Publications by authors named "Shilpy Sharma"

41 Publications

A robotic pill for oral delivery of biotherapeutics: safety, tolerability, and performance in healthy subjects.

Drug Deliv Transl Res 2021 Feb 19. Epub 2021 Feb 19.

Rani Therapeutics, LLC, 2051 Ringwood Ave, San Jose, CA, 95131, USA.

Biotherapeutics are highly efficacious, but the pain and inconvenience of chronic injections lead to poor patient compliance and compromise effective disease management. Despite innumerable attempts, oral delivery of biotherapeutics remains unsuccessful due to their degradation in the gastrointestinal (GI) environment and poor intestinal absorption. We have developed an orally ingestible robotic pill (RP) for drug delivery, which protects the biotherapeutic drug payload from digestion in the GI tract and auto-injects it into the wall of the small intestine as a safe, pain-free injection since the intestines are insensate to sharp stimuli. The payload is delivered upon inflation of a balloon folded within the RP, which deflates immediately after drug delivery. Here we present results from two clinical studies demonstrating the safety, tolerability and performance of the RP in healthy humans. In the first study, three versions of the RP (A, B and C) were evaluated, which were identical in all respects except for the diameter of the balloon. The RP successfully delivered a biotherapeutic (octreotide) in 3 out of 12 subjects in group A, 10 out of 20 subjects in group B and 16 out of 20 subjects in group C, with a mean bioavailability of 65 ± 9% (based on successful drug deliveries in groups A and B). Thus,  reliability of drug delivery with the RP ranged from 25 to 80%, with success rate directly related to balloon size. In a separate study, the deployment of the RP was unaffected by fed or fasting conditions suggesting that the RP may be taken with or without food. These promising clinical data suggest that biotherapeutics currently administered parenterally may be safely and reliably delivered via this versatile, orally ingestible drug delivery platform.
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http://dx.doi.org/10.1007/s13346-021-00938-1DOI Listing
February 2021

Human islet amyloid polypeptide (hIAPP) - a curse in type II diabetes mellitus: insights from structure and toxicity studies.

Biol Chem 2021 01 4;402(2):133-153. Epub 2020 Sep 4.

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, Maharashtra, India.

The human islet amyloid polypeptide (hIAPP) or amylin, a neuroendocrine peptide hormone, is known to misfold and form amyloidogenic aggregates that have been observed in the pancreas of 90% subjects with Type 2 Diabetes Mellitus (T2DM). Under normal physiological conditions, hIAPP is co-stored and co-secreted with insulin; however, under chronic hyperglycemic conditions associated with T2DM, the overexpression of hIAPP occurs that has been associated with the formation of amyloid deposits; as well as the death and dysfunction of pancreatic β-islets in T2DM. Hitherto, various biophysical and structural studies have shown that during this process of aggregation, the peptide conformation changes from random structure to helix, then to β-sheet, subsequently to cross β-sheets, which finally form left-handed helical aggregates. The intermediates, formed during this process, have been shown to induce higher cytotoxicity in the β-cells by inducing cell membrane disruption, endoplasmic reticulum stress, mitochondrial dysfunction, oxidative stress, islet inflammation, and DNA damage. As a result, several research groups have attempted to target both hIAPP aggregation phenomenon and the destabilization of preformed fibrils as a therapeutic intervention for T2DM management. In this review, we have summarized structural aspects of various forms of hIAPP viz. monomer, oligomers, proto-filaments, and fibrils of hIAPP. Subsequently, cellular toxicity caused by toxic conformations of hIAPP has been elaborated upon. Finally, the need for performing structural and toxicity studies to fill in the gap between the structural and cellular aspects has been discussed.
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http://dx.doi.org/10.1515/hsz-2020-0174DOI Listing
January 2021

Myricetin protects pancreatic β-cells from human islet amyloid polypeptide (hIAPP) induced cytotoxicity and restores islet function.

Biol Chem 2021 01 16;402(2):179-194. Epub 2020 Sep 16.

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, 400076Mumbai, Maharashtra, India.

The aberrant misfolding and self-assembly of human islet amyloid polypeptide (hIAPP)-a hormone that is co-secreted with insulin from pancreatic β-cells-into toxic oligomers, protofibrils and fibrils has been observed in type 2 diabetes mellitus (T2DM). The formation of these insoluble aggregates has been linked with the death and dysfunction of β-cells. Therefore, hIAPP aggregation has been identified as a therapeutic target for T2DM management. Several natural products are now being investigated for their potential to inhibit hIAPP aggregation and/or disaggregate preformed aggregates. In this study, we attempt to identify the anti-amyloidogenic potential of Myricetin (MYR)- a polyphenolic flavanoid, commonly found in fruits (like ). Our results from biophysical studies indicated that MYR supplementation inhibits hIAPP aggregation and disaggregates preformed fibrils into non-toxic species. This protection was accompanied by inhibition of oxidative stress, reduction in lipid peroxidation and the associated membrane damage and restoration of mitochondrial membrane potential in INS-1E cells. MYR supplementation also reversed the loss of functionality in hIAPP exposed pancreatic islets via restoration of glucose-stimulated insulin secretion. Molecular dynamics simulation studies suggested that MYR molecules interact with the hIAPP pentameric fibril model at the amyloidogenic core region and thus prevents aggregation and distort the fibrils.
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http://dx.doi.org/10.1515/hsz-2020-0176DOI Listing
January 2021

Cold storage reveals distinct metabolic perturbations in processing and non-processing cultivars of potato (Solanum tuberosum L.).

Sci Rep 2020 04 14;10(1):6268. Epub 2020 Apr 14.

Department of Chemistry, Indian Institute of Science Education and Research, Pune, 411008, India.

