. The most common method to determine IFN-I signature is measuring the gene expression of several IFN-α-inducible genes (IFIGs) in blood samples and calculating a score. Optimal selection of IFIGs improves the sensitivity, specificity, and accuracy of the diagnosis of SLE. We describe the mechanisms of the immunopathogenesis of IFN-I signature (IFNα production) and its clinical consequences in SLE. In addition, we explore the association between IFN-I signature, the presence of autoantibodies, disease activity, medical therapy, and ethnicity. We discuss the presence of IFN-I signature in some patients with other autoimmune diseases, including rheumatoid arthritis, systemic and multiple sclerosis, Sjogren's syndrome, and dermatomyositis. Prospective studies are required to assess the role of IFIG and the best combination of IFIGs to monitor SLE disease activity and drug treatments.

Neuroglobin Facilitates Neuronal Oxygenation through Tropic Migration under Hypoxia or Anemia in Rat: How Does the Brain Breathe?

The discovery of neuroglobin (Ngb), a brain- or neuron-specific member of the hemoglobin family, has revolutionized our understanding of brain oxygen metabolism. Currently, how Ngb plays such a role remains far from clear. Here, we report a novel mechanism by which Ngb might facilitate neuronal oxygenation upon hypoxia or anemia. We found that Ngb was present in, co-localized to, and co-migrated with mitochondria in the cell body and neurites of neurons. Hypoxia induced a sudden and prominent migration of Ngb towards the cytoplasmic membrane (CM) or cell surface in living neurons, and this was accompanied by the mitochondria. In vivo, hypotonic and anemic hypoxia induced a reversible Ngb migration toward the CM in cerebral cortical neurons in rat brains but did not alter the expression level of Ngb or its cytoplasm/mitochondria ratio. Knock-down of Ngb by RNA interference significantly diminished respiratory succinate dehydrogenase (SDH) and ATPase activity in neuronal N2a cells. Over-expression of Ngb enhanced SDH activity in N2a cells upon hypoxia. Mutation of Ngb at its oxygen-binding site (His64) significantly increased SDH activity and reduced ATPase activity in N2a cells. Taken together, Ngb was physically and functionally linked to mitochondria. In response to an insufficient oxygen supply, Ngb migrated towards the source of oxygen to facilitate neuronal oxygenation. This novel mechanism of neuronal respiration provides new insights into the understanding and treatment of neurological diseases such as stroke and Alzheimer's disease and diseases that cause hypoxia in the brain such as anemia.

Surface charge induced bioelectricity generation from freshwater macroalgae Pithophora

A new concept of energy harvesting from the interaction of water molecule with macroalgal surface charges has been developed. The formation of electrical double layers on the charged surface of algae was utilized to generate the voltage and current between two aluminum electrodes along the length of the biofilm. In this study, a facile and efficient algal hydrovoltaic device based on the macroalgae Pithophora roettleri was demonstrated which can generate continuous electric power with a current of ∼4.4 μA and voltage ∼0.32 V. Seven series connected devices can glow an LED light, without any external energy. The superior performance of the device is attributed to the intrinsic properties of the Pithophora algal film, including its hydrophilicity, filamentous structure, conductivity and more. This study is providing a new way to research on hydrovoltaic power generation integrated with electrostatic charges present on algal cytoplasmic membrane or surface.

PGEX2-T based β defensin primer designing and isolation of neutrophil, macrophage, bone marrow and mesenchymal stem cells

Defensins are antimicrobial peptides that act mainly by disrupting the structure of bacterial cell membranes and are found in many compartments of the body. Defensins play a central role in defense against pathogens and they are considered part of the innate immune response. Material and methods-Reagents: Animal cell culture media - Growth medium D-MEM, RPMI and alpha -MEM was purchased from gibco, CA, USA. Fetal bovine serum (FBS)was obtained from Invitrogen, CA, USA. DAPI, agarose was purchased from SRL, India. Penicillium streptomycin solution was obtained from himedia, Mumbai, India. Animal modal: In bred populations of Swiss albino mice of either sex and at 6-8 weeks of age were used. Mice obtained from the Animal House, All India Institute of Medical Science, NewDelhi-110029 and India Standardization, Isolation and purification and characterization of bone marrow from Swiss albino mice. Bone marrow is consisting of multiple type of stem cells such as -Mesenchymal stem cells (MSCs) which are multipotent stem cells that have the potential to self-renew and differentiate into a variety of specialized cell types such as osteoblasts, chondrocytes, adipocytes, and neurons. Result: Cells were harvested by adherence purification, the adherence of cells which is a symbol of macrophage and acentric nucleus is visible in DAPI stain. The extended cytoplasmic surface and CD14 positive staining show the purified macrophage as 98% purity. BM-MSC were harvested by adherence purification, the adherence of cells to the plastic culture dish surface is a symbol of Mesenchymal stem cell and nucleusis visible in DAPI stain. Conclusion: This procedure is widely used to study macrophage biology since moderate numbers of resident, unstimulated macrophages can easily be obtained fromthe peritonealcavity.

