@article { ISI:000259509600003, title = {Albumin competitively inhibits glycation of less abundant proteins}, journal = {Protein and Peptide Letters}, volume = {15}, number = {7}, year = {2008}, month = {JUL}, pages = {663-667}, publisher = {BENTHAM SCIENCE PUBL LTD}, type = {Article}, address = {EXECUTIVE STE Y26, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES}, abstract = {

Glycation, a non-enzymatic reaction between glucose and protein is the primary cause of diabetic complications. Albumin, the most abundant plasma protein undergoes glycation both in vivo and in vitro. The influence of albumin on glycation of less abundant proteins has not been addressed. For the first time, we show that albumin competitively inhibits the glycation of less abundant proteins. This study suggests that at least in the initial stages of diabetes, albumin may protect other proteins from glycation.

}, keywords = {Diabetes, glucose, insulin, MALDI-TOF-MS, vascular complication}, issn = {0929-8665}, doi = {10.2174/092986608785133690}, author = {Bhonsle, Hemangi S. and Singh, Sameer Kumar and Srivastava, Ghanshyam and Boppana, Ramanamurthy and Kulkarni, Mahesh J.} } @article { ISI:000298860100004, title = {Proteomic analysis of glycated proteins from streptozotocin-induced diabetic rat kidney}, journal = {Molecular Biotechnology}, volume = {50}, number = {1}, year = {2012}, month = {JAN}, pages = {28-38}, publisher = {HUMANA PRESS INC}, address = {999 RIVERVIEW DRIVE SUITE 208, TOTOWA, NJ 07512 USA}, abstract = {

Glycation of proteins leading to formation of advanced glycation end products (AGEs) has been considered as one of the important causes of diabetic nephropathy. Therefore, in this study, glycated proteins were detected by anti-AGE antibodies from kidney of streptozotocin-induced diabetic rat showing nephropathic symptoms, by using two dimensional electrophoresis and western blot analysis. These glycated proteins were identified and characterized by using combination of peptide mass finger printing and tandem mass spectrometric approaches. Glycated proteins identified included proteins from metabolic pathways, oxidative stress, cell signaling, and transport. Several of the proteins modified by glycation were involved in glucose metabolism. The extent of glycation was higher in diabetes compared to control, in the glycated proteins that were common to both control and diabetic kidney. Two dimensional electrophoresis proteins profiling of glycated proteins suggest that four of the glycated proteins were significantly up regulated in diabetes.

}, keywords = {AGE, Diabetes, Glucose toxicity, Nephropathy, Post-translation modification}, issn = {1073-6085}, doi = {10.1007/s12033-011-9409-3}, author = {Chougale, Ashok D. and Bhat, Shweta P. and Bhujbal, Swapnil V. and Zambare, Mandar R. and Puntambekar, Shraddha and Somani, Rahul S. and Boppana, Ramanamurthy and Giri, Ashok P. and Kulkarni, Mahesh J.} } @article { ISI:000315745600001, title = {Zoom-in - a targeted database search for identification of glycation modifications analyzed by untargeted tandem mass spectrometry}, journal = {European Journal of Mass Spectrometry}, volume = {18}, number = {6}, year = {2012}, month = {JAN}, pages = {475-481}, publisher = {IM PUBLICATIONS}, type = {Article}, address = {6 CHARLTON MILL, CHARLTON, CHICHESTER,, W SUSSEX PO18 0HY, ENGLAND}, abstract = {

Post-translational modifications (PTMs) are very important to biological function, however their identification and characterization is technically challenging. In this study, we have identified glycation modifications by nano LC-MSE, a data independent acquisition work flow, followed by database search using the Protein Lynx Global Server (PLGS). PLGS search with a complete human protein database hardly identified glycation modifications in a glycated human serum albumin (HSA), which was detected to be glycated by western blotting with advanced glycation end products (AGE) antibody and fluorescence spectroscopy. To overcome this difficulty, {\textquoteleft}{\textquoteleft}Zoom-In{\textquoteright}{\textquoteright} approach, a targeted database search was used to identify glycation modifications in a glycated HSA, which were further manually validated. This approach was useful for identification of glycation modifications from untargeted tandem mass spectrometry workflow such as MSE, but may require the development of a new algorithm or an upgrade of the existing software.

