mutant proteins
Recently Published Documents


TOTAL DOCUMENTS

1051
(FIVE YEARS 170)

H-INDEX

81
(FIVE YEARS 7)

2022 ◽  
Author(s):  
Xuye Yuan ◽  
Tatsuhiko Kadowaki

Deformed wing virus (DWV) is the most prevalent Iflavirus that is infecting honey bees worldwide. However, the mechanisms of its infection and replication in host cells are poorly understood. In this study, we analyzed the structure and function of DWV 3C protease (3Cpro), which is necessary for the cleavage of the polyprotein to synthesize mature viral proteins. We found that the 3Cpros of DWV and picornaviruses share common enzymatic properties, including sensitivity to the same inhibitors, such as rupintrivir. The predicted structure of DWV 3Cpro by AlphaFold2, the predicted rupintrivir binding domain, and the protease activities of mutant proteins revealed that it has a Cys-His-Asn catalytic triad. Moreover, 3Cpros of other Iflaviruses and Dicistrovirus appear to contain Asn, Ser, Asp, or Glu as the third residue of the catalytic triad, suggesting diversity in insect RNA viruses. Both precursor 3Cpro with RNA-dependent RNA polymerase and mature 3Cpro are present in DWV-infected cells, suggesting that they may have different enzymatic properties and functions. DWV 3Cpro is the first 3Cpro characterized among insect RNA viruses, and our study uncovered both the common and unique characteristics among 3Cpros of Picornavirales. Furthermore, the specific inhibitors of DWV 3Cpro could be used to control DWV infection in honey bees.


2021 ◽  
Vol 14 (1) ◽  
pp. 11-33
Author(s):  
Sui Sawaguchi ◽  
Kenji Tago ◽  
Hiroaki Oizumi ◽  
Katsuya Ohbuchi ◽  
Masahiro Yamamoto ◽  
...  

Hypomyelinating leukodystrophy 7 (HLD7) is an autosomal recessive oligodendroglial cell-related myelin disease, which is associated with some nucleotide mutations of the RNA polymerase 3 subunit a (polr3a) gene. POLR3A is composed of the catalytic core of RNA polymerase III synthesizing non-coding RNAs, such as rRNA and tRNA. Here, we show that an HLD7-associated nonsense mutation of Arg140-to-Ter (R140X) primarily localizes POLR3A proteins as protein aggregates into lysosomes in mouse oligodendroglial FBD−102b cells, whereas the wild type proteins are not localized in lysosomes. Expression of the R140X mutant proteins, but not the wild type proteins, in cells decreased signaling through the mechanistic target of rapamycin (mTOR), controlling signal transduction around lysosomes. While cells harboring the wild type constructs exhibited phenotypes with widespread membranes with myelin marker protein expression following the induction of differentiation, cells harboring the R140X mutant constructs did not exhibit them. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), which is also known as an mTOR signaling activator, ameliorated defects in differentiation with myelin marker protein expression and the related signaling in cells harboring the R140X mutant constructs. Collectively, HLD7-associated POLR3A mutant proteins are localized in lysosomes where they decrease mTOR signaling, inhibiting cell morphological differentiation. Importantly, ibuprofen reverses undifferentiated phenotypes. These findings may reveal some of the pathological mechanisms underlying HLD7 and their amelioration at the molecular and cellular levels.


Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3419
Author(s):  
Sunny Xia ◽  
Zoltán Bozóky ◽  
Michelle Di Paola ◽  
Onofrio Laselva ◽  
Saumel Ahmadi ◽  
...  

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.


2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Valeria Calvaresi ◽  
Line T. Truelsen ◽  
Sidsel B. Larsen ◽  
Nikolaj H. T. Petersen ◽  
Thomas Kirkegaard ◽  
...  