Involvement of alpha-subunit N-termini in the mechanism and regulation of the Na+,K+- and H+,K+-ATPases.

The cytoplasmic lysine-rich N-termini of the Na+,K+- and H+,K+-ATPases have been designated as R domains because of their possible role in regulation of ion pumping activity. Prior to the publication of any crystal structures of either protein, it was suggested, based on mutagenesis studies, that regulation of the Na+,K+-ATPase could involve the formation and breakage of a salt bridge between Lys30 of the N-terminus and Glu233 in the first M2-M3 cytoplasmic loop of the alpha subunit. In structural studies of the Na+,K+-ATPase, the N-terminus has never been able to be resolved, probably because it is intrinsically disordered. Nevertheless, after theoretically predicting the N-terminus conformation, and adding it to the structure, the distance between residues Lys30 and Glu233 makes it doubtful that a direct long-lived interaction between them is involved in pump regulation. As an alternative hypothesis, we propose that regulation could occur via an interaction of the positively charged lysine residues of the N-terminus with negatively charged lipid headgroups (notably phosphatidylserine) on the cytoplasmic surface of the surrounding membrane, and that such an interaction could potentially be regulated by an electrostatic switch mechanism, whereby serine and tyrosine residues of the N-terminus are phosphorylated by protein kinases. Phosphorylation of these residues would decrease the positive charge of the N-terminus, allowing its release from the membrane. Experimental and theoretical data will be presented which support this hypothesis.

Structural basis for excited light transfer within bacterial photosynthetic supercomplex.

Single-particle cryogenic electron microscopy (cryo-EM) enables the visualization of membrane protein supercomplexes in their native-like conditions. In green sulfur bacteria (GSB), the photochemical reaction center (RC) features a homodimeric architecture for charge separation across the membrane. We extracted the photosynthetic supercomplex from Chlorobaculum tepidum cells with a mild detergent and determined high-resolution structures of the supercomplex using single-particle cryo-EM. In the cryo-EM density, the supercomplex is assembled based on a symmetric RC core with two Fenna-Matthews-Olson (FMO) trimers, two cytochrome c subunits (PscC), and small membrane subunits. The two FMO trimers bind to the dimeric RC cytoplasmic surface with different interfaces, resulting in an asymmetric feature of the supercomplex. The cryo-EM density also reveals two accessory protein subunits, PscE and PscF, which have not been identified before. We also discovered additional pigments within the complex, especially a linker bacteriochlorophyll (BChl) located in one of the two FMO-PscA interfaces, possibly mediating the exciton transfer from the FMO trimer to the RC core. We further computed the fluorescence resonance energy transfer (FRET) rate between determined pigments and found that the excited light energy transfer is biased on one of the two FMO-PscA branches. Our structure of the GSB photosynthetic supercomplex provides mechanistic insights into the routes for light excitation energy transfer and a possible evolutionary transition intermediate of the bacterial photosynthetic supercomplex from the primitive homodimeric RC.

Small molecule inhibitors of intestinal epithelial anion exchanger SLC26A3 (DRA) with a luminal, extracellular site of action

The anion exchanger protein SLC26A3 (down-regulated in adenoma, DRA) is expressed in the luminal membrane of intestinal epithelial cells in colon, where it facilitates the absorption of Cl− and oxalate. We previously identified a 4,8-dimethylcoumarin class of SLC26A3 inhibitors that act from the SLC26A3 cytoplasmic surface, and demonstrated their efficacy in mouse models of constipation and hyperoxaluria. Here, screening of 50,000 new compounds and 1740 chemical analogs of active compounds from the primary screen produced five novel classes of SLC26A3-selective inhibitors (1,3-dioxoisoindoline-amides; N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamides; thiazolo-pyrimidin-5-ones; 3-carboxy-2-phenylbenzofurans and benzoxazin-4-ones) with IC50 down to 100 nM. Kinetic washout and onset of action studies revealed an extracellular site of action for the thiazolo-pyrimidin-5-one and 3-carboxy-2-phenylbenzofuran inhibitors. Molecular docking computations revealed putative binding sites for these inhibitors. In a loperamide model of constipation in mice, orally administered 7-(2-chloro-phenoxymethyl)-3-phenyl-thiazolo [3,2-a]pyrimidin-5-one (3a) significantly increased stool weight, pellet number and water content. SLC26A3 inhibitors with an extracellular site of action offer the possibility of creating non-absorbable, luminally acting inhibitors with minimal systemic exposure following oral administration. Our findings also suggest that inhibitors of related SLC26 anion transporters with an extracellular site of action might be identified for pharmacological modulation of selected epithelial ion transport processes.