}, keywords = {advanced glycation end products (AGEs), data independent acquisition, Diabetes, post-translational modifications}, issn = {1469-0667}, doi = {10.1255/ejms.1203}, author = {Bhonsle, Hemangi S. and Korwar, Arvind M. and Kesavan, Suresh K. and Bhosale, Santosh D. and Bansode, Sneha B. and Kulkarni, Mahesh J.} } @article { ISI:000313903700015, title = {Glycated proteome: from reaction to intervention}, journal = {Proteomics Clinical Applications}, volume = {7}, number = {1-2, SI}, year = {2013}, month = {JAN}, pages = {155-170}, publisher = {WILEY-V C H VERLAG GMBH}, address = {POSTFACH 101161, 69451 WEINHEIM, GERMANY}, abstract = {

Glycation, a nonenzymatic reaction between reducing sugars and proteins, is a proteome wide phenomenon, predominantly observed in diabetes due to hyperglycemia. Glycated proteome of plasma, kidney, lens, and brain are implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative diseases, cancer, and aging. This review discusses the strategies to characterize protein glycation, its functional implications in different diseases, and intervention strategies to protect the deleterious effects of protein glycation.

}, keywords = {aging, Diabetes, Glycation, RAGE}, issn = {1862-8346}, doi = {10.1002/prca.201200101}, author = {Kulkarni, Mahesh J. and Korwar, Arvind M. and Mary, Sheon and Bhonsle, Hemangi S. and Giri, Ashok P.} } @article { ISI:000376479400017, title = {Dietary flaxseed oil and fish oil ameliorates renal oxidative stress, protein glycation, and inflammation in streptozotocin-nicotinamide-induced diabetic rats}, journal = {Journal of Physiology and Biochemistry}, volume = {72}, number = {2}, year = {2016}, month = {JUN}, pages = {327-336}, publisher = {SERVICIO PUBLICACIONES UNIVERSIDAD NAVARRA}, address = {CAMPUS UNIV, CARRETERA DEL SADAR S-N, APARTADO 177, 31080 PAMPLONA, SPAIN}, abstract = {

Protective and prophylactic effects of omega-3 fatty acids on oxidative stress and inflammation are well known. We assessed beneficial effects of flaxseed oil and fish oil on streptozotocin (65 mg/kg; i.p.)-nicotinamide (110 mg/kg; i.p.) induced diabetic rats by studying renal expression of antioxidant and inflammatory genes. Diabetic rats given 10 \% flaxseed oil or 10 \% fish oil diet for 35 days showed significant decrease in renal lipid peroxidation. Flaxseed oil diet resulted in up-regulation of renal superoxide dismutase-1 (SOD-1) (activity and expression) and glutathione peroxidase-1 (GPx-1) expression. Furthermore, both diets up-regulated catalase (CAT) (activity and expression) and down-regulated heme oxygenase-1 (HO-1) expression. Both diets were able to limit the renal advanced glycation end products (AGEs) formation and reduced receptor of AGE (RAGE) protein expression significantly. Expressions of interleukin-6 (IL-6) and NF-kappa B p65 subunit were down-regulated significantly by flaxseed oil or fish oil diet. The histological tubular injuries were also lowered by both diets. These results suggest that dietary omega-3 fatty acids may slow the progression of diabetic nephropathy (DN) associated with oxidative stress, glycation, and inflammation in the kidney.

