scholarly journals A Class III PDZ Binding Motif in the Myotilin and FATZ Families Binds Enigma Family Proteins: a Common Link for Z-Disc Myopathies

2008 ◽  
Vol 29 (3) ◽  
pp. 822-834 ◽  
Author(s):  
Pernilla von Nandelstadh ◽  
Mohamed Ismail ◽  
Chiara Gardin ◽  
Heli Suila ◽  
Ivano Zara ◽  
...  
Keyword(s):  
Family Members ◽  
In Vivo Studies ◽  
Binding Motif ◽  
High Homology ◽  
Class Iii ◽  
Pdz Domains ◽  
Muscle Disorders ◽  
C Terminus

ABSTRACT Interactions between Z-disc proteins regulate muscle functions and disruption of these interactions results in muscle disorders. Mutations in Z-disc components myotilin, ZASP/Cypher, and FATZ-2 (calsarcin-1/myozenin-2) are associated with myopathies. We report here that the myotilin and the FATZ (calsarcin/myozenin) families share high homology at their final C-terminal five amino acids. This C-terminal E[ST][DE][DE]L motif is present almost exclusively in these families and is evolutionary conserved. We show by in vitro and in vivo studies that proteins from the myotilin and FATZ (calsarcin/myozenin) families interact via this novel type of class III PDZ binding motif with the PDZ domains of ZASP/Cypher and other Enigma family members: ALP, CLP-36, and RIL. We show that the interactions can be modulated by phosphorylation. Calmodulin-dependent kinase II phosphorylates the C terminus of FATZ-3 (calsarcin-3/myozenin-3) and myotilin, whereas PKA phosphorylates that of FATZ-1 (calsarcin-2/myozenin-1) and FATZ-2 (calsarcin-1/myozenin-1). This is the first report of a binding motif common to both the myotilin and the FATZ (calsarcin/myozenin) families that is specific for interactions with Enigma family members.

scholarly journals The Biological and Clinical Relevance of Inhibitor of Growth (ING) Genes in Non-Small Cell Lung Cancer

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1118 ◽  
Author(s):  
Elisabeth Smolle ◽  
Nicole Fink-Neuboeck ◽  
Joerg Lindenmann ◽  
Freyja Smolle-Juettner ◽  
Martin Pichler
Keyword(s):  
Lung Cancer ◽  
Family Members ◽  
In Vivo Studies ◽  
Action Mode ◽  
Inhibition Of Growth ◽  
Molecular Mechanism Of Action ◽  
Anchor Points ◽  
Lung Cancer Detection

Carcinogenic mutations allow cells to escape governing mechanisms that commonly inhibit uncontrolled cell proliferation and maintain tightly regulated homeostasis between cell death and survival. Members of the inhibition of growth (ING) family act as tumor suppressors, governing cell cycle, apoptosis and cellular senescence. The molecular mechanism of action of ING genes, as well as their anchor points in pathways commonly linked to malignant transformation of cells, have been studied with respect to a variety of cancer specimens. This review of the current literature focuses specifically on the action mode of ING family members in lung cancer. We have summarized data from in vitro and in vivo studies, highlighting the effects of varying levels of ING expression in cancer cells. Based on the increasing insight into the function of these proteins, the use of ING family members as clinically useful biomarkers for lung cancer detection and prognosis will probably become routine in everyday clinical practice.

scholarly journals Post-translational incorporation of the antiproliferative agent azatyrosine into the C-terminus of α-tubulin

2003 ◽  
Vol 375 (1) ◽  
pp. 121-129 ◽  
Author(s):  
Silvia A. PURRO ◽  
C. Gastón BISIG ◽  
María A. CONTIN ◽  
Héctor S. BARRA ◽  
Carlos A. ARCE
Keyword(s):  
Cultured Cells ◽  
In Vivo Studies ◽  
Brain Extract ◽  
Similar Degree ◽  
C6 Cells ◽  
Post Translational Modification ◽  
Α Subunit ◽  
C Terminus

