Flagellar microtubule doublet assembly in vitro reveals a regulatory role of tubulin C-terminal tails

Science ◽  
2019 ◽  
Vol 363 (6424) ◽  
pp. 285-288 ◽  
Author(s):  
M. Schmidt-Cernohorska ◽  
I. Zhernov ◽  
E. Steib ◽  
M. Le Guennec ◽  
R. Achek ◽  
...  
Keyword(s):  
Intraflagellar Transport ◽  
Structural Basis ◽  
Ciliary Beating ◽  
Protein Partners ◽  
A Cell ◽  
Carboxyl Terminal ◽  
Microtubule Doublet

Microtubule doublets (MTDs), consisting of an incomplete B-microtubule at the surface of a complete A-microtubule, provide a structural scaffold mediating intraflagellar transport and ciliary beating. Despite the fundamental role of MTDs, the molecular mechanism governing their formation is unknown. We used a cell-free assay to demonstrate a crucial inhibitory role of the carboxyl-terminal (C-terminal) tail of tubulin in MTD assembly. Removal of the C-terminal tail of an assembled A-microtubule allowed for the nucleation of a B-microtubule on its surface. C-terminal tails of only one A-microtubule protofilament inhibited this side-to-surface tubulin interaction, which would be overcome in vivo with binding protein partners. The dynamics of B-microtubule nucleation and its distinctive isotropic elongation was elucidated by using live imaging. Thus, inherent interaction properties of tubulin provide a structural basis driving flagellar MTD assembly.

scholarly journals PapRIV, a BV-2 microglial cell activating quorum sensing peptide

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yorick Janssens ◽  
Nathan Debunne ◽  
Anton De Spiegeleer ◽  
Evelien Wynendaele ◽  
Marta Planas ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Cell Model ◽  
Dependent Manner ◽  
Communication Signals ◽  
Gram Positive Bacteria ◽  
A Cell ◽  
Gastro Intestinal Tract

AbstractQuorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood–brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction.

The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1302-1313 ◽  
Author(s):  
Cheng-Hsiang Kuo ◽  
Po-Ku Chen ◽  
Bi-Ing Chang ◽  
Meng-Chen Sung ◽  
Chung-Sheng Shi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Egf Receptor ◽  
Tube Formation ◽  
Endothelial Tube Formation ◽  
A Cell ◽  
Lewis Y ◽  
Tm Domain

AbstractLewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM—TM domain 1 (rTMD1)—in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.

scholarly journals Recombinant Listeria monocytogenes Expressing a Cell Wall-Associated Listeriolysin O Is Weakly Virulent but Immunogenic

2009 ◽  
Vol 77 (10) ◽  
pp. 4371-4382 ◽  
Author(s):  
Javier A. Carrero ◽  
Boris Calderon ◽  
Hector Vivanco-Cid ◽  
Emil R. Unanue
Keyword(s):  
Listeria Monocytogenes ◽  
Listeriolysin O ◽  
Wild Type ◽  
Positive Bacterium ◽  
Cell Responses ◽  
A Cell ◽  
Weakly Virulent

ABSTRACT Listeriolysin O (LLO) is an essential virulence factor for the gram-positive bacterium Listeria monocytogenes. Our goal was to determine if altering the topology of LLO would alter the virulence and toxicity of L. monocytogenes in vivo. A recombinant strain was generated that expressed a surface-associated LLO (sLLO) variant secreted at 40-fold-lower levels than the wild type. In culture, the sLLO strain grew in macrophages, translocated to the cytosol, and induced cell death. However, the sLLO strain showed decreased infectivity, reduced lymphocyte apoptosis, and decreased virulence despite a normal in vitro phenotype. Thus, the topology of LLO in L. monocytogenes was a factor in the pathogenesis of the infection and points to a role of LLO secretion during in vivo infection. The sLLO strain was cleared by severe combined immunodeficient (SCID) mice. Despite the attenuation of virulence, the sLLO strain was immunogenic and capable of eliciting protective T-cell responses.

scholarly journals Structural basis of peptidoglycan endopeptidase regulation

2020 ◽  
Vol 117 (21) ◽  
pp. 11692-11702 ◽  
Author(s):  
Jung-Ho Shin ◽  
Alan G. Sulpizio ◽  
Aaron Kelley ◽  
Laura Alvarez ◽  
Shannon G. Murphy ◽  
...  
Keyword(s):  
Cell Wall ◽  
Structural Basis ◽  
Bacterial Cells ◽  
Cell Wall Degradation ◽  
Open Conformation ◽  
Poor Understanding ◽  
A Cell ◽  
Inhibitory Domain

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogenVibrio cholerae. Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogensNeisseria gonorrheaeandEscherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart

2014 ◽  
Vol 70 (2) ◽  
pp. 242-252 ◽  
Author(s):  
Sonia Fieulaine ◽  
Michel Desmadril ◽  
Thierry Meinnel ◽  
Carmela Giglione
Keyword(s):  
Sequence Similarity ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Human Counterpart ◽  
Low Activity

Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of theN-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeablein vivoand display similar propertiesin vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

scholarly journals OXPHOS Promotes Apoptotic Resistance and Persistence in TH17 cells

2021 ◽  
Author(s):  
Hanna S. Hong ◽  
Nneka E. Mbah ◽  
Mengrou Shan ◽  
Kristen Loesel ◽  
Lin Lin ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Cell Fate ◽  
Apoptotic Cell ◽  
Metabolic Phenotype ◽  
Tumor Studies ◽  
A Cell

