scholarly journals Effects of flanking sequences and cellular context on subcellular behavior and pathology of mutant HTT

2020 ◽  
Vol 29 (4) ◽  
pp. 674-688 ◽  
Author(s):  
Anjalika Chongtham ◽  
Douglas J Bornemann ◽  
Brett A Barbaro ◽  
Tamas Lukacsovich ◽  
Namita Agrawal ◽  
...  
Keyword(s):  
Structural Parameters ◽  
Subcellular Location ◽  
Full Length ◽  
Aggregation State ◽  
Cellular Processes ◽  
Structural Context ◽  
Comparison Of Experiments ◽  
Cellular Context ◽  
Flanking Sequences ◽  
Polyq Domain

Abstract Huntington’s disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the huntingtin protein (HTT) that is necessary to cause pathology and formation of HTT aggregates. Here we ask whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, we compare the effects of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ.

scholarly journals Biosensors Monitor Ligand-Selective Effects at Kappa Opioid Receptors

2021 ◽  
Author(s):  
Lucie Oberhauser ◽  
Miriam Stoeber
Keyword(s):  
Subcellular Location ◽  
Receptor Activation ◽  
Cellular Level ◽  
Two Dimensions ◽  
Kappa Opioid Receptor ◽  
Kappa Opioid ◽  
Downstream Signaling ◽  
Regulator Protein ◽  
Cellular Context ◽  
Selective Effects

AbstractThe kappa opioid receptor (KOR) has emerged as a promising therapeutic target for pain and itch treatment. There is growing interest in biased agonists that preferentially activate select signaling pathways downstream of KOR activation on the cellular level due to their therapeutic promise in retaining the analgesic and antipruritic effects and eliminating the sedative and dysphoric effects of KOR signaling on the physiological level. The concept of ligand-selective signaling includes that biased ligands promote KOR to selectively recruit one transducer or regulator protein over another, introducing bias into the signaling cascade at the very receptor-proximal level. Measuring agonist effects directly at the receptor has remained challenging and previous studies have focused on inferring agonist-selective KOR engagement with G protein relative to β-arrestin based on downstream signaling readouts. Here we discuss novel strategies to directly assess ligand-selective effects on receptor activation using KOR-interacting biosensors. The conformation-specific cytoplasmic biosensors are disconnected from the endogenous signaling machinery and provide a direct receptor-proxy readout of ligand effects in living cells. Receptor–biosensor interaction is ligand concentration dependent and can be used to determine relative ligand potency and efficacy. In addition, the biosensors reveal the existence of two dimensions of agonist bias in the cellular context: Firstly, agonists can selectively produce discrete protein-engaged KOR states and secondly, agonists can differ in the precise subcellular location at which they activate KOR. We discuss the value and the limitations of using orthogonal receptor-interacting biosensors in the quest to understand functional selectivity amongst KOR agonists in the cellular context.

scholarly journals Reverse Genetic Analysis of the Transcription Regulatory Sequence of the Coronavirus Transmissible Gastroenteritis Virus

2004 ◽  
Vol 78 (11) ◽  
pp. 6061-6066 ◽  
Author(s):  
Kristopher M. Curtis ◽  
Boyd Yount ◽  
Amy C. Sims ◽  
Ralph S. Baric
Keyword(s):  
Genetic Analysis ◽  
Full Length ◽  
Transmissible Gastroenteritis Virus ◽  
Regulatory Sequence ◽  
Reverse Genetic ◽  
Conserved Sequence ◽  
Gastroenteritis Virus ◽  
Flanking Sequences ◽  
Transmissible Gastroenteritis ◽  
Discontinuous Transcription

ABSTRACT Coronavirus discontinuous transcription uses a highly conserved sequence (CS) in the joining of leader and body RNAs. Using a full-length infectious construct of transmissable gastroenteritis virus, the present study demonstrates that subgenomic transcription is heavily influenced by upstream flanking sequences and supports a mechanism of transcription attenuation that is regulated in part by a larger domain composed of primarily upstream flanking sequences which select appropriately positioned CS elements for synthesis of subgenomic RNAs.

A possible biochemical link between NADPH oxidase (Nox) 1 redox-signalling and ERp72

2008 ◽  
Vol 416 (1) ◽  
pp. 55-63 ◽  
Author(s):  
Wei Chen ◽  
Wei Hao Shang ◽  
Yoshifumi Adachi ◽  
Kunitaka Hirose ◽  
David M. Ferrari ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Glutathione Transferase ◽  
Mutational Analysis ◽  
Reductase Activity ◽  
Signalling Pathways ◽  
Oxygen Species ◽  
Cellular Processes ◽  
Nadph Oxidase 1 ◽  
Endoplasmic Reticulum Protein ◽  
Cellular Context