Cold-induced sweetening (CIS) causes considerable losses to the potato processing industry wherein the selection of potato genotypes using biochemical information has found to be advantageous. Here, H NMR spectroscopy was performed to identify metabolic perturbations from tubers of five potato cultivars (Atlantic, Frito Lay-1533, Kufri Jyoti, Kufri Pukhraj, and PU1) differing in their CIS ability and processing characteristics at harvest and after cold storage (4 °C). Thirty-nine water-soluble metabolites were detected wherein significantly affected metabolites after cold storage were categorized into sugars, sugar alcohols, amino acids, and organic acids. Multivariate statistical analysis indicated significant differences in the metabolic profiles among the potato cultivars. Pathway enrichment analysis revealed that carbohydrates, amino acids, and organic acids are the key players in CIS. Interestingly, one of the processing cultivars, FL-1533, exhibited a unique combination of metabolites represented by low levels of glucose, fructose, and asparagine accompanied by high citrate levels. Conversely, non-processing cultivars (Kufri Pukhraj and Kufri Jyoti) showed elevated glucose, fructose, and malate levels. Our results indicate that metabolites such as glucose, fructose, sucrose, asparagine, glutamine, citrate, malate, proline, 4-aminobutyrate can be potentially utilized for the prediction, selection, and development of potato cultivars for long-term storage, nutritional, as well as processing attributes.
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http://dx.doi.org/10.1038/s41598-020-63329-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156394PMC
April 2020

A Glutathione Activatable Ion Channel Induces Apoptosis in Cancer Cells by Depleting Intracellular Glutathione Levels.

Angew Chem Int Ed Engl 2020 05 12;59(20):7944-7952. Epub 2020 Mar 12.

Department of Chemistry, Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road, Pashan, Pune, 411008, Maharashtra, India.

Cancer cells use elevated glutathione (GSH) levels as an inner line of defense to evade apoptosis and develop drug resistance. In this study, we describe a novel 2,4-nitrobenzenesulfonyl (DNS) protected 2-hydroxyisophthalamide system that exploits GSH for its activation into free 2-hydroxyisophthalamide forming supramolecular M /Cl channels. Better permeation of the DNS protected compound into MCF-7 cells compared to the free 2-hydroxyisophthalamide and GSH-activatable ion transport resulted in higher cytotoxicity, which was associated with increased oxidative stress that further reduced the intracellular GSH levels and altered mitochondrial membrane permeability leading to the induction of the intrinsic apoptosis pathway. The GSH-activatable transport-mediated cell death was further validated in rat insulinoma cells (INS-1E); wherein the intracellular GSH levels showed a direct correlation to the resulting cytotoxicity. Lastly, the active compound was found to restrict the growth and proliferation of 3D spheroids of MCF-7 cells with efficiency similar to that of the anticancer drug doxorubicin.
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http://dx.doi.org/10.1002/anie.202000961DOI Listing
May 2020

Metabolic signatures suggest o-phosphocholine to UDP-N-acetylglucosamine ratio as a potential biomarker for high-glucose and/or palmitate exposure in pancreatic β-cells.

Metabolomics 2019 03 29;15(4):55. Epub 2019 Mar 29.

Department of Chemistry, Indian Institute of Science Education and Research (IISER) Pune, Dr. Homi Bhabha Road, Pune, Maharashtra, 411008, India.

Introduction: Chronic exposure to high-glucose and free fatty acids (FFA) alone/or in combination; and the resulting gluco-, lipo- and glucolipo-toxic conditions, respectively, have been known to induce dysfunction and apoptosis of β-cells in Diabetes. The molecular mechanisms and the development of biomarkers that can be used to predict similarities and differences behind these conditions would help in easier and earlier diagnosis of Diabetes.

Objectives: This study aims to use metabolomics to gain insight into the mechanisms by which β-cells respond to excess-nutrient stress and identify associated biomarkers.

Methods: INS-1E cells were cultured in high-glucose, palmitate alone/or in combination for 24 h to mimic gluco-, lipo- and glucolipo-toxic conditions, respectively. Biochemical and cellular experiments were performed to confirm the establishment of these conditions. To gain molecular insights, abundant metabolites were identified and quantified using H-NMR.

Results: No loss of cellular viability was observed in high-glucose while exposure to FFA alone/in combination with high-glucose was associated with increased ROS levels, membrane damage, lipid accumulation, and DNA double-strand breaks. Forty-nine abundant metabolites were identified and quantified using H-NMR. Chemometric pair-wise analysis in glucotoxic and lipotoxic conditions, when compared with glucolipotoxic conditions, revealed partial overlap in the dysregulated metabolites; however, the dysregulation was more significant under glucolipotoxic conditions.

Conclusion: The current study compared gluco-, lipo- and glucolipotoxic conditions in parallel and elucidated differences in metabolic pathways that play major roles in Diabetes. o-phosphocholine and UDP-N-acetylglucosamine were identified as common dysregulated metabolites and their ratio was proposed as a potential biomarker for these conditions.
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http://dx.doi.org/10.1007/s11306-019-1516-3DOI Listing
March 2019

Azadirachtin inhibits amyloid formation, disaggregates pre-formed fibrils and protects pancreatic β-cells from human islet amyloid polypeptide/amylin-induced cytotoxicity.

Biochem J 2019 03 15;476(5):889-907. Epub 2019 Mar 15.

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, Maharashtra 400076, India

The human islet amyloid polypeptide (hIAPP) or amylin is the major constituent of amyloidogenic aggregates found in pancreatic islets of type 2 diabetic patients that have been associated with β-cell dysfunction and/or death associated with type 2 diabetes mellitus (T2DM). Therefore, developing and/or identifying inhibitors of hIAPP aggregation pathway and/or compound that can mediate disaggregation of preformed aggregates holds promise as a medical intervention for T2DM management. In the current study, the anti-amyloidogenic potential of Azadirachtin (AZD)-a secondary metabolite isolated from traditional medicinal plant Neem ()-was investigated by using a combination of biophysical and cellular assays. Our results indicate that AZD supplementation not only inhibits hIAPP aggregation but also disaggregates pre-existing hIAPP fibrils by forming amorphous aggregates that are non-toxic to pancreatic β-cells. Furthermore, AZD supplementation in pancreatic β-cells () resulted in inhibition of oxidative stress; along with restoration of the DNA damage, lipid peroxidation and the associated membrane damage, endoplasmic reticulum stress and mitochondrial membrane potential. AZD treatment also restored glucose-stimulated insulin secretion from pancreatic islets exposed to hIAPP. All-atom molecular dynamics simulation studies on full-length hIAPP pentamer with AZD suggested that AZD interacted with four possible binding sites in the amyloidogenic region of hIAPP. In summary, our results suggest AZD to be a promising candidate for combating T2DM and related amyloidogenic disorders.
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http://dx.doi.org/10.1042/BCJ20180820DOI Listing
March 2019

H, C and N resonance assignment of domain 1 of trans-activation response element (TAR) RNA binding protein isoform 1 (TRBP2) and its comparison with that of isoform 2 (TRBP1).

