scholarly journals FAMOSS, a conserved 41-aa peptide involved in plant tip growth regulation

2021 ◽  
Author(s):  
Anna Mamaeva ◽  
Andrey Kniazev ◽  
Ilia Sedlov ◽  
Nina Golub ◽  
Daria Kharlampieva ◽  
...  
Keyword(s):  
Growth Regulation ◽  
Opposite Effect ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Small Gtpase ◽  
Reading Frame ◽  
Functional Studies ◽  
Non Coding Rnas ◽  
Small Open Reading Frame

Recent evidence shows that small open reading frame (smORF; <100 codons)-encoded peptides (SEPs) containing transmembrane domains are preadapted to be progenitors of novel functional genes. A dozen of such SEPs translated from long non-coding RNAs (lncRNAs) are already functionally characterised in animals. However, functional plant lncRNA-smORF-coded peptides are not yet described. Here, we report detailed functional characterization of a 41-aa peptide encoded by lncRNA-smORFs in the moss Physcomitrium patens, which was named "FAst-growing MOSS" (FAMOSS). We found that the FAMOSS interacts with the Rab-type small GTPase proteins and its overexpression leads to faster moss growth rate and more intensive vesicular transport in apical cells, while its knockout results in the opposite effect. The FAMOSS contains a predicted transmembrane domain and possible orthologs from streptophyta algae to flowering plants have a very conserved structure. Thus, the FAMOSS peptide is a previously unknown conserved player of Rab-mediated processes in plants. Our findings are in line with functional studies of transmembrane SEPs in animals and prove the principles of SEPs evolution. This study provides new insights into functions of plant lncRNA-smORFs.

scholarly journals Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscle fibers

2015 ◽  
Vol 308 (2) ◽  
pp. C123-C138 ◽  
Author(s):  
E. Giacomello ◽  
M. Quarta ◽  
C. Paolini ◽  
R. Squecco ◽  
P. Fusco ◽  
...  
Keyword(s):  
Structural Damage ◽  
Skeletal Muscles ◽  
Functional Characterization ◽  
Sr Proteins ◽  
Endurance Test ◽  
Tubular Aggregates ◽  
Functional Studies ◽  
Old Mice ◽  
Contractile Performance

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4–6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12–15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγcharge and L-type Ca2+current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.

Cloning and functional characterization of an uncoupling protein homolog in hummingbirds

2001 ◽  
Vol 5 (3) ◽  
pp. 137-145 ◽  
Author(s):  
CLAUDIA R. VIANNA ◽  
THILO HAGEN ◽  
CHEN-YU ZHANG ◽  
ERIC BACHMAN ◽  
OLIVIER BOSS ◽  
...  
Keyword(s):  
Uncoupling Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Pectoral Muscle ◽  
Mitochondrial Fraction ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame ◽  
Rapid Amplification

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5′ and 3′ ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being ∼72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3′,3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room’s temperature to 12–14°C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

scholarly journals Cloning and functional characterization of the human fractalkine receptor promoter regions

2002 ◽  
Vol 368 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Alexandre GARIN ◽  
Philippe PELLET ◽  
Philippe DETERRE ◽  
Patrice DEBRÉ ◽  
Christophe COMBADIÈRE
Keyword(s):  
Functional Characterization ◽  
Cell Types ◽  
Promoter Regions ◽  
Exon 2 ◽  
Reading Frame ◽  
Fractalkine Receptor ◽  
A Cell ◽  
Exon 4 ◽  
Coronary Events

We have previously shown that reduced expression of the fractalkine receptor, CX3CR1, is correlated with rapid HIV disease progression and with reduced susceptibility to acute coronary events. In order to elucidate the mechanisms underlying transcriptional regulation of CX3CR1 expression, we structurally and functionally characterized the CX3CR1 gene. It consists of four exons and three introns spanning over 18kb. Three transcripts are produced by splicing the three untranslated exons with exon 4, which contains the complete open reading frame. The transcript predominantly found in leucocytes corresponds to the splicing of exon 2 with exon 4. Transcripts corresponding to splicing of exons 1 and 4 are less abundant in leucocytes and splicing of exons 3 and 4 are rare longer transcripts. A constitutive promoter activity was found in the regions extending upstream from untranslated exons 1 and 2. Interestingly, exons 1 and 2 enhanced the activity of their respective promoters in a cell-specific manner. These data show that the CX3CR1 gene is controlled by three distinct promoter regions, which are regulated by their respective untranslated exons and that lead to the transcription of three mature messengers. This highly complex regulation may allow versatile and precise expression of CX3CR1 in various cell types.