Emerging evidence indicates that Nox (NADPH oxidase) 1-generated ROS (reactive oxygen species) play critical regulatory roles in various cellular processes, yet little is known of direct targets for the oxidase. In the present study we show that one of the proteins selectively oxidized in response to Nox1-generated ROS was ERp72 (endoplasmic reticulum protein 72 kDa) with TRX (thioredoxin) homology domains. Oxidation of ERp72 by Nox1 resulted in an inhibition of its reductase activity. EGF treatment of cells stimulated the Nox1 activity and the activated Nox1 subsequently mediated EGF-induced suppression of the ERp72 reductase activity. Co-immunoprecipitation, GST (glutathione transferase) pulldown assays and mutational analysis, indicated that Nox1 associates with ERp72, which involves its N-terminus encompassing a Ca2+-binding site and the first TRX-like motif. Furthermore, confocal microscopy showed co-localization between Nox1 and ERp72 at the plasma membrane. These results suggest that Nox1 functionally associates with ERp72, regulating redox-sensitive signalling pathways in a cellular context.

scholarly journals Application of the Subtractive Genomics and Molecular Docking Analysis for the Identification of Novel Putative Drug Targets against Salmonella enterica subsp. enterica serovar Poona

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Tanvir Hossain ◽  
Mohammad Kamruzzaman ◽  
Talita Zahin Choudhury ◽  
Hamida Nooreen Mahmood ◽  
A. H. M. Nurun Nabi ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Salmonella Enterica ◽  
Drug Targets ◽  
Subcellular Location ◽  
Essential Genes ◽  
Scoring Functions ◽  
Essential Proteins ◽  
Cellular Processes ◽  
Subtractive Genomics ◽  
Resistance Patterns

The emergence of novel pathogenic strains with increased antibacterial resistance patterns poses a significant threat to the management of infectious diseases. In this study, we aimed at utilizing the subtractive genomic approach to identify novel drug targets against Salmonella enterica subsp. enterica serovar Poona strain ATCC BAA-1673. We employed in silico bioinformatics tools to subtract the strain-specific paralogous and host-specific homologous sequences from the bacterial proteome. The sorted proteome was further refined to identify the essential genes in the pathogenic bacterium using the database of essential genes (DEG). We carried out metabolic pathway and subcellular location analysis of the essential proteins of the pathogen to elucidate the involvement of these proteins in important cellular processes. We found 52 unique essential proteins in the target proteome that could be utilized as novel targets to design newer drugs. Further, we investigated these proteins in the DrugBank databases and 11 of the unique essential proteins showed druggability according to the FDA approved drug bank databases with diverse broad-spectrum property. Molecular docking analyses of the novel druggable targets with the drugs were carried out by AutoDock Vina option based on scoring functions. The results showed promising candidates for novel drugs against Salmonella infections.

scholarly journals Identification of a Novel LXXLL Motif in α-Actinin 4-spliced Isoform That Is Critical for Its Interaction with Estrogen Receptor α and Co-activators

2012 ◽  
Vol 287 (42) ◽  
pp. 35418-35429 ◽  
Author(s):  
Simran Khurana ◽  
Sharmistha Chakraborty ◽  
Xuan Zhao ◽  
Yu Liu ◽  
Dongyin Guan ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Nuclear Receptors ◽  
Actin Filaments ◽  
Estrogen Receptor Α ◽  
Full Length ◽  
Cross Linking ◽  
Cytoskeletal Organization ◽  
Activation Domains ◽  
Lxxll Motif ◽  
Flanking Sequences

α-Actinins (ACTNs) are a family of proteins cross-linking actin filaments that maintain cytoskeletal organization and cell motility. Recently, it has also become clear that ACTN4 can function in the nucleus. In this report, we found that ACTN4 (full length) and its spliced isoform ACTN4 (Iso) possess an unusual LXXLL nuclear receptor interacting motif. Both ACTN4 (full length) and ACTN4 (Iso) potentiate basal transcription activity and directly interact with estrogen receptor α, although ACTN4 (Iso) binds ERα more strongly. We have also found that both ACTN4 (full length) and ACTN4 (Iso) interact with the ligand-independent and the ligand-dependent activation domains of estrogen receptor α. Although ACTN4 (Iso) interacts efficiently with transcriptional co-activators such as p300/CBP-associated factor (PCAF) and steroid receptor co-activator 1 (SRC-1), the full length ACTN4 protein either does not or does so weakly. More importantly, the flanking sequences of the LXXLL motif are important not only for interacting with nuclear receptors but also for the association with co-activators. Taken together, we have identified a novel extended LXXLL motif that is critical for interactions with both receptors and co-activators. This motif functions more efficiently in a spliced isoform of ACTN4 than it does in the full-length protein.