The structural integrity of the membrane-embedded bacterial division complex FtsQBL studied with molecular dynamics simulations

The FtsQBL is an essential molecular complex sitting midway through bacterial divisome assembly. To visualize and understand its structure, and the consequences of its membrane anchorage, we produced a model of the E. coli complex using the deep-learning prediction utility, AlphaFold 2. The heterotrimeric model was inserted into a 3-lipid model membrane and subjected to a 500-ns atomistic molecular dynamics simulation. The model is superb in quality and captures most experimentally derived structural features, at both the secondary structure and the side-chain levels. The model consists of a uniquely interlocking module contributed by the C-terminal regions of all three proteins. The functionally important constriction control domain residues of FtsB and FtsL are located at a fixed vertical position of ∼43–49 Å from the membrane surface. While the periplasmic domains of all three proteins are well-defined and rigid, the single transmembrane helices of each are flexible and their collective twisting and bending contribute to most structural variations, according to principal component analysis. Considering FtsQ only, the protein is more flexible in its free state relative to its complexed state—with the biggest structural changes located at the elbow between the transmembrane helix and the α-domain. The disordered N-terminal domains of FtsQ and FtsL associate with the cytoplasmic surface of the inner membrane instead of freely venturing into the solvent. Contact network analysis highlighted the formation of the interlocking trimeric module in FtsQBL as playing a central role in mediating the overall structure of the complex.

Bioinformatic Analysis of Na+, K+-ATPase Regulation through Phosphorylation of the Alpha-Subunit N-Terminus.

The Na+, K+-ATPase is an integral membrane protein which uses the energy of ATP hydrolysis to pump Na+ and K+ ions across the plasma membrane of all animal cells. It plays crucial roles in numerous physiological processes, such as cell volume regulation, nutrient reabsorption in the kidneys, nerve impulse transmission, and muscle contraction. Recent data suggest that it is regulated via an electrostatic switch mechanism involving the interaction of its lysine-rich N-terminus with the cytoplasmic surface of its surrounding lipid membrane, which can be modulated through the regulatory phosphorylation of the conserved serine and tyrosine residues on the protein's N-terminal tail. Prior data indicate that the kinases responsible for phosphorylation belong to the protein kinase C (PKC) and Src kinase families. To provide indications of which particular enzyme of these families might be responsible, we analysed them for evidence of coevolution via the mirror tree method, utilising coevolution as a marker for a functional interaction. The results obtained showed that the most likely kinase isoforms to interact with the Na+, K+-ATPase were the θ and η isoforms of PKC and the Src kinase itself. These theoretical results will guide the direction of future experimental studies.

Mechanotransduction in the pathogenesis of non-alcoholic fatty liver disease.

Mechanobiology is a domain of interdisciplinary research that aims to explore the impact of physical force, applied externally or internally, on cell and tissue function, including development, growth, and differentiation. Mechanotransduction is a term that describes how cells sense physical forces (such as compression, stretch, and shear stress), convert them into biochemical signals, and mount adaptive responses integrated by the nucleus. There is accumulating evidence that mechanical forces extensively inform the biological behaviour of liver cells in health and disease. Recent research has elucidated many cellular and molecular mechanisms involved in this process including the pleiotropic control and diverse effects of the paralogous transcription co-activators YAP/TAZ, which play a prominent role in mechanotransduction. The liver sinusoids represent a unique microenvironment in which cells are exposed to mechanical cues originating in the cytoskeleton and at interfaces with adjacent cells, the extracellular matrix, and vascular or interstitial fluids. In non-alcoholic fatty liver disease (NAFLD), hepatocellular lipid accumulation and ballooning, activation of inflammatory responses, dysfunction of liver sinusoidal endothelial cells, and transdifferentiation of hepatic stellate cells into a pro-contractile and pro-fibrotic phenotype have been associated with aberrant cycles of mechanosensing and mechanoresponses. The downstream consequences of disrupted mechanical homeostasis likely contribute to the progression of NAFLD and promote the development of portal hypertension, cirrhosis, and hepatocellular carcinoma. Identification of molecular targets involved in pathogenic mechanotransduction will allow for the development of novel strategies to prevent the progression of liver disease in NAFLD.

