scholarly journals The dimeric form of CXCL12 binds to atypical chemokine receptor 1

2021 ◽  
Vol 14 (696) ◽  
pp. eabc9012
Author(s):  
Julia C. Gutjahr ◽  
Kyler S. Crawford ◽  
Davin R. Jensen ◽  
Prachi Naik ◽  
Francis C. Peterson ◽  
...  
Keyword(s):  
Tumor Metastasis ◽  
Mdck Cells ◽  
Titration Calorimetry ◽  
Resonance Spectroscopy ◽  
Biological Functions ◽  
Duffy Antigen ◽  
Chemokine Cxcl12 ◽  
Dimeric Form

The pleiotropic chemokine CXCL12 is involved in diverse physiological and pathophysiological processes, including embryogenesis, hematopoiesis, leukocyte migration, and tumor metastasis. It is known to engage the classical receptor CXCR4 and the atypical receptor ACKR3. Differential receptor engagement can transduce distinct cellular signals and effects as well as alter the amount of free, extracellular chemokine. CXCR4 binds both monomeric and the more commonly found dimeric forms of CXCL12, whereas ACKR3 binds monomeric forms. Here, we found that CXCL12 also bound to the atypical receptor ACKR1 (previously known as Duffy antigen/receptor for chemokines or DARC). In vitro nuclear magnetic resonance spectroscopy and isothermal titration calorimetry revealed that dimeric CXCL12 bound to the extracellular N terminus of ACKR1 with low nanomolar affinity, whereas the binding affinity of monomeric CXCL12 was orders of magnitude lower. In transfected MDCK cells and primary human Duffy-positive erythrocytes, a dimeric, but not a monomeric, construct of CXCL12 efficiently bound to and internalized with ACKR1. This interaction between CXCL12 and ACKR1 provides another layer of regulation of the multiple biological functions of CXCL12. The findings also raise the possibility that ACKR1 can bind other dimeric chemokines, thus potentially further expanding the role of ACKR1 in chemokine retention and presentation.

scholarly journals Phosphorylation of Serine 11 and Serine 92 as New Positive Regulators of Human Snail1 Function: Potential Involvement of Casein Kinase-2 and the cAMP-activated Kinase Protein Kinase A

2010 ◽  
Vol 21 (2) ◽  
pp. 244-253 ◽  
Author(s):  
Matthew Reid MacPherson ◽  
Patricia Molina ◽  
Serhiy Souchelnytskyi ◽  
Christer Wernstedt ◽  
Jorge Martin-Pérez ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Tumor Metastasis ◽  
Epithelial Mesenchymal Transition ◽  
Cop9 Signalosome ◽  
Mesenchymal Transition ◽  
Depth Analysis ◽  
Positive Regulators

Snail1 is a major factor for epithelial-mesenchymal transition (EMT), an important event in tumor metastasis and in other pathologies. Snail1 is tightly regulated at transcriptional and posttranscriptional levels. Control of Snail1 protein stability and nuclear export by GSK3β phosphorylation is important for Snail1 functionality. Stabilization mechanisms independent of GSK3β have also been reported, including interaction with LOXL2 or regulation of the COP9 signalosome by inflammatory signals. To get further insights into the role of Snail1 phosphorylation, we have performed an in-depth analysis of in vivo human Snail1 phosphorylation combined with mutational studies. We identify new phosphorylation sites at serines 11, 82, and 92 and confirmed previously suggested phosphorylations at serine 104 and 107. Serines 11 and 92 participate in the control of Snail1 stability and positively regulate Snail1 repressive function and its interaction with mSin3A corepressor. Furthermore, serines 11 and 92 are required for Snail1-mediated EMT and cell viability, respectively. PKA and CK2 have been characterized as the main kinases responsible for in vitro Snail1 phosphorylation at serine 11 and 92, respectively. These results highlight serines 11 and 92 as new players in Snail1 regulation and suggest the participation of CK2 and PKA in the modulation of Snail1 functionality.