Detyrosination/tyrosination of tubulin is a post-translational modification that occurs at the C-terminus of the α-subunit, giving rise to microtubules rich in either tyrosinated or detyrosinated tubulin which coexist in the cell. We hereby report that the tyrosine analogue, azatyrosine, can be incorporated into the C-terminus of α-tubulin instead of tyrosine. Azatyrosine is structurally identical to tyrosine except that a nitrogen atom replaces carbon-2 of the phenolic group. Azatyrosine competitively excluded incorporation of [14C]tyrosine into tubulin of soluble brain extract. A newly developed rabbit antibody specific to C-terminal azatyrosine was used to study incorporation of azatyrosine in cultured cells. When added to the culture medium (Ham's F12K), azatyrosine was incorporated into tubulin of glioma-derived C6 cells. This incorporation was reversible, i.e. after withdrawal of azatyrosine, tubulin lost azatyrosine and reincorporated tyrosine. Azatyrosinated tubulin self-assembled into microtubules to a similar degree as total tubulin both in vitro and in vivo. Studies by other groups have shown that treatment of certain types of cultured cancer cells with azatyrosine leads to reversion of phenotype to normal, and that administration of azatyrosine into animals harbouring human proto-oncogenic c-Ha-ras prevents tumour formation. These interesting observations led us to study this phenomenon in relation to tubulin status. Under conditions in which tubulin was mostly azatyrosinated, C6 cells remained viable but did not proliferate. After 7–10 days under these conditions, morphology changed from a fused, elongated shape to a rounded soma with thin processes. Incorporation of azatyrosine into the C-terminus of α-tubulin is proposed as one possible cause of reversion of the malignant phenotype.

scholarly journals Polyphenols and Their Effects on Muscle Atrophy and Muscle Health

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 4887
Author(s):  
Takeshi Nikawa ◽  
Anayt Ulla ◽  
Iori Sakakibara
Keyword(s):  
Muscle Atrophy ◽  
Polyphenolic Compounds ◽  
Bioactive Molecules ◽  
In Vivo Studies ◽  
Skeletal Muscle Atrophy ◽  
Physiological Factors ◽  
Muscle Disorders ◽  
Muscle Health

Skeletal muscle atrophy is the decrease in muscle mass and strength caused by reduced protein synthesis/accelerated protein degradation. Various conditions, such as denervation, disuse, aging, chronic diseases, heart disease, obstructive lung disease, diabetes, renal failure, AIDS, sepsis, cancer, and steroidal medications, can cause muscle atrophy. Mechanistically, inflammation, oxidative stress, and mitochondrial dysfunction are among the major contributors to muscle atrophy, by modulating signaling pathways that regulate muscle homeostasis. To prevent muscle catabolism and enhance muscle anabolism, several natural and synthetic compounds have been investigated. Recently, polyphenols (i.e., natural phytochemicals) have received extensive attention regarding their effect on muscle atrophy because of their potent antioxidant and anti-inflammatory properties. Numerous in vitro and in vivo studies have reported polyphenols as strongly effective bioactive molecules that attenuate muscle atrophy and enhance muscle health. This review describes polyphenols as promising bioactive molecules that impede muscle atrophy induced by various proatrophic factors. The effects of each class/subclass of polyphenolic compounds regarding protection against the muscle disorders induced by various pathological/physiological factors are summarized in tabular form and discussed. Although considerable variations in antiatrophic potencies and mechanisms were observed among structurally diverse polyphenolic compounds, they are vital factors to be considered in muscle atrophy prevention strategies.

scholarly journals The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

1999 ◽  
Vol 10 (7) ◽  
pp. 2119-2129 ◽  
Author(s):  
Marcus P. Cooper ◽  
Adayabalam S. Balajee ◽  
Vilhelm A. Bohr
Keyword(s):  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Binding Domain ◽  
In Vivo Studies ◽  
C Terminus ◽  
Nucleotide Excision ◽  
N Terminus

The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase δ. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.

scholarly journals Retinoblastoma Protein Contains a C-terminal Motif That Targets It for Phosphorylation by Cyclin-cdk Complexes