}, keywords = {Diabetes, Fish oil, Flaxseed oil, Glycation, Kidney, Streptozotocin}, issn = {1138-7548}, doi = {10.1007/s13105-016-0482-8}, author = {Jangale, Nivedita M. and Devarshi, Prasad P. and Bansode, Sneha B. and Kulkarni, Mahesh J. and Harsulkar, Abhay M.} } @article { ISI:000381180000001, title = {Targeted quantification of N-1-(carboxymethyl) valine and N-1-(carboxyethyl) valine peptides of beta-hemoglobin for better diagnostics in diabetes}, journal = {Clinical Proteomics}, volume = {13}, year = {2016}, month = {MAR}, pages = {Article Number: 7}, publisher = {BIOMED CENTRAL LTD}, address = {236 GRAYS INN RD, FLOOR 6, LONDON WC1X 8HL, ENGLAND}, abstract = {

Background: N-1-(Deoxyfructosyl) valine (DFV) beta-hemoglobin (beta-Hb), commonly referred as HbA1c, is widely used diagnostic marker in diabetes, believed to provide glycemic status of preceding 90-120 days. However, the turnover of hemoglobin is about 120 days, the DFV-beta-Hb, an early and reversible glycation product eventually may undergo irreversible advanced glycation modifications such as carboxymethylation or carboxyethylation. Hence quantification of N-1-(carboxymethyl) valine (CMV) and N-1-(carboxyethyl) valine (CEV) peptides of beta-Hb would be useful in assessing actual glycemic status. Results: Fragment ion library for synthetically glycated peptides of hemoglobin was generated by using high resolution-accurate mass spectrometry (HR/AM). Using parallel reaction monitoring, deoxyfructosylated, carboxymethylated and carboxyethylated peptides of hemoglobin were quantified in clinical samples from healthy control, pre-diabetes, diabetes and poorly controlled diabetes. For the first time, we report N-1-beta-valine undergoes carboxyethylation and mass spectrometric quantification of CMV and CEV peptides of beta-hemoglobin. Carboxymethylation was found to be the most abundant modification of N-1-beta-valine. Both CMV-beta-Hb and CEV-beta-Hb peptides showed better correlation with severity of diabetes in terms of fasting glucose, postprandial glucose and microalbuminuria. Conclusions: This study reports carboxymethylation as a predominant modification of N-1-beta-valine of Hb, and quantification of CMV-beta-Hb and CEV-beta-Hb could be useful parameter for assessing the severity of diabetes.

}, keywords = {Diabetes, Diagnosis, Glycation, Glycemic control, HbA1c, Mass spectrometry}, issn = {1542-6416}, doi = {10.1186/s12014-016-9108-y}, author = {Jagadeeshaprasad, Mashanipalya G. and Batkulwar, Kedar B. and Meshram, Nishita N. and Tiwari, Shalbha and Korwar, Arvind M. and Unnikrishnan, Ambika G. and Kulkarni, Mahesh J.} } @article {46723, title = {Possible role of glycation in the regulation of amyloid beta precursor protein processing leading to amyloid beta accumulation}, journal = {Medical Hypotheses}, volume = {142}, year = {2020}, month = {SEP}, pages = {109799}, type = {Article}, abstract = {

Alzheimer{\textquoteright}s disease (AD) is one of the most common forms of neurodegenerative diseases amongst the aged population. The disease is multifactorial, and diabetes has been considered as one of the major risk factors for the development of AD. Chronic hyperglycemic condition in diabetes promotes non-enzymatic protein modification by glucose termed as glycation, which affects protein structure and function. Previous studies have shown that many of the enzymes, including proteases, are affected by glycation. Conversely, glycated proteins are known to become resistant to protease action. In these hypotheses, we have extended these two concepts to the regulation of amyloid-beta protein precursor (A beta PP) by secretases leading to amyloid-beta (Af3) accumulation. The first hypothesis deals with the glycation of alpha-secretases leading to its reduced activity, while in the second hypothesis, A beta PP glycation may prevent a-secretases action, rendering its processing by beta secretase. As diabetes is a risk factor for the development of AD, either or both these pathways may operate, leading to the manifestation of AD.

