Pericentriolar matrix integrity relies on cenexin and Polo-Like Kinase (PLK)1

Polo-Like-Kinase (PLK) 1 activity is associated with maintaining the functional and physical properties of the centrosome's pericentriolar matrix (PCM). In this study, we use a multimodal approach of human cells (HeLa) and zebrafish embryos in parallel to phylogenic analysis to test the role of a PLK1 binding protein, cenexin, in regulating the PCM. Our studies identify that cenexin is required for tempering microtubule nucleation and that a conserved C-terminal PLK1 binding site between humans, zebrafish, and out to cnidaria is required for PCM maintenance through PLK1-dependent substrate phosphorylation events. PCM architecture in cenexin-depleted zebrafish embryos was rescued with wild-type human cenexin, but not with a C-terminal cenexin mutant (S796A) deficient in PLK1 binding. We propose a model where cenexin's C-terminus acts in a conserved manner in eukaryotes, excluding nematodes and arthropods, to anchor PLK1 moderating its potential to phosphorylate PCM substrates required for PCM maintenance and function.

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A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels

Molecular Brain ◽

10.1186/s13041-020-00725-y ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Eder Gambeta ◽

Maria A. Gandini ◽

Ivana A. Souza ◽

Laurent Ferron ◽

Gerald W. Zamponi

Keyword(s):

Trigeminal Neuralgia ◽

Missense Mutation ◽

Neurotransmitter Release ◽

Trigeminal System ◽

Wild Type ◽

Calcium Dependent ◽

Whole Cell Patch Clamp ◽

C Terminus ◽

Dependent Inactivation

AbstractA novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

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Regulation of hair follicle development by the TNF signal ectodysplasin and its receptor Edar

Development ◽

10.1242/dev.129.10.2541 ◽

2002 ◽

Vol 129 (10) ◽

pp. 2541-2553 ◽

Cited By ~ 4

Author(s):

Johanna Laurikkala ◽

Johanna Pispa ◽

Han-Sung Jung ◽

Pekka Nieminen ◽

Marja Mikkola ◽

...

Keyword(s):

Signaling Pathway ◽

Hair Follicles ◽

Wild Type ◽

Mutant Mouse Embryos ◽

Anhidrotic Ectodermal Dysplasia ◽

Embryonic Morphogenesis ◽

Hair Follicle Development ◽

And Function ◽

Insight Into

X-linked and autosomal forms of anhidrotic ectodermal dysplasia syndromes (HED) are characterized by deficient development of several ectodermal organs, including hair, teeth and exocrine glands. The recent cloning of the genes that underlie these syndromes, ectodysplasin (ED1) and the ectodysplasin A receptor (EDAR), and their identification as a novel TNF ligand-receptor pair suggested a role for TNF signaling in embryonic morphogenesis. In the mouse, the genes of the spontaneous mutations Tabby (Ta) and downless (dl) were identified as homologs of ED1 and EDAR, respectively. To gain insight into the function of this signaling pathway in development of skin and hair follicles, we analyzed the expression and regulation of Eda and Edar in wild type as well as Tabby and Lef1 mutant mouse embryos. We show that Eda and Edar expression is confined to the ectoderm and occurs in a pattern that suggests a role of ectodysplasin/Edar signaling in the interactions between the ectodermal compartments and the formation and function of hair placodes. By using skin explant cultures, we further show that this signaling pathway is intimately associated with interactions between the epithelial and mesenchymal tissues. We also find that Ta mutants lack completely the placodes of the first developing tylotrich hairs, and that they do not show patterned expression of placodal genes, including Bmp4, Lef1, Shh, Ptch and Edar, and the genes for β-catenin and activin A. Finally, we identified activin as a mesenchymal signal that stimulates Edar expression and WNT as a signal that induces Eda expression, suggesting a hierarchy of distinct signaling pathways in the development of skin and hair follicles. In conclusion, we suggest that Eda and Edar are associated with the onset of ectodermal patterning and that ectodysplasin/edar signaling also regulates the morphogenesis of hair follicles.