Integration of Transcriptome and Metabolome Analyses Reveals the Mechanistic Basis for Cadmium Accumulation in Maize.

Cadmium (Cd) pollution in soil has become a major environmental issue worldwide. However, the underlying molecular mechanism of low grain-Cd accumulation (GCA) in maize is still largely unknown. Herein, we report the mechanistic basis for low GCA in maize by a multiomics approach. The low GCA genotype L63 showed normal vacuolar formation and a lower capacity of xylem loading of Cd than the high-accumulator L42 under Cd stress. Transcriptomic sequencing identified 84 low-GCA-associated genes which are mainly involved in the S-adenosylmethionine (SAM) cycle, metal transport, and vacuolar sequestration. A metabolome analysis revealed that L63 plants had a more active SAM cycle and a greater capacity for terpenoid synthesis and phenylalanine metabolism than L42. Combining the analysis of transcriptome and metabolome characterized several genes as key genes involved in the determination of Cd accumulation. Our study identifies a mechanistic basis for low Cd accumulation in maize grains and provides candidate genes for genetic improvement of crops.

Butein suppresses PD-L1 expression via downregulating STAT1 in non-small cell lung cancer

PD-L1 (programmed cell death ligand 1) is frequently up-regulated in tumors and is critical in tumor immune escape. In addition to antibodies that block the interaction between PD-L1 and PD-1 (programmed cell death protein 1), small-molecule compounds that suppress PD-L1 expression also exhibit significant anti-tumor effects, emerging as a new strategy targeting PD-L1. By using a cell-based screening model, we found that butein, a natural chalcone compound, significantly reduced the cytoplasm and cell surface expression of PD-L1. This effect was further validated in various non-small cell lung cancer (NSCLC) cell lines and primary cells derived from clinical NSCLC tissues. Butein inhibited PD-L1 transcription, but not the half-life of PD-L1 protein. Butein reduced STAT1 level and butein-induced PD-L1 suppression was eliminated by the absence of STAT1. By co-culture system, butein improved tumor elimination by increasing the killing ability of CD8+ T cells. By in vivo study, we further confirmed that butein downregulated PD-L1 expression and improved infiltration of CD8+ T cells in tumor tissues. Taken together, our study suggested that butein could suppress the transcription of PD-L1 via downregulating STAT1, providing a theoretical basis for the application of butein in anti-tumor therapy.

Ultrafast Two-Dimensional Infrared Spectroscopy Resolved a Structured Lysine 159 on the Cytoplasmic Surface of the Microbial Photoreceptor Bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven microbial receptor, and lysine 159 (K159) is a charged residue on the cytoplasmic (CP) side of its E-F loop. However, its conformation and function remain unknown due to fast surface dynamics. By utilizing a 13C, 15N-labeled lysine (K) as an isotope probe, we created a network of site-specific amide-I vibrational signatures (backbone carbonyl stretch) to identify the frequency contribution of the labeled residues to the amide-I excitonic band structure. Thus, the red-shifted amide-I frequency in the 13C, 15N-lysine-labeled bR (uK-bR) to the unlabeled bR (WT-bR) could be differentiated and examined by ultrafast two-dimensional vibrational echo infrared (2D IR) spectroscopy. Our results showed that the backbone carbonyl of K159 is located at a high frequency of ca. 1693 cm-1 and has a vibrational excited-state relaxation time shorter than the bulk helical amide-I mode at the same frequency, suggesting that K159 may possess a hydrogen-bonded γ-turn structure with E161, one of the carboxylate residues on the CP surface of bR. The 2D solid-state NMR study of uK-bR also revealed conformational dependent lysine residues, from which K159 was found to involve the turn motif. This γ-turn structure maintained by K159 may help to stabilize the E-F loop and support E161 in attracting protons from the bulk during the late stage of the bR photocycle. The combined spectroscopic approach illustrated in this work may be applied to map residue-specific local structures and dynamics of other receptors and large proteins.