Cloning and Recombinant Expression of Chitosanase Gene from Bacillus Sp. S-1

2011 ◽  
Vol 343-344 ◽  
pp. 994-999
Author(s):  
Yu Ying Sun ◽  
Shu Jun Wang ◽  
Ji Quan Zhang
Keyword(s):  
Recombinant Expression ◽  
Full Length ◽  
Genome Walking ◽  
Predicted Amino Acid Sequence ◽  
Accession Number ◽  
Physiological Properties ◽  
A Genome ◽  
Full Length Sequence ◽  
Flanking Sequences

In this work, we report the characterization of a chitosanase-producing bacterium isolated from soil. This strain was grouped under the genus Bacillus by virtue of its morphological, physiological properties and 16S rDNA gene sequence and named it Bacillus sp. S-1. According to the information of chitosanase full-length sequences deposited in NCBI, a pair of degenerated primes was designed and a partial sequence of chitosanase gene was obtained by polymerase chain reaction (PCR) using Bacillus sp. S-1 genome DNA as the template. A genome walking library was constructed followed as the protocol provided by CLONTECH Company. The flanking sequences of the 5’ and 3’ terminal was obtained by genome walking method and two-step PCR technique. After overlapped and confirmed, the full-length sequence of chitosanase from Bacillus sp. S-1 was achieved and it contained 1362 bp coding 453 amino acids (accession number is EU924147). The predicted amino acid sequence was 96% similar to that of Bacillus cereus ATCC 14579 (accession number is NC_004722). The fusion protein containing BSCHITO was produced in Escherichia coli and purified using Ni-NTA affinity chromatography. The purified rBSCHITO degraded the chitosan (the degree of deacetylation of 99%) to produce mixture of chitooligosaccharides. The BSCHITO is thus an endo-chitosanase.

scholarly journals Role of the RAB7 Protein in Tumor Progression and Cisplatin Chemoresistance

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1096 ◽  
Author(s):  
Guerra ◽  
Bucci
Keyword(s):  
Cancer Progression ◽  
Extracellular Vesicle ◽  
Endocytic Pathway ◽  
Cellular Processes ◽  
Molecular Events ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Cellular Context ◽  
Guanosine Triphosphatase

RAB7 is a small guanosine triphosphatase (GTPase) extensively studied as regulator of vesicular trafficking. Indeed, its role is fundamental in several steps of the late endocytic pathway, including endosome maturation, transport from early endosomes to late endosomes and lysosomes, clustering and fusion of late endosomes and lysosomes in the perinuclear region and lysosomal biogenesis. Besides endocytosis, RAB7 is important for a number of other cellular processes among which, autophagy, apoptosis, signaling, and cell migration. Given the importance of RAB7 in these cellular processes, the interest to study the role of RAB7 in cancer progression is widely grown. Here, we describe the current understanding of oncogenic and oncosuppressor functions of RAB7 analyzing cellular context and other environmental factors in which it elicits pro and/or antitumorigenic effects. We also discuss the role of RAB7 in cisplatin resistance associated with its ability to regulate the late endosomal pathway, lysosomal biogenesis and extracellular vesicle secretion. Finally, we examined the potential cancer therapeutic strategies targeting the different molecular events in which RAB7 is involved.

scholarly journals Identification of downstream-initiated c-Myc proteins which are dominant-negative inhibitors of transactivation by full-length c-Myc proteins.

1997 ◽  
Vol 17 (3) ◽  
pp. 1459-1468 ◽  
Author(s):  
G D Spotts ◽  
S V Patel ◽  
Q Xiao ◽  
S R Hann
Keyword(s):  
Dominant Negative ◽  
Full Length ◽  
Transactivation Domain ◽  
Translational Initiation ◽  
Dna Binding Domains ◽  
Cellular Processes ◽  
Binding Domains ◽  
Inhibitory Function ◽  
Myc Gene ◽  
S Proteins

The c-myc gene has been implicated in multiple cellular processes including proliferation, differentiation, and apoptosis. In addition to the full-length c-Myc 1 and 2 proteins, we have found that human, murine, and avian cells express smaller c-Myc proteins arising from translational initiation at conserved downstream AUG codons. These c-Myc short (c-Myc S) proteins lack most of the N-terminal transactivation domain but retain the C-terminal protein dimerization and DNA binding domains. As with full-length c-Myc proteins, the c-Myc S proteins appear to be localized to the nucleus, are relatively unstable, and are phosphorylated. Significant levels of c-Myc S, often approaching the levels of full-length c-Myc, are transiently observed during the rapid growth phase of several different types of cells. Optimization of the upstream initiation codons resulted in greatly reduced synthesis of the c-Myc S proteins, suggesting that a "leaky scanning" mechanism leads to the translation of these proteins. In some hematopoietic tumor cell lines having altered c-myc genes, the c-Myc S proteins are constitutively expressed at levels equivalent to that of full-length c-Myc. As predicted, the c-Myc S proteins are unable to activate transcription and inhibited transactivation by full-length c-Myc proteins, suggesting a dominant-negative inhibitory function. While these transcriptional inhibitors would not be expected to function as full-length c-Myc, the occurrence of tumors which express constitutive high levels of c-Myc S and their transient synthesis during rapid cell growth suggest that these proteins do not interfere with the growth-promoting functions of full-length c-Myc.

