Squalene epoxidase (SQE) is a crucial regulatory enzyme for the biosynthesis of several important classes of compounds including sterols and triterpenoids. The present paper identified and ...characterised five SQE genes (
GgSQE1
to
GgSQE5
) from
Glycyrrhiza glabra
through transcriptome data mining and homology-based cloning, for the first time
.
The phylogenetic analysis implied their functional divergence
.
The ORF corresponding to one of the five SQEs, namely,
GgSQE1
, was cloned and studied for its function in a heterologous system, following transient and stable expressions. The transient expression followed by Gg
SQE1
encoding protein purification suggested approximately 58.0-kDa protein following the predicted molecular mass of the deduced protein. The gene expression profiling based on qRT-PCR indicated its highest expression (6.4-folds) in the 10-month-old roots. Furthermore, ABA (12.4-folds) and GA
3
(2.47) treatments upregulated the expression of
GgSQE1
in the shoots after 10 and 12 hours, respectively, which was also reflected in glycyrrhizin accumulation. The inductive effects of ABA and GA
3
over
GgSQE1
expression were also confirmed through functional analysis of
GgSQE1
promoters using GUS fusion construct. Stable constitutive expression of
GgSQE1
in
Nicotiana tabacum
modulated the sterol contents. The study could pave the way for understanding the metabolic flux regulation concerning biosynthesis of related sterols and triterpenoids.
The Wnt/β‐catenin pathway is implicated in the pathogenesis of hepatocellular cancer (HCC). We developed a transgenic mouse (TG) in the FVB strain that overexpresses Ser45‐mutated‐β‐catenin in ...hepatocytes to study the effects on liver regeneration and cancer. In the two independent TG lines adult mice show elevated β‐catenin at hepatocyte membrane with no increase in the Wnt pathway targets cyclin‐D1 or glutamine synthetase. However, TG hepatocytes upon culture exhibit a 2‐fold increase in thymidine incorporation at day 5 (D5) when compared to hepatocytes from wildtype FVB mice (WT). When subjected to partial hepatectomy (PH), dramatic increases in the number of hepatocytes in S‐phase are evident in TG at 40 and WT at 72 hours. Coincident with the earlier onset of proliferation, we observed nuclear translocation of β‐catenin along with an increase in total and nuclear cyclin‐D1 protein at 40 hours in TG livers. To test if stimulation of β‐catenin induces regeneration, we used hydrodynamic delivery of Wnt‐1 naked DNA to control mice, which prompted an increase in Wnt‐1, β‐catenin, and known targets, glutamine synthetase (GS) and cyclin‐D1, along with a concomitant increase in cell proliferation. β‐Catenin‐overexpressing TG mice, when followed up to 12 months, showed no signs of spontaneous tumorigenesis. However, intraperitoneal delivery of diethylnitrosamine (DEN), a known carcinogen, induced HCC at 6 months in TG mice only. Tumors in TG livers showed up‐regulation of β‐catenin, cyclin‐D1, and unique genetic aberrations, whereas other canonical targets were unremarkable. Conclusion: β‐Catenin overexpression offers growth advantage during liver regeneration. Also, whereas no spontaneous HCC is evident, β‐catenin overexpression makes TG mice susceptible to DEN‐induced HCC. HEPATOLOGY 2010
Alzheimer’s disease (AD) is a neurodegenerative disorder that causes progressive loss of cognitive functions like thinking, memory, reasoning, behavioral abilities, and social skills thus affecting ...the ability of a person to perform normal daily functions independently. There is no definitive cure for this disease, and treatment options available for the management of the disease are not very effective as well. Based on histopathology, AD is characterized by the accumulation of insoluble deposits of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs). Although several molecular events contribute to the formation of these insoluble deposits, the aberrant post-translational modifications (PTMs) of AD-related proteins (like APP, Aβ, tau, and BACE1) are also known to be involved in the onset and progression of this disease. However, early diagnosis of the disease as well as the development of effective therapeutic approaches is impeded by lack of proper clinical biomarkers. In this review, we summarized the current status and clinical relevance of biomarkers from cerebrospinal fluid (CSF), blood and extracellular vesicles involved in onset and progression of AD. Moreover, we highlight the effects of several PTMs on the AD-related proteins, and provide an insight how these modifications impact the structure and function of proteins leading to AD pathology. Finally, for disease-modifying therapeutics, novel approaches, and targets are discussed for the successful treatment and management of AD.
Serratiopeptidase is a proteolytic enzyme extensively used as an anti-inflammatory and analgesic drug. Present work reports a thermoactive serratiopeptidase from
Serratia marcescens
AD-W2, a soil ...isolate from the North-Western Himalayan region of India. The extracellular metalloprotease has been purified by a simple two-step procedure resulting in a specific activity of 20,492 Units/mg protein with 5.28-fold purification. The molecular mass of the metalloprotease, as determined by SDS-PAGE was ~ 51 kDa. The purified serratiopeptidase presented optimum activity at pH 9.0, temperature 50 °C and stability in wide pH and temperature range. Critical temperature of 50 °C confirmed the thermoactivity of the purified serratiopeptidase. The kinetic studies of the purified serratiopeptidase revealed V
max
and K
m
of 57,256 Units/mL and 1.57 mg/mL, respectively, for casein. The purified serratiopeptidase from
S. marcescens
AD-W2 was found to be 100% identical to serralysin from
Serratia marcescens
ATCC 21074/E-15. The catalytic domain comprising of Zn coordinated with three histidine residues (His192, His196, His202), along with glutamate (Glu193) and tyrosine (Tyr232) residues, further confirmed that the purified protein is identical to serralysin.