1999 ◽  
Vol 19 (2) ◽  
pp. 1068-1080 ◽  
Author(s):  
Peter D. Adams ◽  
Xiaotong Li ◽  
William R. Sellers ◽  
Kayla B. Baker ◽  
Xiaohong Leng ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
C Terminus ◽  
Sequence Elements ◽  
Stable Association ◽  
Retinoblastoma Tumor

ABSTRACT Stable association of certain proteins, such as E2F1 and p21, with cyclin-cdk2 complexes is dependent upon a conserved cyclin-cdk2 binding motif that contains the core sequence ZRXL, where Z and X are usually basic. In vitro phosphorylation of the retinoblastoma tumor suppressor protein, pRB, by cyclin A-cdk2 and cyclin E-cdk2 was inhibited by a short peptide spanning the cyclin-cdk2 binding motif present in E2F1. Examination of the pRB C terminus revealed that it contained sequence elements related to ZRXL. Site-directed mutagenesis of one of these sequences, beginning at residue 870, impaired the phosphorylation of pRB in vitro. A synthetic peptide spanning this sequence also inhibited the phosphorylation of pRB in vitro. pRB C-terminal truncation mutants lacking this sequence were hypophosphorylated in vitro and in vivo despite the presence of intact cyclin-cdk phosphoacceptor sites. Phosphorylation of such mutants was restored by fusion to the ZRXL-like motif derived from pRB or to the ZRXL motifs from E2F1 or p21. Phospho-site-specific antibodies revealed that certain phosphoacceptor sites strictly required a C-terminal ZRXL motif whereas at least one site did not. Furthermore, this residual phosphorylation was sufficient to inactivate pRB in vivo, implying that there are additional mechanisms for directing cyclin-cdk complexes to pRB. Thus, the C terminus of pRB contains a cyclin-cdk interaction motif of the type found in E2F1 and p21 that enables it to be recognized and phosphorylated by cyclin-cdk complexes.

scholarly journals First Report of Fenpyrazamine Resistance in Botrytis cinerea from Strawberry Fields in Spain

2018 ◽  
Vol 19 (1) ◽  
pp. 45-45
Author(s):  
Dolores Fernández-Ortuño ◽  
Alejandra Vielba-Fernández ◽  
Alejandro Pérez-García ◽  
Juan A. Torés ◽  
Antonio de Vicente
Keyword(s):  
Botrytis Cinerea ◽  
Gray Mold ◽  
In Vivo Studies ◽  
Cross Resistance ◽  
In Vitro Tests ◽  
Class Iii ◽  
Single Spore ◽  
First Report

Botrytis cinerea Pers. is an important fungal pathogen responsible for gray mold, one of the most economically important diseases of strawberry (Fragaria × ananassa) worldwide. The primary disease management strategy involves the application of different classes of fungicides, including the sterol biosynthesis inhibitor class III fungicide fenpyrazamine. In 2014 and 2015, strawberries affected with gray mold symptoms were collected from eight locations in Huelva, where fenhexamid had been used extensively. Twenty-five B. cinerea single-spore isolates were examined to determine EC50 values and to determine a discriminatory dose to monitor fenpyrazamine resistance in the field in future studies. The in vitro tests divided the isolates into two groups: 15 sensitive (EC50 from 0.02 to 1.3 μg/ml) and 10 resistant (EC50 from 50.1 to 172.6 μg/ml), which showed cross-resistance with fenhexamid. Performance of fenpyrazamine in in vivo studies was also carried out. Only the fenpyrazamine-resistant isolates developed gray mold on the fungicide-treated fruit. This is the first report of fenpyrazamine resistance in B. cinerea from strawberry fields in Spain and cross-resistance with fenhexamid.

scholarly journals Peptidic degron in EID1 is recognized by an SCF E3 ligase complex containing the orphan F-box protein FBXO21