Biomol NMR Assign 2018 04 14;12(1):189-194. Epub 2018 Feb 14.

Department of Chemistry, Indian Institute of Science Education & Research, Dr Homi Bhabha Road, Pashan, Pune, 411008, India.

TAR RNA binding protein (TRBP) is a double-stranded RNA binding protein involved in various biological processes like cell growth, development, death, etc. The protein exists as two isoforms TRBP2 and TRBP1. TRBP2 contains additional 21 amino acids at its N-terminus, which are proposed to be involved in its membrane localization, when compared to TRBP1. The resonance assignment (19-228) of the double-stranded RNA binding domains (dsRBD 1 and 2) of TRBP2 has been reported earlier. Here, we report H, C and N resonance assignment for dsRBD1 of TRBP2 (1-105) containing the additional N-terminal residues. This assignment will provide deeper insights to understand the effect of these residues on the structure and dynamics of TRBP2 and would therefore help in further elucidating the differences in the role of these isoforms.
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http://dx.doi.org/10.1007/s12104-018-9807-6DOI Listing
April 2018

Recombinant human islet amyloid polypeptide forms shorter fibrils and mediates β-cell apoptosis via generation of oxidative stress.

Biochem J 2017 11 16;474(23):3915-3934. Epub 2017 Nov 16.

Department of Biosciences and Bioengineering, Indian Institute of Technology, Mumbai 400076, India

Protein misfolding and aggregation play an important role in many human diseases including Alzheimer's, Parkinson's and type 2 diabetes mellitus (T2DM). The human islet amyloid polypeptide (hIAPP) forms amyloid plaques in the pancreas of T2DM subjects (>95%) that are involved in deteriorating islet function and in mediating βcell apoptosis. However, the detailed mechanism of action, structure and nature of toxic hIAPP species responsible for this effect remains elusive to date mainly due to the high cost associated with the chemical synthesis of pure peptide required for these studies. In the present work, we attempted to obtain structural and mechanistic insights into the hIAPP aggregation process using recombinant hIAPP (rhIAPP) isolated from Results from biophysical and structural studies indicate that the rhIAPP self-assembled into highly pure, β-sheet-rich amyloid fibrils with uniform morphology. rhIAPP-mediated apoptosis in cells was associated with increased oxidative stress and changes in mitochondrial membrane potential. The transcript levels of apoptotic genes - and were found to be up-regulated, while the levels of the anti-apoptotic gene - were down-regulated in rhIAPP-treated cells. Additionally, the expression levels of genes involved in combating oxidative stress namely , and were down-regulated. rhIAPP exposure also affected glucose-stimulated insulin secretion from isolated pancreatic islets. The aggregation of rhIAPP also occurred significantly faster when compared with that of the chemically synthesized peptide. We also show that the rhIAPP fibrils were shorter and more cytotoxic. In summary, our study is one among the few to provide comprehensive evaluation of structural, biophysical and cytotoxic properties of rhIAPP.
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http://dx.doi.org/10.1042/BCJ20170323DOI Listing
November 2017

miRNAs: Nanomachines That Micromanage the Pathophysiology of Diabetes Mellitus.

Adv Clin Chem 2017;82:199-264. Epub 2017 Aug 8.

Indian Institute of Science Education and Research (IISER), Pune, Maharashtra, India.

Diabetes mellitus (DM) refers to a combination of heterogeneous complex metabolic disorders that are associated with episodes of hyperglycemia and glucose intolerance occurring as a result of defects in insulin secretion, action, or both. The prevalence of DM is increasing at an alarming rate, and there exists a need to develop better therapeutics and prognostic markers for earlier detection and diagnosis. In this review, after giving a brief introduction of diabetes mellitus and microRNA (miRNA) biogenesis pathway, we first describe various in vitro and animal model systems that have been developed to study diabetes. Further, we elaborate on the significant roles played by miRNAs as regulators of gene expression in the context of development of diabetes and its secondary complications. The different approaches to quantify miRNAs and their potential to be used as therapeutic targets for alleviation of diabetes have also been discussed.
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http://dx.doi.org/10.1016/bs.acc.2017.06.003DOI Listing
April 2019

miRNAs: early prognostic biomarkers for Type 2 diabetes mellitus?

Biomark Med 2015 6;9(10):1025-40. Epub 2015 Oct 6.

Department of Biotechnology, Savitribai Phule Pune University (Formerly University of Pune), Ganeshkhind, Pune 411007, India.

Type 2 diabetes mellitus (T2DM) has reached epidemic proportions and is associated with peripheral insulin resistance. The currently used therapies aim to delay progression of T2DM. Their efficacy could drastically be improved if implemented at earlier stages. Classical diagnostic markers (blood glucose and HbA1C) are generally detected once metabolic imbalance has already set in. Therefore, development of biomarkers for early diagnosis would help identify individuals at risk for developing T2DM. Along with genetic predisposition, epigenetics also plays a major role in T2DM development. In this review, we discuss the potential role of early diagnostic markers such as circulating miRNAs, studies done so far and challenges to be considered while taking into account the novel role of miRNAs as prognostic biomarkers.
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http://dx.doi.org/10.2217/bmm.15.69DOI Listing
August 2016

Effect of prednisone on nasal symptoms and peripheral blood T-cell function in chronic rhinosinusitis.

