scholarly journals Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin

2018 ◽  
Author(s):  
Jacob M Daane ◽  
Jennifer Lanni ◽  
Ina Rothenberg ◽  
Guiscard Seebohm ◽  
Charles W Higdon ◽  
...  
Keyword(s):  
Ion Channel ◽  
Relative Size ◽  
Calcineurin Inhibitors ◽  
Common Mechanism ◽  
Allometric Growth ◽  
Wild Type ◽  
Form And Function ◽  
C Terminus ◽  
And Function ◽  
Calcineurin Signaling

AbstractThe establishment of relative size of organs and structures is paramount for attaining final form and function of an organism. Importantly, variation in the proportions of structures frequently underlies adaptive change in morphology in evolution and maybe a common mechanism underlying selection. However, the mechanism by which growth is integrated within tissues during development to achieve proper proportionality is poorly understood. We have shown that signaling by potassium channels mediates coordinated size regulation in zebrafish fins. Recently, calcineurin inhibitors were shown to elicit changes in zebrafish fin allometry as well. Here, we identify the potassium channel kcnk5b as a key player in integrating calcineurin’s growth effects, in part through regulation of the cytoplasmic C-terminus of the channel. We propose that the interaction between Kcnk5b and calcineurin acts as a signaling node to regulate allometric growth. Importantly, we find that this regulation is epistatic to inherent mechanisms instructing overall size as inhibition of calcineurin is able to bypass genetic instruction of size as seen in sof and wild-type fins, however, it is not sufficient to re-specify positional memory of size of the fin. These findings integrate classic signaling mediators such as calcineurin with ion channel function in the regulation of size and proportion during growth.

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

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.

scholarly journals A unique kinesin-8 surface loop provides specificity for chromosome alignment

2014 ◽  
Vol 25 (21) ◽  
pp. 3319-3329 ◽  
Author(s):  
Haein Kim ◽  
Cindy Fonseca ◽  
Jason Stumpff
Keyword(s):  
Motor Domain ◽  
Common Mechanism ◽  
Surface Loop ◽  
C Terminus ◽  
Chromosome Alignment ◽  
Spindle Microtubules ◽  
And Function ◽  
Positively Charged ◽  
Mitotic Chromatin

Microtubule length control is essential for the assembly and function of the mitotic spindle. Kinesin-like motor proteins that directly attenuate microtubule dynamics make key contributions to this control, but the specificity of these motors for different subpopulations of spindle microtubules is not understood. Kif18A (kinesin-8) localizes to the plus ends of the relatively slowly growing kinetochore fibers (K-fibers) and attenuates their dynamics, whereas Kif4A (kinesin-4) localizes to mitotic chromatin and suppresses the growth of highly dynamic, nonkinetochore microtubules. Although Kif18A and Kif4A similarly suppress microtubule growth in vitro, it remains unclear whether microtubule-attenuating motors control the lengths of K-fibers and nonkinetochore microtubules through a common mechanism. To address this question, we engineered chimeric kinesins that contain the Kif4A, Kif18B (kinesin-8), or Kif5B (kinesin-1) motor domain fused to the C-terminal tail of Kif18A. Each of these chimeric kinesins localizes to K-fibers; however, K-fiber length control requires an activity specific to kinesin-8s. Mutational studies of Kif18A indicate that this control depends on both its C-terminus and a unique, positively charged surface loop, called loop2, within the motor domain. These data support a model in which microtubule-attenuating kinesins are molecularly “tuned” to control the dynamics of specific subsets of spindle microtubules.

scholarly journals NSC348884 cytotoxicity is not mediated by inhibition of nucleophosmin oligomerization

2020 ◽  
Author(s):  
Markéta Šašinková ◽  
Petr Heřman ◽  
Aleš Holoubek ◽  
Dita Strachotová ◽  
Petra Otevřelová ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cytotoxic Effect ◽  
Leukemia Cells ◽  
Live Cells ◽  
Wild Type ◽  
C Terminus ◽  
Interaction Partners ◽  
And Function ◽  
Acute Myeloid

