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1.
Int J Biol Macromol ; 275(Pt 1): 133634, 2024 Jul 02.
Article in English | MEDLINE | ID: mdl-38964690

ABSTRACT

Traditional medicines have reportedly treated SARS-CoV-2 infection. Substantial evidence shows that fish oil supplements promote human immune function, suggesting they may lessen susceptibility to SARS-CoV-2 infection and suppress viral replication by inducing interferon. Fish oil was subjected to partition chromatography and separated into two compounds (EP01 and DH01). Isolated compounds were purified and characterized using UV, FTIR, NMR, and mass spectrometry to confirm their identity. Molecular docking was studied on the SARS CoV-2 variants of concern; SARS CoV-2 WT (PDB: 6VXX), SARS CoV-2 Alpha variant (PDB: 7LWS), SARS CoV-2 Delta variant (PDB: 7TOU), SARS CoV-2 Gamma variant (PDB: 7V78), SARS CoV-2 Kappa variant (PDB: 7VX9), and SARS CoV-2 Omicron variant (PDB: 7QO7) and TMPRSS2 (PDB: 7Y0E). Further selected protein-ligand complexes were subjected to 100 ns MD simulations to predict their biological potential in the SARS-CoV-2 treatment. In-vitro biological studies were carried out to support in-silico findings. Isolated compounds EP01 and DH01 were identified as 5-Tridecyltetrahydro-2H-pyran-2-one and 5-Heptadecyltetrahydro-2H-pyran-2-one, respectively. The compound EP01 significantly reduced (93.24 %) the viral RNA copy number with an IC50 of ~8.661 µM. EP01 proved to be a potent antiviral by in-vitro method against the SARS-CoV-2 clinical isolate, making it a promising antiviral candidate, with a single dose capable of preventing viral replication.

3.
NPJ Aging ; 9(1): 27, 2023 Dec 05.
Article in English | MEDLINE | ID: mdl-38052822

ABSTRACT

Supplementation with S-adenosylhomocysteine (SAH) extends the lifespan of model organisms. To explore the impact of SAH on aging, we generated a Caenorhabditis elegans model by introducing the S-adenosylhomocysteine hydrolase (AHCY-1) variant Y145C, corresponding to the human AHCY Y143C pathogenic mutation. This mutation is anticipated to impair SAH hydrolysis, resulting in its increased levels. Our findings revealed that animals with this endogenous mutation exhibited delayed aging, accompanied by decreased S-adenosylmethionine (SAM) and moderately increased SAH levels. The extended lifespan of these worms depends on the AMP-activated protein kinase (AMPK), its activator Vaccinia virus-related kinase (VRK-1), and the DAF-16 transcription factor. The results underline the complex nature of SAH's influence on aging, proposing that the balance between SAM and SAH might play a pivotal role in defining the lifespan of C. elegans. Moreover, our partial AHCY-1 deficiency model offers a tool for studying the intersection of methionine metabolism and aging.

4.
iScience ; 26(11): 108344, 2023 Nov 17.
Article in English | MEDLINE | ID: mdl-38026164

ABSTRACT

The ubiquitin-proteasome system (UPS) governs the degradation of proteins by ubiquitinating their lysine residues. Our study focuses on lysine deserts - regions in proteins conspicuously low in lysine residues - in averting ubiquitin-dependent proteolysis. We spotlight the prevalence of lysine deserts among bacteria leveraging the pupylation-dependent proteasomal degradation, and in the UPS of eukaryotes. To further scrutinize this phenomenon, we focused on human receptors VHL and SOCS1 to ascertain if lysine deserts could limit their ubiquitination within the cullin-RING ligase (CRL) complex. Our data indicate that the wild-type and lysine-free variants of VHL and SOCS1 maintain consistent turnover rates, unaltered by CRL-mediated ubiquitination, hinting at a protective mechanism facilitated by lysine deserts. Nonetheless, we noted their ubiquitination at non-lysine sites, alluding to alternative regulation by the UPS. Our research underscores the role of lysine deserts in limiting CRL-mediated ubiquitin tagging while promoting non-lysine ubiquitination, thereby advancing our understanding of proteostasis.

5.
EMBO J ; 41(15): e109566, 2022 08 01.
Article in English | MEDLINE | ID: mdl-35762422

ABSTRACT

CHIP (C-terminus of Hsc70-interacting protein) and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin-proteasome system (UPS). CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN-1-UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitylate and regulate S-adenosylhomocysteinase (AHCY-1), a key enzyme in the S-adenosylmethionine (SAM) regeneration cycle, which is essential for SAM-dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.