2015 ◽  
Vol 112 (50) ◽  
pp. 15372-15377 ◽  
Author(s):  
Cuiyan Zhang ◽  
Xiaotong Li ◽  
Guillaume Adelmant ◽  
Jessica Dobbins ◽  
Christoph Geisen ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Family Members ◽  
Binding Motif ◽  
Retinoblastoma Tumor Suppressor ◽  
Ubiquitin Ligase Complex ◽  
Retinoblastoma Tumor ◽  
F Box Protein ◽  
Necessary And Sufficient

EP300-interacting inhibitor of differentiation 1 (EID1) belongs to a protein family implicated in the control of transcription, differentiation, DNA repair, and chromosomal maintenance. EID1 has a very short half-life, especially in G0 cells. We discovered that EID1 contains a peptidic, modular degron that is necessary and sufficient for its polyubiquitylation and proteasomal degradation. We found that this degron is recognized by an Skp1, Cullin, and F-box (SCF)-containing ubiquitin ligase complex that uses the F-box Only Protein 21 (FBXO21) as its substrate recognition subunit. SCFFBXO21 polyubiquitylates EID1 both in vitro and in vivo and is required for the efficient degradation of EID1 in both cycling and quiescent cells. The EID1 degron partially overlaps with its retinoblastoma tumor suppressor protein-binding domain and is congruent with a previously defined melanoma-associated antigen-binding motif shared by EID family members, suggesting that binding to retinoblastoma tumor suppressor and melanoma-associated antigen family proteins could affect the polyubiquitylation and turnover of EID family members in cells.

scholarly journals Immuno informatics Approach in Designing a Novel Vaccine Using Epitopes from All the Structural Proteins of SARS-CoV-2

2020 ◽  
Vol 13 (4) ◽  
pp. 1845-1862
Author(s):  
Leana Rich M. Herrera
Keyword(s):  
Immune Response ◽  
Tertiary Structure ◽  
Cross Reactivity ◽  
Structural Proteins ◽  
In Vivo Studies ◽  
Binding Motif ◽  
Population Coverage ◽  
Viral Attachment

The rapid transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted to the death of hundreds of thousands of people worldwide. With the devastating effects on the economy and healthcare system of many countries, it is crucial to acceleratevaccine development against SARS-CoV-2. Thus, thisworkutilizedimmunoinformaticsto efficiently design a novel multi-epitope vaccine that can potentially induce immune response through the immunogenic, and abundantly expressed structural proteins in SARS-CoV-2. Epitopes were screened and evaluated using various immunoinformatics tools and databases. Antigenicity, allergenicity, and population coverage were assessed. Epitopes were adjoined to form a single vaccine construct (Covax),linked with 50S ribosomal protein as an adjuvant. Physicochemical properties, cross-reactivity, antigenicity,andallergenicityof Covax were evaluated. The tertiary structure of Covax was modeled, refined and validated for docking with toll-like receptor 4 (TLR4). Binding affinity of Covax-TLR4 was estimated and compared with TLR4-adjuvant as control. Lastly,the immune response with Covax was simulated and compared withadjuvant alone. Total of 33 epitopes from S (21), E (3), M (5),and N (4)proteins were merged in Covax. These include epitopes on thereceptor-binding motif (RBM) of S protein known to beessential in the viral attachment. In silico evaluations classified Covax as stable, antigenic, and non-allergenic. Epitopes were estimated to have large worldwide population coverage, especially in areas with high infection rates, indicating broad potential efficacy of Covax as a vaccine for the most affected populations.Results in this work showed that Covax can bind to TLR4 whichindicates potential immunogenicity and superior properties necessary for a successful vaccine. Overall, this work efficiently minimized time, effort and cost in designing a candidate vaccine against SARS-CoV-2. In vitro and in vivo studies on Covax are anticipated.

scholarly journals Regulation of Plant Phototropic Growth by NPH3/RPT2-like Substrate Phosphorylation and 14-3-3 Binding