scholarly journals Novel role of Snail 1 in promoting tumor neoangiogenesis

2019 ◽  
Vol 39 (5) ◽  
Author(s):  
Yi-Kun Zhang ◽  
Hua Wang ◽  
Yu-Wei Guo ◽  
Yang Yue
Keyword(s):  
Tumor Metastasis ◽  
Epithelial To Mesenchymal Transition ◽  
Tube Formation ◽  
Fibroblast Growth ◽  
Quantitative Real Time Pcr ◽  
Potential Therapeutic Target ◽  
Mesenchymal Transition ◽  
Tumor Neoangiogenesis

Abstract Snail1 plays an important role in epithelial to mesenchymal transition (EMT) during tumor metastasis; however, whether Snai1 potentiates the process of neoangiogenesis is completely unknown. In the present study, tube formation assay was used to evaluate neoangiogenesis in vitro. The expression of Snai1 and other pro-neoangiogenic factors was measured by quantitative real time PCR. Tumor derived endothelial cells (TDECs) were stimulated with fibroblast growth factor 1 (FGF1) or VEGF and formed more tubes compared with untreated, whereas cells treated with Sulforaphane had less tube formation. Silencing SNAI1 significantly attenuated tube formation accompanied by decreased CD31, CD34, and VWF expression in TDECs compared with control. In contrast, overexpression of Snai1 led to more CD31, CD34, and VWF expression and tube formation. To determine if the observed effects of SNAI1 on tube formation was a global phenomenon, the same assay was conducted in normal mesenchymal stem cells (MSCs). SNAI1 silencing did not have any effect on tube formation in MSCs. The expression of TIMP2, ENG, and HIF1A was up-regulated 3-fold or higher after silencing SNAI1, and ID1, VEGFA, PLG, LECT1, HPSE were shown down-regulated. Taken together, our study elucidates an important role of EMT inducer Snai1 in regulating tumor neoangiogenesis, suggesting a potential therapeutic target for overcoming tumor EMT.

Phospho-dependent phase separation of FMRP and CAPRIN1 recapitulates regulation of translation and deadenylation

Science ◽  
2019 ◽  
Vol 365 (6455) ◽  
pp. 825-829 ◽  
Author(s):  
Tae Hun Kim ◽  
Brian Tsang ◽  
Robert M. Vernon ◽  
Nahum Sonenberg ◽  
Lewis E. Kay ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Processing ◽  
Intrinsically Disordered Protein ◽  
Resonance Spectroscopy ◽  
Specific Interactions ◽  
Intrinsically Disordered ◽  
Functional Consequences ◽  
Translational Regulators

Membraneless organelles involved in RNA processing are biomolecular condensates assembled by phase separation. Despite the important role of intrinsically disordered protein regions (IDRs), the specific interactions underlying IDR phase separation and its functional consequences remain elusive. To address these questions, we used minimal condensates formed from the C-terminal disordered regions of two interacting translational regulators, FMRP and CAPRIN1. Nuclear magnetic resonance spectroscopy of FMRP-CAPRIN1 condensates revealed interactions involving arginine-rich and aromatic-rich regions. We found that different FMRP serine/threonine and CAPRIN1 tyrosine phosphorylation patterns control phase separation propensity with RNA, including subcompartmentalization, and tune deadenylation and translation rates in vitro. The resulting evidence for residue-specific interactions underlying co–phase separation, phosphorylation-modulated condensate architecture, and enzymatic activity within condensates has implications for how the integration of signaling pathways controls RNA processing and translation.

scholarly journals MPC1 Deficiency Promotes CRC Liver Metastasis via Facilitating Nuclear Translocation of β-Catenin

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Guang-Ang Tian ◽  
Chun-Jie Xu ◽  
Kai-Xia Zhou ◽  
Zhi-Gang Zhang ◽  
Jian-Ren Gu ◽  
...  
Keyword(s):  
Tumor Metastasis ◽  
Nuclear Translocation ◽  
Tca Cycle ◽  
Mitochondrial Pyruvate Carrier ◽  
Human Sample ◽  
Metastatic Capacity ◽  
Tcga Dataset