Subject(s)
Caenorhabditis elegans Proteins , Ubiquitin , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
6.
Bioessays ; 42(1): e1900171, 2020 01.
Article in English | MEDLINE | ID: mdl-31778250

ABSTRACT

The fate of eukaryotic proteins, from their synthesis to destruction, is supervised by the ubiquitin-proteasome system (UPS). The UPS is the primary pathway responsible for selective proteolysis of intracellular proteins, which is guided by covalent attachment of ubiquitin to target proteins by E1 (activating), E2 (conjugating), and E3 (ligating) enzymes in a process known as ubiquitylation. The UPS can also regulate protein synthesis by influencing multiple steps of RNA (ribonucleic acid) metabolism. Here, recent publications concerning the interplay between the UPS and different types of RNA are reviewed. This interplay mainly involves specific RNA-binding E3 ligases that link RNA-dependent processes with protein ubiquitylation. The emerging understanding of their modes of RNA binding, their RNA targets, and their molecular and cellular functions are primarily focused on. It is discussed how the UPS adapted to interact with different types of RNA and how RNA molecules influence the ubiquitin signaling components.


Subject(s)
RNA-Binding Proteins/metabolism , RNA/metabolism , Ubiquitin/metabolism , Animals , Humans , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , RNA Stability , RNA, Long Noncoding/metabolism , RNA, Transfer/metabolism , RNA, Untranslated/metabolism , RNA-Binding Proteins/genetics , Ribosomes/genetics , Ribosomes/metabolism , Signal Transduction , Ubiquitin/genetics , Ubiquitination
7.
Yeast ; 35(8): 499-506, 2018 08.
Article in English | MEDLINE | ID: mdl-29707810

ABSTRACT

Budding yeast Pichia pastoris has highly advanced secretory pathways resembling mammalian systems, an advantage that makes it a suitable model system to study vesicular trafficking. Golgins are large Golgi-resident proteins, primarily reported to play role in cargo vesicle capture, but details of such mechanisms are yet to be deciphered. Golgins that localize to the Golgi via their GRIP domain, a C-terminal Golgi anchoring domain, are known as GRIP domain Golgins. In this present study, we have identified and functionally characterized a homologue of one such GRIP domain Golgin protein, Imh1, from the budding yeast P. pastoris. We have demonstrated that the GRIP domain present at the C-terminal of P. pastoris Imh1 (PpImh1) functions as its Golgi-targeting sequence. Using a combination of yeast two-hybrid analysis, dynamic light scattering and electron microscopy, we have shown that PpImh1 can self-associate and form a homodimer. Analysis of purified recombinant PpImh1 by CD spectroscopy indicates the presence of an 85% α-helical structure, a characteristic of high-content α-helical coiled-coil sequences normally present in other Golgin family proteins. Two-hybrid analysis indicated self-interaction between C-terminal fragments, yet N-terminal fragments do not mediate any such form of self-interaction, suggesting that PpImh1 may form a parallel dimer. Electron microscopy data indicates that PpImh1 forms extended rod-like homo-dimeric molecules with splayed N-terminal end which can act as a tether for capturing vesicles. Our study provides the first evidence in support of the dimeric Y-shaped structure for any Golgin in the budding yeast.


Subject(s)
Fungal Proteins/chemistry , Fungal Proteins/metabolism , Pichia/genetics , Pichia/metabolism , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/metabolism , Amino Acid Sequence , Dimerization , Fungal Proteins/genetics , Golgi Apparatus/genetics , Golgi Apparatus/metabolism , Microscopy, Electron , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Two-Hybrid System Techniques , Vesicular Transport Proteins/genetics
8.
FEBS J ; 285(7): 1251-1276, 2018 04.
Article in English | MEDLINE | ID: mdl-29427328

ABSTRACT

Keratin 8/18, the predominant keratin pair of simple epithelia, is often aberrantly expressed in various squamous cell carcinomas (SCCs) including skin SCC. Its aberrant expression is correlated with increased invasiveness and poor prognosis of the same, although the underlying mechanism is still unclear. A previous report from our laboratory has shown K8-mediated regulation of α6ß4 integrin signaling and thereby tumorigenic potential of oral SCC-derived cells. Another study on transgenic mouse model has shown that during skin carcinogenesis, K8 favors conversion of papillomas toward malignancy. In order to understand the role of K8 and allied mechanism in skin SCC, K8 was stably knocked down in a skin epidermoid carcinoma-derived A431 cells. K8 downregulation significantly reduced the tumorigenic potential of these cells. In agreement with our phenotypic data, differential quantitative proteomics followed by IPA analysis showed altered expression of many proteins associated with biological functions including 'Cancer', 'Cellular movement', 'Cell death and survival', and 'Cellular morphology'. Some of these proteins were TMS1, MARCKSL1, RanBP1, 14-3-3γ, Rho-GDI2, etc. Furthermore, to our surprise, there was a significant reduction in K17 protein stability upon loss of K8, probably due to its caspase-mediated degradation. This was supported by altered TMS1-NF-κB signaling, leading to increased apoptotic sensitivity of A431 cells which in turn affected 'Cell death and survival'. Moreover, MARCKSL1-Paxillin1-Rac axis was found to be deregulated bestowing a possible mechanism behind altered 'Cellular movement' pathway. Altogether our study unravels a much broader regulatory role of K8, governing multiple signaling pathways and consequently regulating oncogenic potential of skin SCC-derived cells. DATABASE: Proteome Xchange Consortium via PRIDE database (dataset identifier PXD007206).


