Functional Characterization of a Novel Frameshift Mutation in the C-terminus of the Nav1.5 Channel Underlying a Brugada Syndrome with Variable Expression in a Spanish Family

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as theSchizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

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Characterization of four conventional dendritic cell subsets in human skin-draining lymph nodes in relation to T-cell activation

Blood ◽

10.1182/blood-2011-03-344838 ◽

2011 ◽

Vol 118 (9) ◽

pp. 2502-2510 ◽

Cited By ~ 56

Author(s):

Rieneke van de Ven ◽

Mari F. C. M. van den Hout ◽

Jelle J. Lindenberg ◽

Berbel J. R. Sluijter ◽

Paul A. M. van Leeuwen ◽

...

Keyword(s):

T Cell ◽

Dendritic Cell ◽

Lymph Nodes ◽

T Cell Activation ◽

Early Stage ◽

Functional Characterization ◽

Primary Melanoma ◽

Lymphoid Tissues ◽

Variable Expression

Abstract To increase (tumor) vaccine efficacy, there is an urgent need for phenotypic and functional characterization of human dendritic cell (DC) subsets residing in lymphoid tissues. In this study we identified and functionally tested 4 human conventional DC (cDC) subsets within skin-draining sentinel lymph nodes (SLNs) from early-stage melanoma patients. These SLNs were all tumor negative and were removed on average 44 days after excision of the primary melanoma. As such, they were considered representative of steady-state conditions. On comparison with skin-migrated cDC, 2 CD1a+ subsets were identified as most likely skin-derived CD11cint Langerhans cells (LC) with intracellular langerin and E-cadherin expression or as CD11chi dermal DCs with variable expression of langerin. Two other CD1a− LN-residing cDC subsets were characterized as CD14−BDCA3hiCD103− and CD14+BDCA3loCD103+, respectively. Whereas the CD1a+ skin-derived subsets displayed greater levels of phenotypic maturation, they were associated with lower levels of inflammatory cytokine release and were inferior in terms of allogeneic T-cell priming and IFNγ induction. Thus, despite their higher maturation state, skin-derived cDCs (and LCs in particular) proved inferior T-cell activators compared with the CD1a− cDC subsets residing in melanoma-draining LNs. These observations should be considered in the design of DC-targeting immunotherapies.

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Purification and Functional Characterization of the C-Terminal Domain of the β-Actin-Binding Protein AIM1 In Vitro

Molecules ◽

10.3390/molecules23123281 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3281 ◽

Cited By ~ 1

Author(s):

Fang Wu ◽

Liangkai Cheng ◽

Qi Yu ◽

Lin Zhang ◽

Hong Li ◽

...

Keyword(s):

Prostate Cancer ◽

Structural Model ◽

Functional Characterization ◽

Actin Binding ◽

Migration And Invasion ◽

Binding Modes ◽

C Terminus ◽

Biology Research

The protein absent in melanoma 1 (AIM1) is a member of the βγ-crystal lens superfamily that is associated with the development of multiple cancers. The binding of AIM1 to β-actin affects the migration and invasion of prostate cancer epithelial cells. The C-terminus of AIM1 is required for the β-actin interaction. However, the characteristics of AIM1 in vitro and the interaction mode between AIM1 and β-actin remain unknown. We describe novel methods to prepare pure recombinant AIM1 and identify possible binding modes between AIM1 and β-actin; we also obtain the crystal of the first two βγ-crystallin domains of AIM1 (g1g2) for future structural biology research. We first express and purify AIM1 after cloning the sequence into a modified pET-28a_psp expression vector. Next, we define the minimum unit formed by the βγ-crystallin domain repeats that bound to β-actin and perform its physiological function. Finally, we made the structural model of the AIM1 g1g2 that can be used to guide future biomedical investigations and prostate cancer research.

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Identification and functional characterization of novel mutations including frameshift mutation in exon 4 of CSF1R in patients with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia

Journal of Neurology ◽

10.1007/s00415-018-9017-2 ◽

2018 ◽

Vol 265 (10) ◽

pp. 2415-2424 ◽

Cited By ~ 5

Author(s):

Takeshi Miura ◽

Naomi Mezaki ◽

Takuya Konno ◽

Akio Iwasaki ◽

Naoyuki Hara ◽

...

Keyword(s):

Frameshift Mutation ◽

Functional Characterization ◽

Adult Onset ◽

Novel Mutations ◽

Axonal Spheroids ◽

Exon 4

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Molecular, ultrastructural and functional characterization of a Spanish family with Hermansky-Pudlak syndrome: role of insC974 in platelet function and clinical relevance

British Journal of Haematology ◽

10.1046/j.1365-2141.2003.04557.x ◽

2003 ◽

Vol 123 (1) ◽

pp. 132-138 ◽

Cited By ~ 10

Author(s):

Rocio González-Conejero ◽

José Rivera ◽

Ginés Escolar ◽

Isabel Zuazu-Jausoro ◽

Vicente Vicente ◽

...

Keyword(s):

Platelet Function ◽

Clinical Relevance ◽

Functional Characterization ◽

Spanish Family ◽

Hermansky Pudlak Syndrome

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Functional characterization of FlgM in the regulation of flagellar synthesis and motility in Yersinia pseudotuberculosis

Microbiology ◽

10.1099/mic.0.026294-0 ◽

2009 ◽

Vol 155 (6) ◽

pp. 1890-1900 ◽

Cited By ~ 18

Author(s):

Lisha Ding ◽

Yao Wang ◽

Yangbo Hu ◽

Steve Atkinson ◽

Paul Williams ◽

...

Keyword(s):

Yersinia Pseudotuberculosis ◽

Functional Characterization ◽

Direct Interaction ◽

Site Directed Mutagenesis ◽

Wild Type ◽

C Terminus ◽

In Trans ◽

Differential Gene ◽

Flagellar Biogenesis

We describe here the functional characterization of the flgM gene in Yersinia pseudotuberculosis. Direct interaction of FlgM with the alternative sigma factor σ 28 (FliA) was first confirmed. A conserved region in the C-terminus of FlgM was found which included the σ 28 binding domain. By site-directed mutagenesis, bacterial two-hybrid analysis and Western blotting, the primary FlgM binding sites with σ 28 were shown to be Ile85, Ala86 and Leu89. A role for FlgM in swimming motility was demonstrated by inactivation of flgM and subsequent complementation in trans. Transcriptional fusion analyses showed differential gene expression of flhDC, fliA, flgM and fliC in the fliA and flgM mutants compared with the wild-type. flhDC expression was not influenced by σ 28 or FlgM while fliA expression was abolished in the fliA mutant and considerably reduced in the flgM mutant when compared to the wild-type, indicating that both FliA and FlgM can activate fliA transcription. Conversely, flgM transcription was higher in the fliA mutant when compared to the wild-type, suggesting that flgM transcription was repressed by σ 28. Interestingly, fliC expression was markedly increased in the flgM mutant, suggesting a negative regulatory role for FlgM in fliC expression. The transcription of other σ-dependent genes (cheW, flgD, flaA, csrA and fliZ) was also examined in fliA and flgM mutant backgrounds and this revealed that other σ-factors apart from σ 28 may be involved in flagellar biogenesis in Y. pseudotuberculosis. Taking together the motility phenotypes and effects of flgM mutation on the regulation of these key motility genes, we propose that the mechanisms regulating flagellar biogenesis in Y. pseudotuberculosis may differ from those described for other bacteria.

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