Accumulating evidence has pointed out that metastasis is the leading cause of death in several malignant tumor, including CRC. During CRC, metastatic capacity is closely correlated with reprogrammed energy metabolism. Mitochondrial Pyruvate Carrier 1 (MPC1), as the carrier of transporting pyruvate into mitochondria, linked the glycolysis and TCA cycle, which would affect the energy production. However, the specific role of MPC1 on tumor metastasis in CRC remains unexplored. Here, by data mining of genes involved in pyruvate metabolism using the TCGA dataset, we found that MPC1 was significantly downregulated in CRC compared to nontumor tissues. Similar MPC1 expression pattern was also found in multiple GEO datasets. IHC staining in both human sample and AOM/DSS induced mouse CRC model revealed significant downregulation of MPC1. What is more, we found that MPC1 expression was gradually decreased in normal tissue, primary CRC, and metastasis CRC. Additionally, poor prognosis emerged in the MPC1 low expression patients, especially in patients with metastasis. Following, functional tests showed that MPC1 overexpression inhibited the motility of CRC cells in vitro and MPC1 silencing enhanced liver metastases in vivo. Furthermore, we uncovered that decreased MPC1 activated the Wnt/β-catenin pathway by promoting nuclear translocation of β-catenin to mediate the expression of MMP7, E-cadherin, Snail1, and myc. Collectively, our data suggest that MPC1 has the potential to be served as a promising biomarker for diagnosis and a therapeutic target in CRC.

scholarly journals Overview of RNA G-quadruplex structures

2017 ◽  
Vol 63 (4) ◽  
Author(s):  
Magdalena Małgowska
Keyword(s):  
Three Dimensional ◽  
Telomere Maintenance ◽  
Structural Studies ◽  
Biological Functions ◽  
Cellular Processes ◽  
Novel Drugs ◽  
G Quadruplex ◽  
Conformational Diversity

G-quadruplexes are non-canonical secondary structures which may be formed by guanine rich sequences, both in vitro and in living cells. The number of biological functions assigned to these structural motifs has grown rapidly since the discovery of their involvement in the telomere maintenance. Knowledge of the three-dimensional structures of G-quadruplexes plays an important role in understanding their conformational diversity, physiological functions, and in the design of novel drugs targeting G-quadruplexes. For the last decades, structural studies have been mainly focused on the DNA G-quadruplexes. Their RNA counterparts gained an increased interest along with still-emerging recognition of the central role of RNA in multiple cellular processes. In this review we focus on structural properties of RNA G-quadruplexes, based on high-resolution structures, available in RCSB PDB data base and on structural models. In addition, we point out to the current challenges in this field of research.

scholarly journals A novel function of M. tuberculosis chaperonin paralog GroEL1 in copper homeostasis

2019 ◽  
Author(s):  
Mohammed Yousuf Ansari ◽  
Sakshi D. Batra ◽  
Hina Ojha ◽  
Ashish ◽  
Jaya S. Tyagi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Functional Divergence ◽  
Cellular Protein ◽  
Copper Stress ◽  
Titration Calorimetry ◽  
Evolutionary Divergence ◽  
Wild Type ◽  
Knock Out

AbstractMycobacterial GroELs namely GroEL1 and GroEL2 belong to the family of molecular chaperones, chaperonins. Chaperonins in Escherichia coli are termed as GroEL and GroES which are encoded by essential genes and are involved in cellular protein folding. GroEL1 has a characteristic Histidine-rich C-terminus contrary to its essential paralog GroEL2 and E. coli GroEL which have hydrophobic (GGM) repeats. Since Histidine richness is likely to be involved in metal binding, in this study we have attempted to decipher the role of GroEL1 protein in chelating metals and the consequent role on M. tuberculosis physiology. Using isothermal titration calorimetry (ITC), we found that GroEL1 binds copper, nickel and cobalt, with the highest binding affinity to copper. Since copper is known to be toxic at higher concentration, we cultured Wild Type M. tuberculosis H37Rv, groEL1 knock-out and groEL1-complemented strain with increasing concentrations of copper. We found that M. tuberculosis groEL1 knock out strain is more sensitive to copper than the wild type. Further hypothesizing that the probable mode of action of copper is by induction of oxidative stress, we attempted to understand the role of GroEL1 in redox silencing and hydroxyl radical mediated DNA damage. We interestingly found through our in vitro studies that GroEL1 is helpful in protection from copper stress through maintaining redox balance and free radical mediated DNA damage. Thus, these results indicate that the duplication of chaperonin genes in M. tuberculosis might have led to their evolutionary divergence and resulted in a functional divergence of chaperonins.