Identification of the structural motif responsible for trimeric assembly of the C-terminal regulatory domains of polycystin channels PKD2L1 and PKD2

2010 ◽  
Vol 429 (1) ◽  
pp. 171-183 ◽  
Author(s):  
Katrina L. Molland ◽  
Anoop Narayanan ◽  
John W. Burgner ◽  
Dinesh A. Yernool
Keyword(s):  
Coiled Coil ◽  
Cd Spectroscopy ◽  
Full Length ◽  
Structural Motif ◽  
Aggregation State ◽  
Regulatory Domains ◽  
Kd Values ◽  
Autosomal Dominant Polycystic Kidney ◽  
Α Helix ◽  
Asymmetric Environment

Polycystin 2-type cation channels PKD2 and PKD2L1 interact with polycystin 1-type proteins PKD1 and PKD1L3 respectively, to form receptor–cation-channel complexes. The PKD2L1–PKD1L3 complex perceives sour taste, whereas disruption of the PKD2–PKD1 complex, responsible for mechanosensation, leads to development of ADPKD (autosomal-dominant polycystic kidney disease). Besides modulating channel activity and related signalling events, the CRDs (C-terminal regulatory domains) of PKD2 and PKD2L1 play a central role in channel oligomerization. The present study investigates the aggregation state of purified full-length PKD2L1-CRD as well as truncations of CRDs from PKD2 channels. Far- and near-UV CD spectroscopy show that the full-length PKD2L1 CRD (PKD2L1-198) and the truncated PKD2 CRD (PKD2-244) are α-helical with no β-sheet, the α-helix content agrees with sequence-based predictions, and some of its aromatic residues are in an asymmetric environment created at least by partially structured regions. Additionally, the CRD truncations exhibit an expected biochemical function by binding Ca2+ in a physiologically relevant range with Kd values of 2.8 μM for PKD2-244 and 0.51 μM for PKD2L1-198. Complimentary biophysical and biochemical techniques establish that truncations of the PKD2 and PKD2L1 CRDs are elongated molecules that assemble as trimers, and the trimeric aggregation state is independent of Ca2+ binding. Finally, we show that a common coiled-coil motif is sufficient and necessary to drive oligomerization of the PKD2 and PKD2L1 CRD truncations under study. Despite the moderate sequence identity (39%) between CRDs of PKD2 and PKD2L1, they both form trimers, implying that trimeric organization of CRDs may be true of all polycystin channels.

scholarly journals Domain Organization in Plant Blue-Light Receptor Phototropin2 of Arabidopsis thaliana Studied by Small-Angle X-ray Scattering

2020 ◽  
Vol 21 (18) ◽  
pp. 6638
Author(s):  
Masayoshi Nakasako ◽  
Mao Oide ◽  
Yuki Takayama ◽  
Tomotaka Oroguchi ◽  
Koji Okajima
Keyword(s):  
Blue Light ◽  
Small Angle ◽  
Structural Changes ◽  
Structural Parameters ◽  
Kinase Domain ◽  
Full Length ◽  
Receptor Protein ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

Phototropin2 (phot2) is a blue-light (BL) receptor protein that regulates the BL-dependent activities of plants for efficient photosynthesis. Phot2 is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) to absorb BL, and a kinase domain. Photo-activated LOV domains, especially LOV2, play a major role in photo-dependent increase in the phosphorylation activity of the kinase domain. The atomic details of the overall structure of phot2 and the intramolecular mechanism to convert BL energy to a phosphorylation signal remain unknown. We performed structural studies on the LOV fragments LOV1, LOV2, LOV2-linker, and LOV2-kinase, and full-length phot2, using small-angle X-ray scattering (SAXS). The aim of the study was to understand structural changes under BL irradiation and discuss the molecular mechanism that enhance the phosphorylation activity under BL. SAXS is a suitable technique for visualizing molecular structures of proteins in solution at low resolution and is advantageous for monitoring their structural changes in the presence of external physical and/or chemical stimuli. Structural parameters and molecular models of the recombinant specimens were obtained from SAXS profiles in the dark, under BL irradiation, and after dark reversion. LOV1, LOV2, and LOV2-linker fragments displayed minimal structural changes. However, BL-induced rearrangements of functional domains were noted for LOV2-kinase and full-length phot2. Based on the molecular model together with the absorption measurements and biochemical assays, we discuss the intramolecular interactions and domain motions necessary for BL-enhanced phosphorylation activity of phot2.

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