2021 ◽  
Author(s):  
Stuart Sullivan ◽  
Thomas Waksman ◽  
Louise Henderson ◽  
Dimitra Paliogianni ◽  
Melanie Lütkemeyer ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Common Mechanism ◽  
Binding Motif ◽  
Growth Responses ◽  
Directional Growth ◽  
Agc Kinases ◽  
C Terminus

Polarity underlies all plant physiology and directional growth responses such as phototropism. Yet, our understanding of how plant tropic responses are established is far from complete. The plasma-membrane associated BTB-containing protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by AGC kinases known as the phototropins (phots). However, the mechanism by which phots initiate phototropic signalling via NPH3, and other NPH3/RPT2-like (NRL) members, has remained unresolved. Here we demonstrate that NPH3 is directly phosphorylated by phot1 both in vitro and in vivo. Light-dependent phosphorylation within a conserved consensus sequence (RxS) located at the extreme C-terminus of NPH3 is necessary to promote its functionality for phototropism and petiole positioning in Arabidopsis. Phosphorylation of this region by phot1 also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Seedlings expressing mutants of NPH3 that are unable to bind or constitutively bind 14-3-3s show compromised functionality that is consistent with a model where signalling outputs arising from a gradient in NPH3 RxS phosphorylation/localisation across the stem are a major contributor to phototropic responsiveness. Our current findings provide further evidence that 14-3-3 proteins are instrumental components regulating auxin-dependent growth and show for the first time that NRL proteins are direct phosphorylation targets for plant AGC kinases. Moreover, the C-terminal phosphorylation site/14-3-3-binding motif of NPH3 is conserved in several members of the NRL family, suggesting a common mechanism of regulation.

scholarly journals Molecular cloning and characterization of PLC-η2

2005 ◽  
Vol 391 (3) ◽  
pp. 667-676 ◽  
Author(s):  
Yixing Zhou ◽  
Michele R. Wing ◽  
John Sondek ◽  
T. Kendall Harden
Keyword(s):  
Splice Variants ◽  
Second Messenger ◽  
Binding Motif ◽  
Rt Pcr ◽  
Specific Expression ◽  
Catalytic Core ◽  
C Terminus ◽  
Rapid Amplification

PLC (phospholipase C) isoenzymes catalyse the conversion of PtdIns(4,5)P2 into the Ca2+-mobilizing second messenger, Ins(1,4,5)P3, and the protein kinase C-activating second messenger, diacylglycerol. With the goal of identifying additional mammalian PLC isoenzymes, we screened the NCBI non-redundant database using a BLAST algorithm for novel sequences with homology with the conserved PLC catalytic core. Two unique sequences corresponding to two unknown PLC isoenzymes were identified, and one of these, designated PLC-η2, was cloned and characterized. Most of the coding sequence of PLC-η2 was constructed from two ESTs (expressed sequence tags), which included an overlapping sequence that was confirmed by multiple ESTs and mRNAs. 5′-RACE (rapid amplification of cDNA ends) also identified an upstream exon not deduced from available EST or mRNA sequences. Sequence analysis of PLC-η2 revealed the canonical domains of a PLC isoenzyme with an additional long C-terminus that contains a class II PDZ-binding motif. Genomic analyses indicated that PLC-η2 is encoded by 23 exons. RT-PCR (reverse transcriptase-PCR) analyses illustrated expression of PLC-η2 in human retina and kidney, as well as in mouse brain, eye and lung. RT-PCR with exon-specific primers also revealed tissue-specific expression of four splice variants in mouse that represent alternative use of sequences in exons 21, 22 and 23. PLC-η2-specific antisera recognized one of these splice variants as an approx. 155 kDa species when expressed in COS-7 cells; PLC-η2 natively expressed in 1321N1 human astrocytoma cells also migrated as an approx. 155 kDa species. PLC activity was observed in vitro and in vivo for three different constructs of PLC-η2, each containing possible alternatively spliced first exons. Co-expression of PLC-η2 with Gβ1γ2 dimers of heterotrimeric G-proteins resulted in marked stimulation of inositol lipid hydrolysis. Thus PLC-η2 may in part function downstream of G-protein-coupled receptors.

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