Int Forum Allergy Rhinol 2014 Aug 21;4(8):609-16. Epub 2014 Apr 21.

Department of Surgery, Section of Otolaryngology-Head and Neck Surgery, The University of Chicago, Chicago, IL, USA; Department of Otorhinolaryngology, Showa University School of Medicine, Tokyo, Japan.

Background: Systemic corticosteroids are the most effective anti-inflammatory drugs used for controlling chronic rhinosinusitis (CRS) symptoms. The potential mechanisms for their beneficial effects include increasing the number and function of T regulatory cells (Tregs), as reported in the local tissue post-intranasal steroid treatment. We investigated the effect of systemic corticosteroids on peripheral blood (PB) Tregs in subjects with CRS.

Methods: Twenty CRS subjects and 19 controls were recruited. PB mononuclear cells (PBMCs) were isolated from CRS subjects before and after systemic corticosteroid administration in the course of clinical treatment. Control subjects received no treatment and were studied at one visit. Nasal symptoms were recorded. CD4(+) CD25(+) Foxp3(+) cells (Tregs) were analyzed by flow cytometry. Messenger RNA (mRNA) levels for interferon γ (IFN-γ), interleukin 4 (IL-4), IL-10, IL-13, IL-17A, transforming growth factor β1 (TGF-β1), forkhead box P3 (FoxP3), and GATA-binding factor 3 (GATA-3) were measured in PBMCs using real-time polymerase chain reaction (PCR).

Results: CRS subjects reported improved nasal symptoms (p = 0.005) and significantly reduced PB Tregs after treatment with corticosteroids (p = 0.042). The transcript levels of IL-4 and GATA-3 were significantly higher in the CRS subjects at their first visit when compared to controls (p = 0.019 and p = 0.05, respectively). Corticosteroid treatment lowered the transcript levels of immunoregulatory transcription factors [FoxP3 (p = 0.048) and GATA-3 (p = 0.012)] and IFN-γ (p = 0.036) in PB.

Conclusion: In contrast to prior work in local nasal tissue, our study reports reduced PB Tregs and decreased T helper 1 (T(H)1) and T(H)2 function after treatment with systemic corticosteroids. These data indicate that corticosteroid effects on Tregs in CRS are complex involving local signals in the tissue that are distinct from those in circulating cells.
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http://dx.doi.org/10.1002/alr.21336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5999408PMC
August 2014

BRCA1 promotes the ubiquitination of PCNA and recruitment of translesion polymerases in response to replication blockade.

Proc Natl Acad Sci U S A 2013 Aug 30;110(33):13558-63. Epub 2013 Jul 30.

Basic Biomedical Science Division, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069, USA.

Breast cancer gene 1 (BRCA1) deficient cells not only are hypersensitive to double-strand breaks but also are hypersensitive to UV irradiation and other agents that cause replication blockade; however, the molecular mechanisms behind these latter sensitivities are largely unknown. Here, we report that BRCA1 promotes cell survival by directly regulating the DNA damage tolerance pathway in response to agents that create cross-links in DNA. We show that BRCA1 not only promotes efficient mono- and polyubiquitination of proliferating cell nuclear antigen (PCNA) by regulating the recruitment of replication protein A, Rad18, and helicase-like transcription factor to chromatin but also directly recruits translesion polymerases, such as Polymerase eta and Rev1, to the lesions through protein-protein interactions. Our data suggest that BRCA1 plays a critical role in promoting translesion DNA synthesis as well as DNA template switching.
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http://dx.doi.org/10.1073/pnas.1306534110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746927PMC
August 2013

The roles of DNA polymerase ζ and the Y family DNA polymerases in promoting or preventing genome instability.

Mutat Res 2013 Mar-Apr;743-744:97-110. Epub 2012 Nov 26.

Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, United States. Electronic address:

Cancer cells display numerous abnormal characteristics which are initiated and maintained by elevated mutation rates and genome instability. Chromosomal DNA is continuously surveyed for the presence of damage or blocked replication forks by the DNA Damage Response (DDR) network. The DDR is complex and includes activation of cell cycle checkpoints, DNA repair, gene transcription, and induction of apoptosis. Duplicating a damaged genome is associated with elevated risks to fork collapse and genome instability. Therefore, the DNA damage tolerance (DDT) pathway is also employed to enhance survival and involves the recruitment of translesion DNA synthesis (TLS) polymerases to sites of replication fork blockade or single stranded DNA gaps left after the completion of replication in order to restore DNA to its double stranded form before mitosis. TLS polymerases are specialized for inserting nucleotides opposite DNA adducts, abasic sites, or DNA crosslinks. By definition, the DDT pathway is not involved in the actual repair of damaged DNA, but provides a mechanism to tolerate DNA lesions during replication thereby increasing survival and lessening the chance for genome instability. However this may be associated with increased mutagenesis. In this review, we will describe the specialized functions of Y family polymerases (Rev1, Polη, Polι and Polκ) and DNA polymerase ζ in lesion bypass, mutagenesis, and prevention of genome instability, the latter due to newly appreciated roles in DNA repair. The recently described role of the Fanconi anemia pathway in regulating Rev1 and Polζ-dependent TLS is also discussed in terms of their involvement in TLS, interstrand crosslink repair, and homologous recombination.
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http://dx.doi.org/10.1016/j.mrfmmm.2012.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616148PMC
September 2013

Peripheral blood and tissue T regulatory cells in chronic rhinosinusitis.

Am J Rhinol Allergy 2012 Sep-Oct;26(5):371-9

Section of Otolaryngology-Head and Neck Surgery, Department of Surgery, The University of Chicago, Chicago, Illinois, USA.