Abstract 4422: Molecular and functional characterization of alpha satellite non-coding RNAs

2019 ◽  
Author(s):  
Rodrigo E. Cáceres ◽  
Marco A. Andonegui ◽  
Diego A. Oliva ◽  
Rodrigo González ◽  
Fernando Luna ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Alpha Satellite ◽  
Non Coding Rnas

scholarly journals Structural and functional characterization of the pore-forming domain of pinholin S2168

2020 ◽  
Vol 117 (47) ◽  
pp. 29637-29646
Author(s):  
Lena M. E. Steger ◽  
Annika Kohlmeyer ◽  
Parvesh Wadhwani ◽  
Jochen Bürck ◽  
Erik Strandberg ◽  
...  
Keyword(s):  
Experimental Data ◽  
Order Parameter ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Azimuthal Angle ◽  
Structural Models ◽  
Lytic Cycle ◽  
Helical Conformation ◽  
Rotational Orientation

Pinholin S2168 triggers the lytic cycle of bacteriophage φ21 in infectedEscherichia coli. Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual “pinholes.” Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state15N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (Smol= 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G40xxxS44xxxG48) together with a patch of H-bonding residues (T51, T54, N55) sideways along TMD2, available for helix–helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

Functional characterization of an abnormal factor XII molecule (F XII Bern)

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 997-1004
Author(s):  
WA Wuillemin ◽  
I Huber ◽  
M Furlan ◽  
B Lammle
Keyword(s):  
Dextran Sulfate ◽  
Functional Characterization ◽  
Healthy Woman ◽  
Chain Molecule ◽  
Molecular Defect ◽  
Factor Xii ◽  
Single Chain ◽  
Functional Studies ◽  
Functional Defect

An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.

scholarly journals Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification

2015 ◽  
Vol 396 (8) ◽  
pp. 903-915 ◽  
Author(s):  
Nazhat Shirzad-Wasei ◽  
Jenny van Oostrum ◽  
Petra H.M. Bovee-Geurts ◽  
Lisanne J.A. Kusters ◽  
Giel J.C.G.M. Bosman ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Partial Purification ◽  
Functional Studies ◽  
Host Membrane ◽  
Lipid Nanodiscs ◽  
Membrane Scaffold ◽  
Rapid Transfer

Abstract Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

scholarly journals RsSOS1 Responding to Salt Stress Might Be Involved in Regulating Salt Tolerance by Maintaining Na+ Homeostasis in Radish (Raphanus sativus L.)

Horticulturae ◽  
2021 ◽  
Vol 7 (11) ◽  
pp. 458
Author(s):  
Wanting Zhang ◽  
Jingxue Li ◽  
Junhui Dong ◽  
Yan Wang ◽  
Liang Xu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Salt Stress ◽  
Salt Tolerance ◽  
Functional Characterization ◽  
Vital Role ◽  
Salt Stress Response ◽  
Reading Frame ◽  
Yield And Quality

Radish is a kind of moderately salt-sensitive vegetable. Salt stress seriously decreases the yield and quality of radish. The plasma membrane Na+/H+ antiporter protein Salt Overly Sensitive 1 (SOS1) plays a crucial role in protecting plant cells against salt stress, but the biological function of the RsSOS1 gene in radish remains to be elucidated. In this study, the RsSOS1 gene was isolated from radish genotype ‘NAU-TR17’, and contains an open reading frame of 3414 bp encoding 1137 amino acids. Phylogenetic analysis showed that RsSOS1 had a high homology with BnSOS1, and clustered together with Arabidopsis plasma membrane Na+/H+ antiporter (AtNHX7). The result of subcellular localization indicated that the RsSOS1 was localized in the plasma membrane. Furthermore, RsSOS1 was strongly induced in roots of radish under 150 mmol/L NaCl treatment, and its expression level in salt-tolerant genotypes was significantly higher than that in salt-sensitive ones. In addition, overexpression of RsSOS1 in Arabidopsis could significantly improve the salt tolerance of transgenic plants. Meanwhile, the transformation of RsSOS1△999 could rescue Na+ efflux function of AXT3 yeast. In summary, the plasma membrane Na+/H+ antiporter RsSOS1 plays a vital role in regulating salt-tolerance of radish by controlling Na+ homeostasis. These results provided useful information for further functional characterization of RsSOS1 and facilitate clarifying the molecular mechanism underlying salt stress response in radish.

scholarly journals Identification of recombinant Fabs for structural and functional characterization of HIV-host factor complexes

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0250318
Author(s):  
Natalia Sevillano ◽  
Evan M. Green ◽  
Jörg Votteler ◽  
Dong Young Kim ◽  
Xuefeng Ren ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Functional Characterization ◽  
Phage Display Library ◽  
Host Factor ◽  
Host Protein ◽  
Rapid Screening ◽  
Functional Studies ◽  
Negative Stain ◽  
Immunization Strategies

Viral infection and pathogenesis is mediated by host protein—viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-β-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12–419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the Fabs have overlapping or independent epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins.

Sign in / Sign up

Export Citation Format

Share Document