Subject(s)
Keratin-18 , Keratin-8 , Signal Transduction/genetics , Carcinogenesis/genetics , Cell Survival/genetics , Gene Expression Regulation/genetics , Gene Knockdown Techniques , Humans , Immunohistochemistry , Keratin-18/genetics , Keratin-18/metabolism , Keratin-8/genetics , Keratin-8/metabolism , Proteomics
9.
Int J Biol Macromol ; 104(Pt A): 1338-1344, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28684355

ABSTRACT

Fanconi anemia (FA), a cancer predisposition syndrome exhibits hallmark feature of radial chromosome formation, and hypersensitivity to DNA crosslinking agents. A set of FA pathway proteins mainly FANCI, FANCD2 and BRCA2 are expressed to repair the covalent crosslink between the dsDNA. However, FA, BRCA pathways play an important role in DNA ICL repair as well as in homologous recombination repair, but the presumptive role of FA-BRCA proteins has not clearly explored particularly in context to function associated protein-protein interactions (PPIs). Here, in-vivo, in-vitro and in-silico studies have been performed for functionally relevant domains of FANCI, FANCD2 and BRCA2. To our conclusion, FANCI ARM repeat interacts with FANCD2 CUE domain and BRCA2 C-terminal region. Interestingly, FANCD2 CUE domain also interacts strongly with BRCA2 C-terminal region. Interactions between BRCA2 CTR and functionally relevant mutations Ser222Ala (cell cycle checkpoint mutant) and Leu231Arg (DNA ICL repair mutant) present in FANCD2 CUE domain have been analysed. To our finding, these mutations abrogate the binding between FANCD2 CUE domain and BRCA2 CTR. Furthermore, (1) different domain of FANCI, FANCD2 and BRCA2 are playing important role in PPIs, (2) mutations cause the impairment in the PPIs which in turn may disrupt the DNA ICL repair mechanism.


Subject(s)
DNA Repair , Fanconi Anemia Complementation Group Proteins/metabolism , Protein Interaction Mapping , Fanconi Anemia Complementation Group Proteins/chemistry , Fanconi Anemia Complementation Group Proteins/genetics , Mutation , Protein Domains , Repetitive Sequences, Amino Acid
10.
Sci Rep ; 7(1): 3849, 2017 06 20.
Article in English | MEDLINE | ID: mdl-28634376

ABSTRACT

BRCA1 associated ring domain protein 1(BARD1) is a tumor suppressor protein having a wide role in cellular processes like cell-cycle checkpoint, DNA damage repair and maintenance of genomic integrity. Germ-line mutation Gln 564 His discovered in linker region of BARD1 leads to loss of binding to Cleavage stimulating factor (CstF50), which in turn instigates the premature mRNA transcript formation and apoptosis. We have studied the dynamics of ARD domain present in the BARD1 wild-type and mutant protein in association with CstF50 using biophysical, biochemical and molecular dynamics simulations. It has been observed that the ARD domain is relatively more flexible than the BRCT domain of BARD1. Further relative orientations of both the ARD and BRCT domains varies due to the highly flexible nature of the connecting linker region present between the domains. It has been observed that mutant ARD domain is more dynamic in nature compared to wild-type protein. Molecular docking studies between BARD1 Gln 564 His mutant and CstF50 shows the loss of interactions. Furthermore, domain motion of ARD present in BARD1 was stabilized when complexed with CstF50.


Subject(s)
Cleavage Stimulation Factor/metabolism , Protein Domains , Tumor Suppressor Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein Multimerization , Protein Stability , Structure-Activity Relationship , Thermodynamics , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism
11.
Proteomics ; 17(7)2017 04.
Article in English | MEDLINE | ID: mdl-28176443

ABSTRACT

Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine73 (head-domain) and Serine431 (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed tandem mass tag-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead, and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative, and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1T14,Y15 , EIF4EBP1T46,T50 , EIF4BS422 , AKT1S1T246,S247 , CTTN1T401,S405,Y421 , and CAP1S307/309 in K8-S73A/D mutant and CTTN1T401,S405,Y421 , BUB1BS1043 , and CARHSP1S30,S32 in K8-S431A/D mutants as well as some anonymous phosphosites including MYCS176 , ZYXS344 , and PNNS692 could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site-specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies.