Background: The pathogenesis of chronic rhinosinusitis (CRS) has not been fully elucidated. Increased inflammatory cell infiltration and decreased numbers and/or impaired function of T regulatory cells (Tregs) have been reported. This study aimed to determine the role of Tregs in CRS in peripheral blood (PB) and sinus tissue.

Methods: Sinus tissue was obtained from 16 CRS subjects and 5 controls. PB from additional 16 CRS subjects and total 20 controls was obtained. Immunohistochemical analysis (CD3(+), CD4(+), CD8(+), and Treg [CD4(+)-FoxP3(+) and CD25(+)-FoxP3(+)] cells) of sinus tissue was performed. Percentage of PB Tregs (CD4(+)-CD25(+)-FoxP3(+) cells) was analyzed by flow cytometry. Spontaneous and phytohemagglutinin (PHA)-induced release of cytokines (IL-6, IL-4, IL-10, interferon gamma, transforming growth factor [TGF] beta1, and TNF-alpha) from PB mononuclear cells (PBMCs) was determined.

Results: PB flow cytometric analysis revealed a lower percentage of Tregs in subjects with CRS compared with healthy controls (p = 0.0003). Although no differences in the PB Treg counts were observed between the CRS subjects with nasal polyposis (CRSwNP) and without nasal polyposis (CRSsNP), immunohistochemical analysis performed on sinus tissue revealed a higher proportion of Tregs in CRSwNP subjects compared with CRSsNP (p < 0.05). Additionally, we failed to detect any Tregs from control sphenoid sinus tissue. Lower levels of regulatory cytokines (IL-10 and TGF-β1) and higher levels of proinflammatory cytokines (TNF-α and IL-6) were found from PBMCs from CRS subjects compared with controls (p < 0.05).

Conclusion: Our findings suggest that CRS subjects exhibit a decreased percentage of PB Tregs compared with normal controls. PBMCs from CRS subjects show a more proinflammatory and less regulatory phenotype.
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http://dx.doi.org/10.2500/ajra.2012.26.3800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972732PMC
May 2013

REV1 and DNA polymerase zeta in DNA interstrand crosslink repair.

Environ Mol Mutagen 2012 Dec 13;53(9):725-40. Epub 2012 Oct 13.

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109-2200, USA.

DNA interstrand crosslinks (ICLs) are covalent linkages between two strands of DNA, and their presence interferes with essential metabolic processes such as transcription and replication. These lesions are extremely toxic, and their repair is essential for genome stability and cell survival. In this review, we will discuss how the removal of ICLs requires interplay between multiple genome maintenance pathways and can occur in the absence of replication (replication-independent ICL repair) or during S phase (replication-coupled ICL repair), the latter being the predominant pathway used in mammalian cells. It is now well recognized that translesion DNA synthesis (TLS), especially through the activities of REV1 and DNA polymerase zeta (Polζ), is necessary for both ICL repair pathways operating throughout the cell cycle. Recent studies suggest that the convergence of two replication forks upon an ICL initiates a cascade of events including unhooking of the lesion through the actions of structure-specific endonucleases, thereby creating a DNA double-stranded break (DSB). TLS across the unhooked lesion is necessary for restoring the sister chromatid before homologous recombination repair. Biochemical and genetic studies implicate REV1 and Polζ as being essential for performing lesion bypass across the unhooked crosslink, and this step appears to be important for subsequent events to repair the intermediate DSB. The potential role of Fanconi anemia pathway in the regulation of REV1 and Polζ-dependent TLS and the involvement of additional polymerases, including DNA polymerases kappa, nu, and theta, in the repair of ICLs is also discussed in this review.
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http://dx.doi.org/10.1002/em.21736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543726PMC
December 2012

Association of the -1072G/A polymorphism in the LTC4S gene with asthma in an Indian population.

Int Arch Allergy Immunol 2012 21;159(3):271-7. Epub 2012 Jun 21.

Molecular Immunogenetics Laboratory and Center of Excellence for Translational Research in Asthma and Lung Disease, CSIR-Institute of Genomics and Integrative Biology, Delhi, India.

Background: Atopic asthma, the most common chronic disease affecting children and young adults, is a complex disorder with variable phenotypes. Cysteine leukotrienes (Cys-LTs) are powerful bronchoconstrictors and play a critical role in airway inflammation and remodeling that are characteristic of asthma.

Objective: To investigate the association of ALOX5, LTC4S and CysLTR2 gene polymorphisms with atopic asthma in an Indian population.

Methods: A total of 19 single nucleotide polymorphisms (SNPs) within these genes were genotyped in a family-based cohort (n = 239) and a case-control cohort (139 cases and 194 controls) followed by association analyses.

Results: We found a significant association of the -1072G/A (rs3776944) SNP with atopic asthma in the family-based association analysis (p = 0.0004). These results were also replicated in the case-control cohort (p = 0.009). The allele A was negatively associated with atopic asthma. We also noted a significant association in the two-locus (rs3776944G/A and rs730012A/C) haplotypic analysis of this gene both in the family-based (p = 0.03) and the case-control (p = 0.02) analyses.

Conclusion: This study supports the role of the LTC4S gene polymorphism in genetic susceptibility to atopic asthma in an Indian population.
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http://dx.doi.org/10.1159/000336675DOI Listing
December 2012

DNA polymerase ζ is a major determinant of resistance to platinum-based chemotherapeutic agents.

Mol Pharmacol 2012 Jun 2;81(6):778-87. Epub 2012 Mar 2.