JAK-STAT signalling pathway inhibitors have emerged as promising therapeutic agents for the treatment of hair loss. Among different JAK isoforms, JAK3 has become an ideal target for drug discovery ...because it only regulates a narrow spectrum of γc cytokines. Here, we report the discovery of MJ04, a novel and highly selective 3-pyrimidinylazaindole based JAK3 inhibitor, as a potential hair growth promoter with an IC
of 2.03 nM. During in vivo efficacy assays, topical application of MJ04 on DHT-challenged AGA and athymic nude mice resulted in early onset of hair regrowth. Furthermore, MJ04 significantly promoted the growth of human hair follicles under ex-vivo conditions. MJ04 exhibited a reasonably good pharmacokinetic profile and demonstrated a favourable safety profile under in vivo and in vitro conditions. Taken together, we report MJ04 as a highly potent and selective JAK3 inhibitor that exhibits overall properties suitable for topical drug development and advancement to human clinical trials.
The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular ...proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.
Lens epithelium-derived growth factor (LEDGF) is an important co-factor of human immunodeficiency virus DNA integration; however, its cellular functions are poorly characterized. We now report ...identification of the Cdc7-activator of S-phase kinase (ASK) heterodimer as a novel interactor of LEDGF. Both kinase subunits co-immunoprecipitated with endogenous LEDGF from human cell extracts. Truncation analyses identified the integrase-binding domain of LEDGF as essential and minimally sufficient for the interaction with Cdc7-ASK. Reciprocally, the interaction required autophosphorylation of the kinase and the presence of 50 C-terminal residues of ASK. The kinase phosphorylated LEDGF in vitro, with Ser-206 being the major target, and LEDGF phosphorylated at this residue could be detected during S phase of the cell cycle. LEDGF potently stimulated the enzymatic activity of Cdc7-ASK, increasing phosphorylation of MCM2 in vitro by more than 10-fold. This enzymatic stimulation as well as phosphorylation of LEDGF depended on the protein-protein interaction. Intriguingly, removing the C-terminal region of ASK, involved in the interaction with LEDGF, resulted in a hyperactive kinase. Our results indicate that the interaction with LEDGF relieves autoinhibition of Cdc7-ASK kinase, imposed by the C terminus of ASK.
Purpose
Phosphatidylinositol-3 kinases (PI3Ks) are involved in regulating cell growth, proliferation, differentiation, apoptosis and survival. p110α and p110β, two ubiquitously expressed isoforms of ...PI3K signalling, are involved in growth factor mediated signaling and survival by generating second messengers. Earlier, we have generated GFP-fusion proteins of p110α and p110β and expressed them in normal and cancer cell-lines to investigate their subcellular localization and their role in various activities. Here, we sought to examine the role of p110α and p110β isoforms in protecting MCF-7 breast cancer cells against oxidative stress.
Material methods
We performed cytotoxicity assays, DNA transfection, Plasmid DNA preparation, western blotting, flourscence microscopy and statistical analysis.
Results
To know whether p110α and p110β are involved in protecting MCF-7 breast cancer cells against oxidative stress, we subjected MCF-7 cells to H2O2 treatment and observed a dose dependent decrease in cell viability and a marked increase in the levels of pro-apoptotic markers which include PARP, Bcl-2, Bax and procaspase-9. We then over-expressed recombinant GFP-fusion p110α and p110β proteins in MCF-7 cells and observed a significant decrease in apoptosis and a concomitant increase in pAkt levels.
Conclusion
We report the involvement of p110α and p110β isoforms of Class 1A PI3K signalling in rescue from oxidative stress-induced apoptosis in MCF-7 cells in Akt dependent manner.
Background & Aims Pegylated-Interferon-α2a (peg-IFN), a first line therapy for Hepatitis C virus (HCV) patients, also impacts the recurrence of hepatocellular carcinoma (HCC). The activation of the ...Wnt pathway due to β-catenin gene mutations contributes to the development of a significant subset of HCC. Herein, we explored the effect of peg-IFN on Wnt/β-catenin signaling in vitro and in vivo. Methods Multiple human hepatoma cell lines were treated with Peg-IFN to assess its effect on the Wnt pathway and the mechanisms involved. Transgenic (TG) mice expressing stable β-catenin mutant in the liver were exposed to diethylnitrosamine (DEN) and treated with peg-IFN. Results In vitro , peg-IFN decreased the transcriptional activity of β-catenin/Tcf and did so independently of JAK/Stat signaling. Peg-IFN treatment led to increased mRNA and protein expression of RanBP3, a known β-catenin nuclear export factor, in all hepatoma cells. Co-precipitation studies showed an increased association between RanBP3 and β-catenin after peg-IFN treatment. The siRNA-mediated RanBP3 knockdown abrogated Peg-IFN-induced decrease in TOPFlash reporter activity. In vivo, Peg-IFN treatment led to increased nuclear RanBP3, decreased nuclear β-catenin and cyclin D1, and decreased cytoplasmic glutamine synthetase. Increased association of RanBP3 and β-catenin was also observed in vivo in response to Peg-IFN that led to decreased hepatocyte proliferation. Conclusions Peg-IFN inhibits β-catenin signaling through the up-regulation of RanBP3, which may be a contributory mechanism for the delayed HCC and improved survival in treated HCV patients. This observation might have chemo-preventive or chemo-therapeutic implications in tumor with aberrant Wnt pathway activation.
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