AbstractOligomerization of the nucleolar phosphoprotein nucleophosmin (NPM) is mediated by its N-terminal domain. In acute myeloid leukemia, a frequent NPM mutation occurring at the C-terminus causes NPM delocalization to the cytoplasm. Due to formation of NPM heterooligomers, the wild-type NPM as well as many of NPM interaction partners are also delocalized. Proper localization and function of mislocalized proteins in the cells with mutated NPM may be restored by targeting NPM oligomerization. We introduce a reliable set of complementary methods for monitoring NPM oligomerization in both cell lysates and live cells. Using this methodological background we show that a putative inhibitor of NPM oligomerization, NSC348884, does not prevent formation of NPM oligomers in leukemia cells. Instead, we reveal that the observed cytotoxic effect of NSC348884 is associated with changes in cell adhesion signaling.

scholarly journals Differential Localization and Function of PB1-F2 Derived from Different Strains of Influenza A Virus

2010 ◽  
Vol 84 (19) ◽  
pp. 10051-10062 ◽  
Author(s):  
Chi-Jene Chen ◽  
Guang-Wu Chen ◽  
Ching-Ho Wang ◽  
Chih-Heng Huang ◽  
Yeau-Ching Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Mitochondrial Localization ◽  
Sequence Context ◽  
C Terminus ◽  
Different Strains ◽  
And Function

ABSTRACT PB1-F2 is a viral protein that is encoded by the PB1 gene of influenza A virus by alternative translation. It varies in length and sequence context among different strains. The present study examines the functions of PB1-F2 proteins derived from various human and avian viruses. While H1N1 PB1-F2 was found to target mitochondria and enhance apoptosis, H5N1 PB1-F2, surprisingly, did not localize specifically to mitochondria and displayed no ability to enhance apoptosis. Introducing Leu into positions 69 (Q69L) and 75 (H75L) in the C terminus of H5N1 PB1-F2 drove 40.7% of the protein to localize to mitochondria compared with the level of mitochondrial localization of wild-type H5N1 PB1-F2, suggesting that a Leu-rich sequence in the C terminus is important for targeting of mitochondria. However, H5N1 PB1-F2 contributes to viral RNP activity, which is responsible for viral RNA replication. Lastly, although the swine-origin influenza virus (S-OIV) contained a truncated form of PB1-F2 (12 amino acids [aa]), potential mutation in the future may enable it to contain a full-length product. Therefore, the functions of this putative S-OIV PB1-F2 (87 aa) were also investigated. Although this PB1-F2 from the mutated S-OIV shares only 54% amino acid sequence identity with that of seasonal H1N1 virus, it also increased viral RNP activity. The plaque size and growth curve of the viruses with and without S-OIV PB1-F2 differed greatly. The PB1-F2 protein has various lengths, amino acid sequences, cellular localizations, and functions in different strains, which result in strain-specific pathogenicity. Such genetic and functional diversities make it flexible and adaptable in maintaining the optimal replication efficiency and virulence for various strains of influenza A virus.

scholarly journals Interaction between FliE and FlgB, a Proximal Rod Component of the Flagellar Basal Body ofSalmonella

2000 ◽  
Vol 182 (11) ◽  
pp. 3029-3036 ◽  
Author(s):  
Tohru Minamino ◽  
Shigeru Yamaguchi ◽  
Robert M. Macnab
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Basal Body ◽  
Strong Interactions ◽  
Wild Type ◽  
Body Protein ◽  
C Terminus ◽  
Genes Encoding ◽  
And Function ◽  
Better Than

ABSTRACT FliE is a flagellar basal body protein of Salmonellawhose detailed location and function have not been established. A mutant allele of fliE, which caused extremely poor flagellation and swarming, generated extragenic suppressors, all of which mapped to flgB, one of four genes encoding the basal body rod; the fliE flgB pseudorevertants were better flagellated and swarmed better than the fliE parent, especially when the temperature was reduced from 37 to 30°C. Motility of the pseudorevertants in liquid culture was markedly better than motility on swarm plates; we interpret this to mean that reduced flagellation is less deleterious at low viscous loads. Overproduction of the mutant FliE protein improved the motility of the parentalfliE mutant and its pseudorevertants, though not to wild-type levels. Overproduction of suppressor FlgB (but not wild-type FlgB) in the fliE mutant also resulted in improved motility. The second-site FlgB mutation by itself had no phenotype; cells swarmed as well as wild-type cells. When overproduced, wild-type FliE was dominant over FliE-V99G, but the reverse was not true; that is, overproduced FliE-V99G was not negatively dominant over wild-type FliE. We conclude that the mutant protein has reduced probability of assembly but, if assembled, functions relatively well. Export of the flagellar protein FlgD, which is known to be FliE dependent, was severely impaired by the FliE-V99G mutation but was significantly improved in the suppressor strains. The FliE mutation, V99G, was close to the C terminus of the 104-amino-acid sequence; the suppressing mutations in FlgB were all either G119E or G129D, close to the C terminus of its 138-amino-acid sequence. Affinity blotting experiments between FliE as probe and various basal body proteins as targets and vice versa revealed strong interactions between FliE and FlgB; much weaker interactions between FliE and other rod proteins were observed and probably derive from the known similarities among these proteins. We suggest that FliE subunits constitute a junction zone between the MS ring and the rod and also that the proximal rod structure consists of FlgB subunits.