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Oxaliplatin, satraplatin, and picoplatin are cisplatin analogs that interact with DNA forming intrastrand and interstrand DNA cross-links (ICLs). Replicative bypass of cisplatin DNA adducts requires the cooperative actions of at least three translesion DNA synthesis (TLS) polymerases: Polη, REV1, and Polζ. Because oxaliplatin, satraplatin, and picoplatin contain bulkier chemical groups attached to the platinum core compared with cisplatin, we hypothesized that these chemical additions may impede replicative bypass by TLS polymerases and reduce tolerance to platinum-containing adducts. We examined multiple responses of cancer cells to oxaliplatin, satraplatin, or picoplatin treatment under conditions where expression of a TLS polymerase was limited. Our studies revealed that, although Polη contributes to the tolerance of cisplatin adducts, it plays a lesser role in promoting replication through oxaliplatin, satraplatin, and picoplatin adducts. REV1 and Polζ were necessary for tolerance to all four platinum analogs and prevention of hyperactivation of the DNA damage response after treatment. In addition, REV1 and Polζ were important for the resolution of DNA double-stranded breaks created during replication-associated repair of platinum-containing ICLs. Consistent with ICLs being the predominant cytotoxic lesion, depletion of REV1 or Polζ rendered two different model cell systems extremely sensitive to all four drugs, whereas Polη depletion had little effect. Together, our data suggest that REV1 and Polζ are critical for promoting resistance to all four clinically relevant platinum-based drugs by promoting both translesion DNA synthesis and DNA repair.
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http://dx.doi.org/10.1124/mol.111.076828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362893PMC
June 2012

REV1 and polymerase ζ facilitate homologous recombination repair.

Nucleic Acids Res 2012 Jan 16;40(2):682-91. Epub 2011 Sep 16.

Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

REV1 and DNA Polymerase ζ (REV3 and REV7) play important roles in translesion DNA synthesis (TLS) in which DNA replication bypasses blocking lesions. REV1 and Polζ have also been implicated in promoting repair of DNA double-stranded breaks (DSBs). However, the mechanism by which these two TLS polymerases increase tolerance to DSBs is poorly understood. Here we demonstrate that full-length human REV1, REV3 and REV7 interact in vivo (as determined by co-immunoprecipitation studies) and together, promote homologous recombination repair. Cells lacking REV3 were hypersensitive to agents that cause DSBs including the PARP inhibitor, olaparib. REV1, REV3 or REV7-depleted cells displayed increased chromosomal aberrations, residual DSBs and sites of HR repair following exposure to ionizing radiation. Notably, cells depleted of DNA polymerase η (Polη) or the E3 ubiquitin ligase RAD18 were proficient in DSB repair following exposure to IR indicating that Polη-dependent lesion bypass or RAD18-dependent monoubiquitination of PCNA are not necessary to promote REV1 and Polζ-dependent DNA repair. Thus, the REV1/Polζ complex maintains genomic stability by directly participating in DSB repair in addition to the canonical TLS pathway.
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http://dx.doi.org/10.1093/nar/gkr769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258153PMC
January 2012

Effect of fluticasone furoate on interleukin 6 secretion from adenoid tissues in children with obstructive sleep apnea.

Arch Otolaryngol Head Neck Surg 2011 Jun;137(6):576-82

Section of Otolaryngology-Head and Neck Surgery, The University of Chicago Medical Center, IL 60637, USA.

Objective: To determine the effect of intranasal corticosteroid therapy on T-regulatory cells and other inflammatory cytokines in adenoid tissues in children with obstructive sleep apnea syndrome.

Design: Randomized, prospective, exploratory study.

Setting: Academic pediatric otolaryngology practice in a tertiary care children's hospital.

Patients: Participants included 24 children between the ages of 2 and 12 years who were undergoing adenotonsillectomy for polysomnogram-documented obstructive sleep apnea syndrome.

Intervention: Children were randomized to either no treatment (n = 13) or treatment with fluticasone furoate nasal spray, 55 μg/nostril daily (n = 11), for 2 weeks before adenotonsillectomy. Adenoid tissue was obtained at the time of the procedure.

Main Outcome Measures: The number of tissue T-regulatory cells, as determined by staining with FOXP3, CD4, and CD25, was the primary outcome measure. Staining for interleukin (IL)-10 and transforming growth factor-β protein by immunohistochemistry, and adenoid mononuclear cell spontaneous and induced release of cytokines (IL-10, IL-6, IL-12, IL-13, tumor necrosis factor, and transforming growth factor β) were secondary outcomes.

Results: Cells isolated from fluticasone furoate nasal spray-treated adenoid tissue released significantly less IL-6 spontaneously as well as upon stimulation with anti-CD3 monoclonal antibody (P = .05) compared with nontreated adenoid tissue. There were no significant differences in the number of CD4/FOXP3-, CD25/FOXP3-, or transforming growth factor β-positive cells. Intensity of staining for IL-10 was also comparable between the groups.

Conclusions: In this study, we show reduction of IL-6, a proinflammatory cytokine, in adenoid tissue obtained from children with obstructive sleep apnea syndrome treated with fluticasone furoate nasal spray. This reduction could contribute to the clinical efficacy of this class of medications in the treatment of childhood obstructive sleep apnea syndrome.
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http://dx.doi.org/10.1001/archoto.2011.86DOI Listing
June 2011

Recruitment factors which affect the outcome of a seasonal allergic rhinitis trial.

Allergy Asthma Proc 2011 Jan-Feb;32(1):55-63

Department of Surgery, The University of Chicago, Illinois, USA.

Although intranasal corticosteroids (INSs) are the first-line treatment for seasonal allergic rhinitis (SAR), some patients do not respond adequately, reflecting biological heterogeneity or confounding conditions. The objective of this study was to determine what recruitment factors identify SAR subjects who will be unresponsive to mometasone furoate (MF). We performed a 2-week, double-blind, placebo-controlled, parallel study on 40 subjects with SAR. Each subject underwent a decongestant test using oxymetazoline. Baseline nasal symptoms, nasal peak inspiratory flow (NPIF) and Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) scores were recorded. Next, subjects were randomized to either 200 μg of MF or placebo. Symptom diaries and NPIF measurements were completed twice daily. After 2 weeks, subjects repeated the RQLQ and the global assessment of symptoms. There was a significant reduction in symptoms in the MF group compared with placebo (p ≤ 0.05) in patients with baseline total symptom scores of ≥6. Multivariate analysis showed that treatment (MF versus placebo; p = 0.049) and amount of decongestion (percent change in NPIF after oxymetazoline; p = 0.008) predicted the improvement in total nasal symptoms. In clinical trials, SAR subjects must report multiple symptoms to be responsive to treatment with INSs. Our results also support the use of the decongestant test for choice of appropriate study volunteers, both to ensure participation of potentially responsive subjects and to eliminate those with confounding issues.
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http://dx.doi.org/10.2500/aap.2011.32.3414DOI Listing
December 2011

Genetic polymorphisms in TNF genes and tuberculosis in North Indians.