AbstractThe binding of the major stress-inducible human 70-kDa heat shock protein (Hsp70) to the anionic phospholipid bis-(monoacylglycero)-phosphate (BMP) in the lysosomal membrane is crucial for its impact on cellular pathology in lysosomal storage disorders. However, the conformational features of this protein-lipid complex remain unclear. Here, we apply hydrogen–deuterium exchange mass spectrometry (HDX-MS) to describe the dynamics of the full-length Hsp70 in the cytosol and its conformational changes upon translocation into lysosomes. Using wild-type and W90F mutant proteins, we also map and discriminate the interaction of Hsp70 with BMP and other lipid components of the lysosomal membrane. We identify the N-terminal of the nucleotide binding domain (residues 87–118) as the primary orchestrator of BMP interaction. We show that the conformation of this domain is significantly reorganized in the W90F mutant, explaining its inability to stabilize lysosomal membranes. Overall, our results reveal important new molecular details of the protective effect of Hsp70 in lysosomal storage diseases, which, in turn, could guide future drug development.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sven-A. Freibert ◽  
Michal T. Boniecki ◽  
Claudia Stümpfig ◽  
Vinzent Schulz ◽  
Nils Krapoth ◽  
...  

AbstractSynthesis of iron-sulfur (Fe/S) clusters in living cells requires scaffold proteins for both facile synthesis and subsequent transfer of clusters to target apoproteins. The human mitochondrial ISCU2 scaffold protein is part of the core ISC (iron-sulfur cluster assembly) complex that synthesizes a bridging [2Fe-2S] cluster on dimeric ISCU2. Initial iron and sulfur loading onto monomeric ISCU2 have been elucidated biochemically, yet subsequent [2Fe-2S] cluster formation and dimerization of ISCU2 is mechanistically ill-defined. Our structural, biochemical and cell biological experiments now identify a crucial function of the universally conserved N-terminal Tyr35 of ISCU2 for these late reactions. Mixing two, per se non-functional ISCU2 mutant proteins with oppositely charged Asp35 and Lys35 residues, both bound to different cysteine desulfurase complexes NFS1-ISD11-ACP, restores wild-type ISCU2 maturation demonstrating that ionic forces can replace native Tyr-Tyr interactions during dimerization-induced [2Fe-2S] cluster formation. Our studies define the essential mechanistic role of Tyr35 in the reaction cycle of de novo mitochondrial [2Fe-2S] cluster synthesis.


2021 ◽  
Author(s):  
Thiya Mukherjee ◽  
Bibek Subedi ◽  
Aashima Khosla ◽  
Adara L Warner ◽  
Ruben Lerma-Reyes ◽  
...  

Class IV homeodomain leucine-zipper transcription factors (HD-Zip IV TFs) are key regulators of epidermal differentiation that are characterized by a DNA-binding homeodomain (HD) in conjunction with lipid sensor domain termed START (Steroidogenic Acute Regulatory (StAR)-related lipid Transfer). Previous work demonstrated that the START domain of GLABRA2 (GL2), a HD-Zip IV member from Arabidopsis, is required for transcription factor activity. Here, we address the functions and possible interactions of START and the HD in DNA binding, dimerization, and protein turnover. Deletion analysis of the HD and missense mutations of a conserved lysine (K146) result in phenotypic defects in leaf trichomes, root hairs and seed mucilage, similar to those observed for START mutants, despite nuclear localization of the mutant proteins. Gel shift and ChIP-seq experiments demonstrate that while HD mutations impair binding to target DNA, the START domain is dispensable for DNA binding. Vice versa, yeast two-hybrid assays reveal impaired GL2 dimerization for START domain mutants, but not HD mutants. Using in vivo cycloheximide chase experiments, we provide evidence for the role of START, but not HD, in maintaining protein stability. This work advances our fundamental understanding of HD-Zip TFs as multidomain regulators of epidermal development in plants.


2021 ◽  
Vol 12 ◽  
Author(s):  
Zijing Zhou ◽  
Jinyuan Vero Li ◽  
Boris Martinac ◽  
Charles D. Cox

Missense mutations in the gene that encodes for the mechanically-gated ion channel Piezo1 have been linked to a number of diseases. Gain-of-function variants are linked to a hereditary anaemia and loss-of-function variants have been linked to generalized lymphatic dysplasia and bicuspid aortic valve. Two previously characterized mutations, S217L and G2029R, both exhibit reduced plasma membrane trafficking. Here we show that both mutations also display reduced stability and higher turnover rates than wild-type Piezo1 channels. This occurs through increased ubiquitination and subsequent proteasomal degradation. Congruent with this, proteasome inhibition using N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) reduced the degradation of both mutant proteins. While ALLN treatment could not rescue the function of S217L we show via multiple complementary methodologies that proteasome inhibition via ALLN treatment can not only prevent G2029R turnover but increase the membrane localized pool of this variant and the functional Piezo1 mechanosensitive currents. This data in combination with a precision medicine approach provides a new potential therapeutic avenue for the treatment of Piezo1 mediated channelopathies.