scholarly journals Short Runs for a Long Slide: Principalization in Complex Facial Restoration after Acid Attack Burn Injury

2019 ◽  
Vol 12 (1) ◽  
pp. 75-80
Author(s):  
Christopher D. Hughes ◽  
RobertJaroslaw Dabek ◽  
Johanna N. Riesel ◽  
Nemanja Baletic ◽  
James Chodosh ◽  
...  
Keyword(s):  
Burn Injury ◽  
Burn Injuries ◽  
Common Mechanism ◽  
Acid Attack ◽  
Chemical Burns ◽  
Form And Function ◽  
The Face ◽  
Vulnerable Patient ◽  
And Function ◽  
Lower Income Countries

Burn injuries are responsible for a significant portion of surgically treatable morbidity throughout the world and particularly in underdeveloped and developing countries. Intentional flame, chemical, and contact burns are unfortunately a common mechanism of injury. It is estimated that intentional chemical burns are responsible for between 2 and 20% of burn injuries seen at burn centers in lower income countries. Women are commonly targeted and the perpetrators are often known to the victims. The combination of a high disease prevalence, limited surgical and anesthetic resources, a vulnerable patient population, and largely disfiguring, nonlethal injuries present unique challenges for the reconstructive surgeon who may not encounter such cases regularly. In this article, we present a case of a 16-year-old female who sustained severe, full-thickness burns to the face including eyelids, neck, abdomen, and upper extremities after an intentional acid attack. She began her treatment course with us approximately 1 year after the injury. The deformities of her oral and periorbital regions presented particularly difficult reconstructive problems, including impending visual loss. Using plastic surgical principalization, we provided our patient adequate restoration of facial form and function through numbers of interventions using fundamental and state-of-the-art techniques.

Ontogenetic and Phylogenetic Variation of Form and Function of the Ocular Lens

1989 ◽  
Vol 174 ◽  
Author(s):  
J. G. Sivak
Keyword(s):  
Refractive Index ◽  
Life Histories ◽  
Spherical Aberration ◽  
Focal Length ◽  
Relative Size ◽  
Crystalline Lens ◽  
Optical Quality ◽  
Form And Function ◽  
And Function ◽  
Optical Structure

AbstractThe crystalline lens of the eye is a unique optical structure that continues to develop within the eye throughout life. This process of development results in the formation of a lens with a gradient refractive index that has important optical consequences, particularly in the control of spherical aberration. The optical characteristics of the vertebrate crystalline lens are reviewed in terms of environmental concerns and spherical aberration. Lens shape and relative size are determined by such factors as whether the eye is to be used in air or water and whether it is to be used under scotopic or photopic conditions. The continued growth of the lens through life can be related to whether the lens exhibits positive or negative spherical aberration. In general, spherical aberration, as measured using a split laser beam method with excised lenses, is minimized in species with life histories indicating superior resolution ability. In addition, lens optical quality, as indicated by zonular differences in focal length, deteriorates with lens age. While the embryonic and post-embryonic lens undergoes significant change in size, shape and refractive index distribution, it appears that focal constancy is maintained, at least in certain species. This finding may indicate a means of simplifying the process of emmetropization.

scholarly journals Effects of N-Terminal and C-Terminal Polyhistidine Tag on the Stability and Function of the Thermophilic P450 CYP119