scholarly journals The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients

2020 ◽  
Vol 295 (37) ◽  
pp. 12900-12909
Author(s):  
Zongwei Guo ◽  
Ting Song ◽  
Ziqian Wang ◽  
Donghai Lin ◽  
Keke Cao ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Cell Fate ◽  
Protein Misfolding ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Allosteric Site ◽  
Transverse Relaxation ◽  
Bh3 Domain

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.

scholarly journals The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

2005 ◽  
Vol 389 (3) ◽  
pp. 869-876 ◽  
Author(s):  
Delphine Destoumieux-Garzón ◽  
Sophie Duquesne ◽  
Jean Peduzzi ◽  
Christophe Goulard ◽  
Michel Desmadril ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Size Exclusion ◽  
Receptor Interaction ◽  
Titration Calorimetry ◽  
Scanning Calorimetry ◽  
Recognition Mechanism ◽  
Microcin J25

The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB–ExbB–ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA− strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a Kd of 1.2 μM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand–receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11–Pro16 β-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.

scholarly journals Role of Calcium in Protein Folding and Function of Tva, the Receptor of Subgroup A Avian Sarcoma and Leukosis Virus

2001 ◽  
Vol 75 (5) ◽  
pp. 2051-2058 ◽  
Author(s):  
Qing-Yin Wang ◽  
Klavs Dolmer ◽  
Wen Huang ◽  
Peter G. W. Gettins ◽  
Lijun Rong
Keyword(s):  
Protein Folding ◽  
Calcium Binding ◽  
Titration Calorimetry ◽  
Viral Receptor ◽  
Calcium Affinity ◽  
Binding Residues ◽  
And Function ◽  
Avian Sarcoma

ABSTRACT Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd ) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 μM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.

scholarly journals Salmonella typhimurium LT2 Catabolizes Propionate via the 2-Methylcitric Acid Cycle

1999 ◽  
Vol 181 (18) ◽  
pp. 5615-5623 ◽  
Author(s):  
Alexander R. Horswill ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Salmonella Typhimurium ◽  
Negative Ion ◽  
Resonance Spectroscopy ◽  
Ionization Mass ◽  
Wild Type ◽  
Acid Cycle ◽  
Mutant Strains ◽  
Methylcitrate Synthase

ABSTRACT We previously identified the prpBCDE operon, which encodes catabolic functions required for propionate catabolism inSalmonella typhimurium. Results from13C-labeling experiments have identified the route of propionate breakdown and determined the biochemical role of each Prp enzyme in this pathway. The identification of catabolites accumulating in wild-type and mutant strains was consistent with propionate breakdown through the 2-methylcitric acid cycle. Our experiments demonstrate that the α-carbon of propionate is oxidized to yield pyruvate. The reactions are catalyzed by propionyl coenzyme A (propionyl-CoA) synthetase (PrpE), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (probably PrpD), 2-methylisocitrate hydratase (probably PrpD), and 2-methylisocitrate lyase (PrpB). In support of this conclusion, the PrpC enzyme was purified to homogeneity and shown to have 2-methylcitrate synthase activity in vitro.1H nuclear magnetic resonance spectroscopy and negative-ion electrospray ionization mass spectrometry identified 2-methylcitrate as the product of the PrpC reaction. Although PrpC could use acetyl-CoA as a substrate to synthesize citrate, kinetic analysis demonstrated that propionyl-CoA is the preferred substrate.

Sign in / Sign up

Export Citation Format

Share Document