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TLR7 modulating B-cell immune responses in the spleen of C57BL/6 mice infected with Schistosoma japonicum

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009943 ◽

2021 ◽

Vol 15 (11) ◽

pp. e0009943

Author(s):

Haixia Wei ◽

Hongyan Xie ◽

Jiale Qu ◽

Anqi Xie ◽

Shihao Xie ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Gene Knockout ◽

Innate Immune ◽

Wild Type ◽

Cell Responses ◽

Splenic Cells ◽

And Function ◽

Egg Antigen

B cells played an important role in Schistosoma infection-induced diseases. TLR7 is an intracellular member of the innate immune receptor. The role of TLR7 on B cells mediated immune response is still unclear. Here, C57BL/6 mice were percutaneously infected by S. japonicum for 5–6 weeks. The percentages and numbers of B cells increased in the infected mice (p < 0.05), and many activation and function associated molecules were also changed on B cells. More splenic cells of the infected mice expressed TLR7, and B cells were served as the main cell population. Moreover, a lower level of soluble egg antigen (SEA) specific antibody and less activation associated molecules were found on the surface of splenic B cells from S. japonicum infected TLR7 gene knockout (TLR7 KO) mice compared to infected wild type (WT) mice (p < 0.05). Additionally, SEA showed a little higher ability in inducing the activation of B cells from naive WT mice than TLR7 KO mice (p < 0.05). Finally, the effects of TLR7 on B cells are dependent on the activation of NF-κB p65. Altogether, TLR7 was found modulating the splenic B cell responses in S. japonicum infected C57BL/6 mice.

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The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

Molecular Biology of the Cell ◽

10.1091/mbc.e02-03-0157 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3811-3821 ◽

Cited By ~ 55

Author(s):

Pauli J. Ojala ◽

Ville O. Paavilainen ◽

Maria K. Vartiainen ◽

Roman Tuma ◽

Alan G. Weeds ◽

...

Keyword(s):

Binding Site ◽

Binding Activity ◽

Size Exclusion ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Monomer ◽

Wild Type ◽

Native Page ◽

Exclusion Chromatography

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K D = 0.05 μM) than ATP-G-actin (K D = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k off = 1.8 s−1) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

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PSGL-1 regulates platelet P-selectin-mediated endothelial activation and shedding of P-selectin from activated platelets

Thrombosis and Haemostasis ◽

10.1160/th07-03-0207 ◽

2007 ◽

Vol 98 (10) ◽

pp. 806-812 ◽

Cited By ~ 46

Author(s):

Vandana Dole ◽

Wolfgang Bergmeier ◽

Ian Patten ◽

Junichi Hirahashi ◽

Tanya Mayadas ◽

...

Keyword(s):

Endothelial Activation ◽

Leukocyte Rolling ◽

Wild Type ◽

Platelet Surface ◽

Activated Platelets ◽

And Function ◽

Body Secretion

SummaryWe have previously shown that activated platelets in circulation stimulate release of endothelial Weibel-Palade bodies thus increasing leukocyte rolling in venules. P-selectin on the activated platelets mediates adhesion to leukocytes via PSGL-1 and is rapidly shed into plasma. We were interested in studying the role of PSGL-1 in regulating expression and function of platelet P-selectin. We show here that PSGL-1 is critical for the activation of endothelial cells in venules of mice infused with activated platelets. The interaction of platelet P-selectin with PSGL-1 is also required for P-selectin shedding, as P-selectin was retained significantly longer on the surface of activated platelets infused into PSGL-1-/- compared to wild-type mice. The leukocyte integrin αMβ2 (Mac-1) was not required for P-selectin shedding. In addition to shedding, P-selectin can be downregulated from the platelet surface through internalization and this is the predominant mechanism in the absence of PSGL-1. We demonstrate that leukocyte- neutrophil elastase,known to cleave P-selectin in vitro, is not the major sheddase for P-selectin in vivo. In conclusion, interaction of platelet P-selectin with PSGL-1 is crucial for activation of the endothelium andWeibel-Palade body secretion. The interaction with PSGL-1 also results in rapid shedding of P-selectin thus downregulating the inflammatory potential of the platelet.