BMC Infect Dis 2010 Jun 10;10:165. Epub 2010 Jun 10.

Department of Medicine, All India Institute of Medical Sciences, New Delhi-110029, India.

Background: Pulmonary tuberculosis, the most common clinical form of mycobacterial diseases, is a granulomatous disease of the lungs caused by Mycobaterium tuberculosis. A number of genes have been identified in studies of diverse origins to be important in tuberculosis. Of these, both tumor necrosis factor alpha (TNF-alpha) and lymphotoxin alpha (LT-alpha) play important immunoregulatory roles.

Methods: To investigate the association of TNF polymorphisms with tuberculosis in the Asian Indians, we genotyped five potentially functional promoter polymorphisms in the TNFA gene and a LTA_NcoI polymorphism (+252 position) of the LTA gene in a clinically well-defined cohort of North-Indian patients with tuberculosis (N = 185) and their regional controls (N = 155). Serum TNF-alpha (sTNF-alpha) levels were measured and correlated with genotypes and haplotypes.

Results: The comparison of the allele frequencies for the various loci investigated revealed no significant differences between the tuberculosis patients and controls. Also, when the patients were sub-grouped into minimal, moderately advanced and far advanced disease on the basis of chest radiographs, TST and the presence/absence of cavitary lesions, none of the polymorphisms showed a significant association with any of the patient sub-groups. Although a significant difference was observed in the serum TNF-alpha levels in the patients and the controls, none of the investigated polymorphisms were found to affect the sTNF-alpha levels. Interestingly, it was observed that patients with minimal severity were associated with lower log sTNF-alpha levels when compared to the patients with moderately advanced and far advanced severity. However, none of these differences were found to be statistically significant. Furthermore, when haplotypes were analyzed, no significant difference was observed.

Conclusions: Thus, our findings exclude the TNF genes as major risk factor for tuberculosis in the North Indians.
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http://dx.doi.org/10.1186/1471-2334-10-165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2894837PMC
June 2010

1H, 15N, 13C resonance assignment of 9.7 M urea-denatured state of the GTPase effector domain (GED) of dynamin.

Biomol NMR Assign 2009 Jun 18;3(1):13-6. Epub 2008 Nov 18.

Department of Chemical Sciences, Tata Institute of Fundamental Research, 1, Homi Bhabha Road, Mumbai, 400005, India.

The GTPase effector domain (GED) of dynamin, a multi-domain protein involved in endocytosis, forms a megadalton-sized self-assembly (even at micromolar concentrations) in native conditions in vitro. While such large assemblies have remained inaccessible to detailed NMR structural characterization, till date, a significant recent achievement has been the elucidation of the GED association pathway starting from a Gdn-HCl denatured monomer. Since, the nature of the denaturant has a strong influence on the conformational preferences in the denatured states, and hence on the association pathways, or even on the final assembly, we report here the NMR resonance assignment of 9.7 M urea-denatured GED from Homo sapiens. This will form the basis for the characterization of the association pathways and the final assembly driven by urea dilution.
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http://dx.doi.org/10.1007/s12104-008-9129-1DOI Listing
June 2009

1H, 15N, 13C resonance assignment of folded and 8 M urea-denatured state of SUMO from Drosophila melanogaster.

Biomol NMR Assign 2008 Jun 4;2(1):13-5. Epub 2007 Dec 4.

Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, 400005, India.

SUMO, an important post-translational modifier of variety of substrate proteins, regulates different cellular functions. Here, we report the NMR resonance assignment of the folded and 8 M urea-denatured state of SUMO from Drosophila melanogaster (dsmt3).
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http://dx.doi.org/10.1007/s12104-007-9072-6DOI Listing
June 2008

NMR-derived solution structure of SUMO from Drosophila melanogaster (dSmt3).

Proteins 2009 Jun;75(4):1046-50

Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai 400005, India.

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http://dx.doi.org/10.1002/prot.22389DOI Listing
June 2009

Promoter polymorphism in the MS4A2 gene and asthma in the Indian population.

Int Arch Allergy Immunol 2009 12;149(3):208-18. Epub 2009 Feb 12.

Molecular Immunogenetics Laboratory, Institute of Genomics and Integrative Biology, Delhi, India.

Background: A previous study from our laboratory identified 3-locus risk and protective haplotypes of the MS4A2 gene, a prime candidate for asthma and atopy, that were associated with differential histamine release profiles from basophils and FcepsilonRIbeta transcript levels in the Indian population.

Methods: To explore the role of promoter polymorphisms in the observed association, 4 additional promoter polymorphisms (-752C/T, -654C/T, -426T/C and -109T/C) were investigated in 240 nuclear families with atopic asthma, 237 atopic asthmatics and 221 unrelated controls, all of whom were clinically well characterized. The beta-subunit transcript levels were measured for 15 individuals and were correlated with the associated promoter polymorphisms.

Results: We observed a significant association of the -752C/T and -109T/C polymorphisms with asthma, in addition to the already reported association for the INT2 G/A, intron 5 (CA)(n) and 3'-UTR C/T polymorphisms. Additionally, the allele T of the -109T/C polymorphism was associated with a reduced risk of asthma and lower percent peripheral blood eosinophils, thereby pointing towards a protective role for this allele in asthma. Further, the 7-locus haplotypes C_C_T_C_A_16_C and T_C_T_T_G_18_T were identified as the major risk/susceptibility and protective haplotypes, respectively (p < 0.05). Three-locus sliding-window haplotype analysis also identified the -426T/C, -109T/C and INT2 G/A polymorphisms to be in regions of high priority (p < 0.00001). Indeed, the -109T allele was found to be associated with reduced expression levels for FcepsilonRIbeta.