AbstractApoptotic cell death is a cell-intrinsic, immune tolerance mechanism that regulates the magnitude and resolution of T cell-mediated responses. Evasion of apoptosis is critical for the generation of memory T cells, as well as autoimmune T cells, and knowledge of the mechanisms that enable resistance to apoptosis will provide insight into ways to modulate their activity during protective and pathogenic responses. IL-17-producing CD4 T cells (TH17s) are long-lived, memory cells. These features enable their role in host defense, chronic inflammatory disorders, and anti-tumor immunity. A growing number of reports now indicate that TH17s in vivo require mitochondrial oxidative phosphorylation (OXPHOS), a metabolic phenotype that is poorly induced in vitro. To elucidate the role of OXPHOS in TH17 processes, we developed a system to polarize TH17s that metabolically resembled their in vivo counterparts. We discovered that directing TH17s to use OXPHOS promotes mitochondrial fitness, glutamine anaplerosis, and an anti-apoptotic phenotype marked by high BCL-XL and low BIM. Through competitive co-transfer experiments and tumor studies, we further revealed how OXPHOS protects TH17s from cell death while enhancing their persistence in the periphery and tumor microenvironment. Together, our work demonstrates a non-classical role of metabolism in regulating TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.

scholarly journals Febrile temperatures increase in vitro antibody affinity for malaria and dengue antigens

2018 ◽  
Author(s):  
Razvan C. Stan ◽  
Katia S. Françoso ◽  
Rubens P.S. Alves ◽  
Luís Carlos S. Ferreira ◽  
Irene S. Soares ◽  
...  
Keyword(s):  
Immune Responses ◽  
Binding Affinity ◽  
The Body ◽  
Antibody Affinity ◽  
Positive Role ◽  
Protein Partners ◽  
Adaptive Role

AbstractFever is a regulated elevation in the body setpoint temperature and may arise as a result of infectious and noninfectious causes. While beneficial in modulating immune responses to infection, the potential of febrile temperatures in regulating antigen binding affinity to antibodies has not been explored. We have investigated this process under in vitro conditions using selected malaria or dengue antigens and specific monoclonal antibodies, and observed a marked increase in the affinity of these antibody-antigen complexes at 40°C, compared to physiological (37°C) or pathophysiological temperatures (42°C). Induced thermal equilibration of the protein partners at these temperatures, prior to measurements, further increased their binding affinity. These results may indicate an unexpected beneficial and adaptive role for fever in vivo, and highlight the positive role of thermal priming in enhancing protein-protein affinity for samples of scarce availability.

scholarly journals MED1 is a lipogenesis coactivator required for postnatal adipose expansion

2020 ◽  
Author(s):  
Younghoon Jang ◽  
Young-Kwon Park ◽  
Ji-Eun Lee ◽  
Nhien Tran ◽  
Oksana Gavrilova ◽  
...  
Keyword(s):  
Transcription Factors ◽  
De Novo ◽  
Late Phase ◽  
High Fat ◽  
Maternal Milk ◽  
A Cell ◽  
Coactivator Complex

MED1 often serves as a surrogate of the general transcription coactivator complex Mediator for identifying active enhancers. MED1 is required for phenotypic conversion of fibroblasts to adipocytes in vitro but its role in adipose development and expansion in vivo has not been reported. Here we report that MED1 is dispensable for adipose development in mice. Instead, MED1 is required for postnatal adipose expansion and the induction of de novo lipogenesis (DNL) genes after pups switch diet from high-fat maternal milk to carbohydrate-based chow. During adipogenesis, MED1 is dispensable for induction of lineage-determining transcription factors (TFs) PPARγ and C/EBPα but is required for lipid accumulation in the late phase of differentiation. Mechanistically, MED1 controls the induction of DNL genes by facilitating lipogenic TF ChREBP-dependent recruitment of Mediator to active enhancers. Together, our findings identify a cell- and gene-specific regulatory role of MED1 as a lipogenesis coactivator required for postnatal adipose expansion.

scholarly journals Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo

2012 ◽  
Vol 287 (42) ◽  
pp. 35747-35755 ◽  
Author(s):  
Tanmay Dutta ◽  
Arun Malhotra ◽  
Murray P. Deutscher
Keyword(s):  
Potential Role ◽  
Wild Type ◽  
X Ray ◽  
E Coli ◽  
Phage T4 ◽  
A Cell ◽  
Rnase Z

Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.

scholarly journals How does the NPM1 mutant induce leukemia?

2011 ◽  
Vol 3 (2s) ◽  
pp. 6 ◽  
Author(s):  
Paolo Sportoletti
Keyword(s):  
Myeloid Leukemia ◽  
Tumor Development ◽  
Genetic Alterations ◽  
In Vivo Studies ◽  
Npm1 Mutation ◽  
Protein Partners ◽  
Cellular Pathways

NPM1 is the most frequently mutated gene in AML and the role of the NPM1 mutant in acute myeloid leukemia along with its leukemogenic potential are still under investigation. NPM1 genetic alterations can contribute to leukemogenesis through the direct oncogenic effect of the mutant protein and the concomitant loss of one functional allele. Npm1 loss determines tumor development in the mouse while in human NPM1 maps in a chromosomal region frequently loss in myelodysplastic syndrome (MDS). The NPM1 mutant cytoplasmic delocalization in leukemic blasts alters multiple cellular pathways through either loss or gain of function effects on different protein partners. Here we discuss the most relevant studies on the role of the NPM1 molecule in hematological malignancies and both in vitro and in vivo studies that are trying to elucidate the way by which the NPM1 mutation induces leukemia.

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