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miR-221 and miR-155 regulate human dendritic cell development, apoptosis, and IL-12 production through targeting of p27kip1, KPC1, and SOCS-1

Blood ◽

10.1182/blood-2010-12-322503 ◽

2011 ◽

Vol 117 (16) ◽

pp. 4293-4303 ◽

Cited By ~ 182

Author(s):

Changming Lu ◽

Xin Huang ◽

Xiaoxiao Zhang ◽

Kristin Roensch ◽

Qing Cao ◽

...

Keyword(s):

Cytokine Signaling ◽

Wild Type ◽

Suppressor Of Cytokine Signaling ◽

Hematopoietic Progenitor ◽

Monocyte Differentiation ◽

P27kip1 Protein ◽

And Function ◽

Antigen Presenting ◽

Dc Maturation

Abstract Dendritic cells (DCs) are potent antigen-presenting cells derived from hematopoietic progenitor cells and circulating monocytes. To investigate the role of microRNAs (miRNAs) during DC differentiation, maturation, and function, we profiled miRNA expression in human monocytes, immature DCs (imDCs), and mature DCs (mDCs). Stage-specific, differential expression of 27 miRNAs was found during monocyte differentiation into imDCs and mDCs. Among them, decreased miR-221 and increased miR-155 expression correlated with p27kip1 accumulation in DCs. Silencing of miR-221 or overexpressing of miR-155 in DCs resulted in p27kip1 protein increase and DC apoptosis. Moreover, mDCs from miR-155−/− mice were less apoptotic than those from wild-type mice. Silencing of miR-155 expression had little effect on DC maturation but reduced IL-12p70 production, whereas miR-155 overexpression in mDCs enhanced IL-12p70 production. Kip1 ubiquitination-promoting complex 1, suppressor of cytokine signaling 1, and CD115 (M-CSFR) were functional targets of miR-155. Furthermore, we provide evidence that miR-155 indirectly regulated p27kip1 protein level by targeting Kip1 ubiquitination-promoting complex 1. Thus, our study uncovered miRNA signatures during monocyte differentiation into DCs and the new regulatory role of miR-221 and miR-155 in DC apoptosis and IL-12p70 production.

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Role of the Pilot Protein YscW in the Biogenesis of the YscC Secretin in Yersinia enterocolitica

Journal of Bacteriology ◽

10.1128/jb.186.16.5366-5375.2004 ◽

2004 ◽

Vol 186 (16) ◽

pp. 5366-5375 ◽

Cited By ~ 64

Author(s):

Peter Burghout ◽

Frank Beckers ◽

Emmie de Wit ◽

Ria van Boxtel ◽

Guy R. Cornelis ◽

...

Keyword(s):

Outer Membrane ◽

Yersinia Enterocolitica ◽

Amino Acid Residues ◽

Wild Type ◽

C Terminus ◽

Outer Membrane Lipoprotein ◽

Protein Secretion System ◽

Membrane Lipoprotein ◽

Type Iii Protein Secretion

ABSTRACT The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that ∼30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.