Subject(s)
Epithelial Cells/metabolism , Gene Expression Regulation, Neoplastic , Keratin-8/genetics , Phosphoproteins/genetics , Proteomics/methods , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , CDC2 Protein Kinase , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cortactin/genetics , Cortactin/metabolism , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Epithelial Cells/pathology , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Humans , Keratin-8/metabolism , Mutation , Phosphoproteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Skin/metabolism , Skin/pathology , Transcription Factors/genetics , Transcription Factors/metabolism
12.
J Biomol Struct Dyn ; 35(14): 3032-3042, 2017 Nov.
Article in English | MEDLINE | ID: mdl-27686023

ABSTRACT

Fanconi anemia complementation groups - I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein-protein and protein-DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.


Subject(s)
Fanconi Anemia Complementation Group Proteins/chemistry , Repetitive Sequences, Amino Acid , Circular Dichroism , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/isolation & purification , Mass Spectrometry , Models, Molecular , Protein Conformation , Proteolysis , Recombinant Proteins/chemistry , Spectrometry, Fluorescence , Structure-Activity Relationship
13.
Cell Biochem Biophys ; 74(3): 317-25, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27324042

ABSTRACT

Ribosomal S6 kinases (RSKs) are the major functional components in mitogen-activated protein kinase (MAPK) pathway, and these are activated by upstream Extracellular signal-regulated kinase. Upon activation, RSKs activate a number of substrate molecules involved in transcription, translation and cell-cycle regulation. But how cellular binding partners are engaged in the MAPK pathways and regulate the molecular mechanisms have not been explored. Considering the importance of protein-protein interactions in cell signalling and folding pattern of native protein, functional C-terminal kinase domain of RSK3 has been characterized using in vitro, in silico and biophysical approaches. RSKs discharge different functions by binding to downstream kinase partners. Hence, depending upon cellular binding partners, RSKs translocate between cytoplasm and nucleus. In our study, it has been observed that the refolded C-terminal Kinase domain (CTKD) of RSK 3 has a compact domain structure which is predominantly α-helical in nature by burying the tryptophans deep into the core, which was confirmed by CD, Fluorescence spectroscopy and limited proteolysis assay. Our study also revealed that RSK 3 CTKD was found to be a homotrimer from DLS experiments. A model was also built for RSK 3 CTKD and was further validated using PROCHECK and ProSA webservers.


Subject(s)
Ribosomal Protein S6 Kinases, 90-kDa/chemistry , Circular Dichroism , Cloning, Molecular , Dynamic Light Scattering , Extracellular Signal-Regulated MAP Kinases/chemistry , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Molecular Docking Simulation , Protein Folding , Protein Interaction Domains and Motifs , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Signal Transduction , Spectrometry, Fluorescence
14.
J Biomol Struct Dyn ; 34(7): 1533-44, 2016 Jul.
Article in English | MEDLINE | ID: mdl-26307947

ABSTRACT

BARD1-BRCA1 complex plays an important role in DNA damage repair, apoptosis, chromatin remodeling, and other important processes required for cell survival. BRCA1 and BARD1 heterodimer possess E3 ligase activity and is involved in genome maintenance, by functioning in surveillance for DNA damage, thereby regulating multiple pathways including tumor suppression. BRCT domains are evolutionary conserved domains present in different proteins such as BRCA1, BARD1, XRCC, and MDC1 regulating damage response and cell-cycle control through protein-protein interactions. Nonetheless, the role of BARD1BRCT in the recruitment of DNA repair mechanism and structural integrity with BRCA1 complex is still implicit. To explicate the role of BARD1BRCT in the DNA repair mechanism, in silico, in vitro, and biophysical approach were applied to characterize BARD1 BRCT wild-type and Arg658Cys and Ile738Val mutants. However, no drastic secondary and tertiary structural changes in the mutant proteins were observed. Thermal and chemical denaturation studies revealed that mutants Arg658Cys and Ile738Val have a decrease in Tm and ∆G than the wild type. In silico studies of BARD1 BRCT (568-777) and mutant protein indicate loss in structural compactness on the Ile738Val mutant. Comparative studies of wild-type and mutants will thus be helpful in understanding the basic role of BARD1BRCT in DNA damage repair.


Subject(s)
Codon , Models, Molecular , Mutant Proteins , Tumor Suppressor Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , Amino Acid Substitution , Humans , Protein Conformation , Protein Denaturation , Protein Folding , Protein Stability , Spectrum Analysis , Structure-Activity Relationship , Thermodynamics , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism
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