2021 ◽  
Vol 12 ◽  
Author(s):  
Kun Chu ◽  
Yi He ◽  
Ziyuan Li ◽  
Zhongxin Jiang ◽  
Liang Wang ◽  
...  

Premature ovarian insufficiency (POI) affects about 1% of women under 40 years and leads most often to definitive infertility with adverse health outcomes. Genetic factor has been reported to play an important role in POI. However, the genetic etiology remains unknown in the majority of the POI patients. Whole-exome sequencing and variant analysis were carried out in a POI pedigree. In vitro studies of the wild-type and mutant proteins were conducted in primary granulosa cells (GCs) and granulosa cell line. The result showed that the patients carried compound heterozygous nonsynonymous mutations (c.245C > T and c.181C > G) in LAT gene, which were identified to be transmitted from their parents. The two variants were assessed to affect residues that were conserved across different species examined, and were predicted to be deleterious by software predictions. Protein structure predicting result indicated that the two variants could alter their interactions with surrounding residues, which may change the internal structure of the LAT protein. Moreover, LAT protein expression in GCs was demonstrated for the first time, and further functional assays suggested that this mutation could reduce LAT expression and influence GC survival, which may contribute to the etiology of POI. In summary, we detect novel LAT pathogenic variants in a POI pedigree and report for the first time that LAT is present and functional in the GCs of the ovary. Our findings not only shed new light on the role of LAT in GCs, but also broaden the spectrum of genetic causes of POI.


Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1197
Author(s):  
Éva Bulyáki ◽  
Judit Kun ◽  
Tamás Molnár ◽  
Alexandra Papp ◽  
András Micsonai ◽  
...  

β2-microglobulin (β2m), the light chain of the MHC-I complex, is associated with dialysis-related amyloidosis (DRA). Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m variant, which showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Here, we investigated the role of the D76N mutation in the amyloid formation of β2m by point mutations affecting the Asp76-Lys41 ion-pair of WT β2m and the charge cluster on Asp38. Using a variety of biophysical techniques, we investigated the conformational stability and partial unfolding of the native state of the variants, as well as their amyloidogenic propensity and the stability of amyloid fibrils under various conditions. Furthermore, we studied the intermolecular interactions of WT and mutant proteins with various binding partners that might have in vivo relevance. We found that, relative to WT β2m, the exceptional amyloidogenicity of the pathogenic D76N β2m variant is realized by the deleterious synergy of diverse effects of destabilized native structure, higher sensitivity to negatively charged amphiphilic molecules (e.g., lipids) and polyphosphate, more effective fibril nucleation, higher conformational stability of fibrils, and elevated affinity for extracellular components, including extracellular matrix proteins.


2021 ◽  
Vol 12 ◽  
Author(s):  
Dongheng Zhou ◽  
Huanhuan Yan ◽  
Shuying Yang ◽  
Yuhong Zhang ◽  
Xiaoyan Xu ◽  
...  

Abnormal accumulation of TDP43-related mutant proteins in the cytoplasm causes amyotrophic lateral sclerosis (ALS). Herein, unbiased drug screening approaches showed that SC75741, a multi-target inhibitor, inhibited inflammation-induced aggregation by inhibiting NF-κB and also degraded already aggregated proteins by inhibiting c-Abl mediated autophagy-lysosomal pathway. We delineate the mechanism that SC75741 could markedly enhance TFEB nuclear translocation by an mTORC1-independent TFEB regulatory pathway. In addition, SC75741 enhanced the interaction between p62 with TDP25 and LC3C, thus promoting TDP25 degradation. Taken together, these findings show that SC75741 has beneficial neuroprotective effects in ALS. Our study elucidates that dual-targeted inhibition of c-Abl and NF-κB may be a potential treatment for TDP43 proteinopathies and ALS.


Sign in / Sign up

Export Citation Format

Share Document