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Yaprak Aslantas ◽  
Nur Basak Surmeli
Keyword(s):  
Active Site ◽  
High Efficiency ◽  
Industrial Applications ◽  
Heme Iron ◽  
Mechanistic Studies ◽  
Wild Type ◽  
His Tag ◽  
C Terminus ◽  
The Stability ◽  
And Function

Biocatalysts are sought-after in synthesis of pharmaceuticals and agrochemicals due to their high regioselectivity and enantioselectivity. Among biocatalysts, heme-containing cytochrome P450 (P450) oxygenases are an attractive target since they catalyze oxidation of “unactivated” carbon-hydrogen bonds with high efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be widely used as a biocatalyst since it shows activity at high temperatures and low pH. Polyhistidine tags (His-tags) are widely used to simplify purification of proteins. However, His-tags can cause changes to protein structure and function. Here, we demonstrate the effects of His-tags on CYP119. To this end, the His-tags were cloned at the N-terminus or C-terminus of the CYP119, and His-tagged proteins were expressed and isolated. The thermostability and peroxidase activity of His-tagged CYP119s were tested and compared to wild type CYP119. Results indicated that while addition of His-tags increased the yield and simplified isolation of CYP119, they also influenced the electronic structure of active site and the activity of the protein. We show that N-terminal His-tagged CYP119 has desirable properties and potential to be used in industrial applications, but mechanistic studies using this protein need careful interpretation since the His-tag affects electronic properties of the active site heme iron.

scholarly journals Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain

2004 ◽  
Vol 381 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Teresa RINALDI ◽  
Elah PICK ◽  
Alessia GAMBADORO ◽  
Stefania ZILLI ◽  
Vered MAYTAL-KIVITY ◽  
...  
Keyword(s):  
Terminal Region ◽  
Proper Function ◽  
Mitochondrial Morphology ◽  
Wild Type ◽  
Polyubiquitin Chains ◽  
Unique Role ◽  
Terminal Domain ◽  
C Terminus ◽  
Tubular System ◽  
And Function

Substrates destined for degradation by the 26 S proteasome are labelled with polyubiquitin chains. Rpn11/Mpr1, situated in the lid subcomplex, partakes in the processing of these chains or in their removal from substrates bound to the proteasome. Rpn11 also plays a role in maintaining mitochondrial integrity, tubular structure and proper function. The recent finding that Rpn11 participates in proteasome-associated deubiquitination focuses interest on the MPN+ (Mpr1, Pad1, N-terminal)/JAMM (JAB1/MPN/Mov34) metalloprotease site in its N-terminal domain. However, Rpn11 damaged at its C-terminus (the mpr1-1 mutant) causes pleiotropic effects, including proteasome instability and mitochondrial morphology defects, resulting in both proteolysis and respiratory malfunctions. We find that overexpression of WT (wild-type) RPN8, encoding a paralogous subunit that does not contain the catalytic MPN+ motif, corrects proteasome conformations and rescues cell cycle phenotypes, but is unable to correct defects in the mitochondrial tubular system or respiratory malfunctions associated with the mpr1-1 mutation. Transforming mpr1-1 with various RPN8–RPN11 chimaeras or with other rpn11 mutants reveals that a WT C-terminal region of Rpn11 is necessary, and more surprisingly sufficient, to rescue the mpr1-1 mitochondrial phenotype. Interestingly, single-site mutants in the catalytic MPN+ motif at the N-terminus of Rpn11 lead to reduced proteasome-dependent deubiquitination connected with proteolysis defects. Nevertheless, these rpn11 mutants suppress the mitochondrial phenotypes associated with mpr1-1 by intragene complementation. Together, these results point to a unique role for the C-terminal region of Rpn11 in mitochondrial maintenance that may be independent of its role in proteasome-associated deubiquitination.

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

2022 ◽  
Author(s):  
Abrar Adnan Aljiboury ◽  
Amra Mujcic ◽  
Erin Curtis ◽  
Thomas Cammerino ◽  
Denise Magny ◽  
...  
Keyword(s):  
Physical Properties ◽  
Binding Site ◽  
Zebrafish Embryos ◽  
Multimodal Approach ◽  
Wild Type ◽  
Microtubule Nucleation ◽  
C Terminus ◽  
Substrate Phosphorylation ◽  
And Function

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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