Conclusions: A promoter-dependent mechanism with altered transcriptional regulation of FcepsilonRIbeta may be involved for its association with asthma. These results, therefore, could be useful in predicting the genetic susceptibility of individuals for developing asthma.
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http://dx.doi.org/10.1159/000199716DOI Listing
July 2009

Conserved structural and dynamics features in the denatured states of drosophila SUMO, human SUMO and ubiquitin proteins: Implications to sequence-folding paradigm.

Proteins 2009 Aug;76(2):387-402

Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India.

We have characterized here the structural and dynamics properties of urea-denatured state of dSmt3 by multidimensional NMR at 27 degrees C and pH 5.6. The various results suggest that hydrophobic clusters as well as different native and non-native secondary structural elements are transiently formed. The backbone in the regions Gln26-Lys31 and Gly47-Gln60 shows conformationally restricted motions. The AABUF profile of the sequence reflected that this region has the highest tendency to undergo hydrophobic clustering and may thus assist the formation of transient structures. The secondary chemical shifts and coupling constants indicated that this region has strong tendency to occupy the broad beta-domain of (phi,psi) space. A number of NMR parameters indicated that the region Asp58-Gln60 (corresponding to beta3-beta4 turn in the folded state) has residual turn-like structure. The present structural and dynamics results on urea-denatured dSmt3 have been compared with the previously published results on denatured states of similar fold proteins e.g. human SUMO-1 (55% homologous), ubiquitin (13.8% homologous) and GB1. Although the sequence homology is rather poor between them, the residual structure in all cases seems to be largely native type. The implications of these to sequence-folding paradigm and initial folding processes have been discussed.
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http://dx.doi.org/10.1002/prot.22354DOI Listing
August 2009

Comparison of NMR structural and dynamics features of the urea and guanidine-denatured states of GED.

Arch Biochem Biophys 2009 Jan 8;481(2):169-76. Epub 2008 Nov 8.

Department of Chemical Sciences, Tata Institute of Fundamental Research, 1, Homi Bhabha Road, Mumbai-400005, India.

Denatured states of proteins, the starting points as well as the intermediates of folding in vivo, play important roles in biological function. In this context, we describe here urea unfolding and characterization of the denatured state of GTPase effector domain (GED) of dynamin created by 9.7 M urea. These are compared with similar data for guanidine induced denaturation reported earlier. The unfolding characteristics in the two cases, as measured by the optical probes, are significantly different, urea unfolding proceeding via an intermediate. The structural and motional characteristics, determined by NMR, of the two denatured states are also strikingly different. The urea-denatured state shows a combination of alpha- and beta-preferences in contrast to the entirely beta-preferences in the guanidine-denatured state. Higher (15)N transverse relaxation rates suggest higher folding propensities in the urea-denatured state. The implications of these to GED folding are discussed.
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http://dx.doi.org/10.1016/j.abb.2008.11.002DOI Listing
January 2009

Equilibrium refolding transitions driven by trifluoroethanol and by guanidine hydrochloride dilution are similar in GTPase effector domain: implications to sequence-self-association paradigm.

Biochemistry 2008 Dec;47(49):12945-53

Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai-400005, India.

Protein folding transitions starting from a denatured state play crucial roles in deciding the final fate of a protein. A fundamental question in this regard is the role of the amino acid sequence of the protein. In this context, we have investigated here the equilibrium refolding to a partially folded state of the GTPase effector domain (GED) of dynamin driven by addition of increasing amounts of trifluoroethanol (TFE) and compared it with that driven by progressive dilution of the guanidine hydrochloride (Gdn-HCl) denaturant, which has been reported recently [ ( 2008 ) Protein Science 17 , 1319 - 1325 ]. The structural and dynamics changes as the molecule refolds starting from the Gdn-HCl denatured state have been monitored by circular dichroism, fluorescence, and NMR. The molecule remains a monomer in the TFE limiting case, whereas in the Gdn-HCl case, the molecule self-associates as the denaturant is removed. Even so, the two equilibrium transitions seem to have many similarities. The limiting helical contents are similar, and the regions of progressive increase in millisecond time scale motions, suggestive of slow conformational transitions, are largely the same. Though in the guanidine dilution case the partially folded molecules self-associate and there is multimer-monomer equilibrium, the very high concentration ( approximately 6 M) of guanidine prevents self-association in the case of TFE created species. Taken together, the observations under the drastically different solvation conditions suggest that the GED sequence is designed to self-assemble via helices leading to formation of a fully folded megadalton size assembly. The present observations may also have implications for the folding and association mechanism of the protein. These are important from the point of view of dynamin function.
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http://dx.doi.org/10.1021/bi801698qDOI Listing
December 2008

Effect of a single point mutation on the stability, residual structure and dynamics in the denatured state of GED: relevance to self-assembly.

Biophys Chem 2008 Sep 17;137(1):13-8. Epub 2008 Jun 17.

Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai-400005, India.

The GTPase effector domain (GED) of dynamin forms large soluble oligomers in vitro, while its mutant--I697A--lacks this property at low concentrations. With a view to understand the intrinsic structural characteristics of the polypeptide chain, the global unfolding characteristics of GED wild type (WT) and I697A were compared using biophysical techniques. Quantitative analysis of the CD and fluorescence denaturation profiles revealed that unfolding occurred by a two-state process and the mutant was less stable than the WT. Even in the denatured state, the mutation caused chemical shift perturbations and significant differences were observed in the 15N transverse relaxation rates (R2), not only at the mutation site but all around. These results demonstrate that the hydrophobic change associated with the mutation perturbs the structural and motional preferences locally, which are then relayed via different folding pathways along the chain and the property of oligomerization in the native state is affected.
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http://dx.doi.org/10.1016/j.bpc.2008.06.005DOI Listing
September 2008