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Cerebrovascular Changes and Neurodegeneration Related to Hyperlipidemia: Characteristics of the Human ApoB-100 Transgenic Mice

Current Pharmaceutical Design ◽

10.2174/1381612826666200218101818 ◽

2020 ◽

Vol 26 (13) ◽

pp. 1486-1494 ◽

Cited By ~ 1

Author(s):

Melinda E. Tóth ◽

Brigitta Dukay ◽

Zsófia Hoyk ◽

Miklós Sántha

Keyword(s):

Transgenic Mouse ◽

Serum Lipid ◽

Lipoprotein Metabolism ◽

Transgenic Mouse Models ◽

Wild Type ◽

Lipid Levels ◽

Cerebrovascular Lesions ◽

Wide Range ◽

And Function

Serum lipid levels are closely related to the structure and function of blood vessels. Chronic hyperlipidemia may lead to damage in both the cardio- and the cerebrovascular systems. Vascular dysfunctions, including impairments of the blood-brain barrier, are known to be associated with neurodegenerative diseases. A growing number of evidence suggests that cardiovascular risk factors, such as hyperlipidemia, may increase the likelihood of developing dementia. Due to differences in lipoprotein metabolism, wild-type mice are protected against dietinduced hypercholesterolemia, and their serum lipid profile is different from that observed in humans. Therefore, several transgenic mouse models have been established to study the role of different apolipoproteins and their receptors in lipid metabolism, as well as the complications related to pathological lipoprotein levels. This minireview focused on a transgenic mouse model overexpressing an apolipoprotein, the human ApoB-100. We discussed literature data and current advancements on the understanding of ApoB-100 induced cardio- and cerebrovascular lesions in order to demonstrate the involvement of this type of apolipoprotein in a wide range of pathologies, and a link between hyperlipidemia and neurodegeneration.

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Factor XII in PMM2-CDG patients: role of N-glycosylation in the secretion and function of the first element of the contact pathway

Orphanet Journal of Rare Diseases ◽

10.1186/s13023-020-01564-9 ◽

2020 ◽

Vol 15 (1) ◽

Author(s):

Raquel López-Gálvez ◽

María Eugenia de la Morena-Barrio ◽

Alberto López-Lera ◽

Monika Pathak ◽

Antonia Miñano ◽

...

Keyword(s):

Factor Xii ◽

Clinical Effects ◽

Wild Type ◽

Vascular Events ◽

Contact Phase ◽

Glycosylation Sites ◽

Expression Model ◽

Contact Pathway ◽

And Function

Abstract Background Congenital disorders of glycosylation (CDG) are rare diseases with impaired glycosylation and multiorgan disfunction, including hemostatic and inflammatory disorders. Factor XII (FXII), the first element of the contact phase, has an emerging role in hemostasia and inflammation. FXII deficiency protects against thrombosis and the p.Thr309Lys variant is involved in hereditary angioedema through the hyperreactivity caused by the associated defective O-glycosylation. We studied FXII in CDG aiming to supply further information of the glycosylation of this molecule, and its functional and clinical effects. Plasma FXII from 46 PMM2-CDG patients was evaluated by coagulometric and by Western Blot in basal conditions, treated with N-glycosydase F or activated by silica or dextran sulfate. A recombinant FXII expression model was used to validate the secretion and glycosylation of wild-type and variants targeting the two described FXII N-glycosylation sites (p.Asn230Lys; p.Asn414Lys) as well as the p.Thr309Lys variant. Results PMM2-CDG patients had normal FXII levels (117%) but high proportions of a form lacking N-glycosylation at Asn414. Recombinant FXII p.Asn230Lys, and p.Asn230Lys&p.Asn414Lys had impaired secretion and increased intracellular retention compared to wild-type, p.Thr309Lys and p.Asn414Lys variants. The hypoglycosylated form of PMM2-CDG activated similarly than FXII fully glycosylated. Accordingly, no PMM2-CDG had angioedema. FXII levels did not associate to vascular events, but hypoglycosylated FXII, like hypoglycosylated transferrin, antithrombin and FXI levels did it. Conclusions N-glycosylation at Asn230 is essential for FXII secretion. PMM2-CDG have high levels of FXII lacking N-glycosylation at Asn414, but this glycoform displays similar activation than fully glycosylated, explaining the absence of angioedema in CDG.

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Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00404-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Mutational Analysis ◽

Wild Type ◽

Content Type ◽

Growth Defects ◽

C Terminus ◽

K 12 ◽

And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

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