Constitutive PRMT5 Expression Drives Spontaneous Lymphoblastic Lymphoma In Vivo

Protein arginine methyltransferase 5 (PRMT5) is a highly conserved enzyme that catalyzes monomethylation and symmetric dimethylation of arginine residues on many target proteins, including histones and non-histone proteins (P53, P65/NFκB, E2F1, AKT and spliceosome proteins). (Shailesh, 2018) PRMT5 is overexpressed in numerous solid and hematologic malignancies. (Yang, 2013) It is associated with several oncogenic mechanisms, from driving the malignant phenotype to promoting tumor cell survival and invasiveness. While preliminary data has shown PRMT5 to be upregulated in T-ALL/LBL, the mechanism by which PRMT5 promotes T-ALL/LBL has yet to be established. (Mei, 2019) Preclinical transgenic models of leukemia and lymphoma driven by a variety of oncogenes (MYC, NOTCH) have identified PRMT5 activity as a critical component for the driver phenotype. (Li, 2015) To test the hypothesis that overexpression of this enzyme is capable of malignant driver activity, we developed a transgenic (Tg) mouse model designed to over-express PRMT5 in the lymphoid compartment. (Figure 1) Tg mice were created by injecting a linearized pBH-Eμ-PRMT5 vector into FVB/N pronuclei and implanted into pseudo-pregnant FVB/N mice. We obtained five founder lines crossed with wild-type FVB/N mice to obtain the F1 generation. RT-PCR for PRMT5 showed preferential overexpression in Tg B lymphocytes compared to control FVB/N mice (p<0.01) but also overexpression in T and NK cell compartments due to leaky Eμ phenotype. We observed a high percentage of F1 progeny from all five founder lines to develop spontaneous lymphoproliferative disease compared to control mice (p<0.001). Immunophenotypic and pathologic evaluation was performed by flow cytometry, immunohistochemistry, and clonality by IgH and TCR Vβ PCR analysis. Pathologic features showed a range of lymphoid neoplasms, with pre-T lymphoblastic immunophenotype being the most common. Tumor-bearing animals demonstrated diffuse involvement of all organs, peripheral blood, and bone marrow. Pre-T lymphoblastic lymphomas (LBL) showed expression of cytoplasmic CD3, surface CD4 (dim), TdT, and variable Ki67 with constitutive overexpression of PRMT5, MYC, CYCLIN D1, Ki67, and CD99. We developed a T-LBL cell line (Tg813) derived from a thymic tumor that displayed clonal TCR Vβ.17 gene rearrangement, intracellular TdT and CD3 expression with dim surface CD4 expression, immunophenotypic characteristics consistent with T-ALL/LBL. This cell line was maintained in culture and successfully passaged in immune-competent FVB mice, which led to disseminated disease, suggesting that PRMT5 drives an immune escape program in vivo. Preliminary data demonstrate that tumor-bearing animals display a prominent population of normal and neoplastic T-cell subsets with strong PD1 expression. PD1 expression in T-ALL/LBL has been linked with oncogenic function (Yang, 2019) and, in normal T-cell subsets, is a marker for exhaustion. (Lee, 2015) In addition, treatment of our Tg813 in vitro model with PRMT5 knockdown (CRISPR CAS9) and selective inhibition with a SAM-competitive inhibitor (C220 and PRT382, Prelude Therapeutics) showed a reduction of global symmetric dimethyl arginine, loss of the PRMT5 epigenetic mark H4R3(Sme2) and decreased proliferation with cell cycle arrest by day 6, findings consistent with PRMT5-dependent driver activity. Engraftment of Tg813 into SCID and immunocompetent FVB/N mice led to disseminated lymphomas (mean: 21 days). Tg813 engrafted animals treated with C220 (10mg/kg) showed improved survival compared to vehicle-treated mice (p<0.001). This preclinical model supports the hypothesis that PRMT5 over-expression provides cancer driver activity and a unique opportunity to study the role of this oncogene in an immune-competent, in vivo model system. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Изменение конформации и распределения гемоглобина при изменении объема эритроцита

The intracellular space of red blood cells (RBC) includes 40% hemoglobin (Hb) molecules and 60% being water molecules. When the intracellular volume changes, which is possible when erythrocyte cells pass through the blood stream, the possibility of redistribution of intracellular components of the cell due to its volume changes is assumed. By methods of optical spectroscopy (non-invasive methods of infrared spectroscopy, Raman-spectroscopy, laser interference microscopy) changes of morphology, conformation and redistribution of Hb have been revealed in the human erythrocyte due to an increase in the ratio of [Na+]in and [K+]in when Na+/K+-ATPase is blocked in the cell. The decrease of activity of Na+/K+-ATPase by a ouabaine (3 mM) has been found to lead not only to an increase in [Na+]in cell but also to an increase in positive charge on the cytoplasmic surface of the RBC membrane. In these conditions, changes in the conformation of both the heme and globine parts of the cytoplasmic Hb have been identified. It has been shown that cell depolarization, together with cell volume change, leads to a decrease in packing density of Hb molecule, which may be related to sorption of intracellular Na+ (or Ca2+) c Hb, as well as increasing the amount of water molecules in the cell and redistributin Hb in the cell. These processes can lead to a change in the conformation of Hb, as well as to a redistribution and alteration of the conformation of the cytoplasmic Hb.