}, keywords = {Alpha-secretase, Alzheimer {\textquoteleft}s disease, Amyloid-beta, Amyloid-beta protein precursor, Beta-secretase, Diabetes, glucose, Glycation, Type-3 diabetes}, issn = {0306-9877}, doi = {10.1016/j.mehy.2020.109799}, author = {Patil, Gouri V. and Joshi, Rakesh S. and Kazi, Rubina S. and Kulsange, Shabda E. and Kulkarni, Mahesh J.} } @article {47095, title = {Molecular investigation of glycated insulin-induced insulin resistance via insulin signaling and AGE-RAGE axis}, journal = {Biochimica ET Biophysica Acta-Molecular Basis of Disease}, volume = {1867}, year = {2021}, month = {FEB }, pages = {166029}, type = {Article}, abstract = {

Hyperglycemic condition in diabetes promotes glycation of various plasma proteins including insulin. Glycation of insulin has been reported to reduce its biological activity. Reduced biological activity of glycated insulin could be either due to reduced affinity for the insulin receptor and impaired insulin signaling, or it can act as a ligand for the receptor for advanced glycation end products (RAGE) and activates oxidative stress and pro-inflammatory pathways leading to insulin resistance. This study investigates the effect of glycated insulin on both insulin and RAGE signaling. Glycated insulin treatment to Chinese hamster ovary (CHO-IR-GLUT4) cells stably expressing insulin receptor (IR) and glucose transporter fused with a green fluorescent protein (GLUT4-GFP) resulted in the impairment of insulin signaling, as the phosphorylation of IR and AKT significantly reduced, which affected GLUT4 translocation and glucose uptake. Moreover, it also activated RAGE signaling as observed by increased expression of NADPH oxidase accompanied by an increase in reactive oxygen species (ROS). Immunofluorescence study indicated the translocation of NF-kappa B to the nucleus upon treatment of glycated insulin. This was associated with increased RAGE expression, Caspase 3, and cell death. Downregulation of RAGE with the losartan treatment restored the impaired insulin signaling and glucose uptake. Additionally, in silico study demonstrated that glycated insulin has reduced binding affinity to insulin receptor and increased binding affinity to RAGE. Overall, this study demonstrates the role of glycated insulin in exacerbating insulin resistance by impairing insulin signaling as well as stimulating AGE-RAGE signaling.

}, keywords = {AGE, Diabetes, Glycation, Hyperglycemia, insulin, RAGE}, issn = {0925-4439}, doi = {10.1016/j.bbadis.2020.166029}, author = {Walke, Prachi B. and Bansode, Sneha B. and More, Nikita P. and Chaurasiya, Arvindkumar H. and Joshi, Rakesh S. and Kulkarni, Mahesh J.} } @article {49132, title = {Mechanistic insights on anserine hydrolyzing activities of human carnosinases}, journal = {Biochemica ET Biophysica Acta- General Subjects }, volume = {1867}, year = {2023}, month = {MAR}, type = {Article}, abstract = {

Anserine and carnosine represent histidine-containing dipeptides that exert a pluripotent protective effect on human physiology. Anserine is known to protect against oxidative stress in diabetes and cardiovascular diseases. Human carnosinases (CN1 and CN2) are dipeptidases involved in the homeostasis of carnosine. In poikilothermic vertebrates, the anserinase enzyme is responsible for hydrolyzing anserine. However, there is no specific anserine hydrolyzing enzyme present in humans. In this study, we have systematically investigated the anserine hydrolyzing activity of human CN1 and CN2. A targeted multiple reaction monitoring (MRM) based approach was employed for studying the enzyme kinetics of CN1 and CN2 using carnosine and anserine as substrates. Surprisingly, both CN1 and CN2 can hydrolyze anserine effectively. The observed catalytic turnover rate (Vmax/[E]t) was 21.6 s-1 and 2.8 s-1 for CN1 and CN2, respectively. CN1 is almost eight-fold more efficient in hydrolyzing anserine compared to CN2, which is comparable to the efficiency of the carnosine hydrolyzing activity of CN2. The Michaelis constant (Km) value for CN1 (1.96 mM) is almost three-fold lower compared to CN2 (6.33 mM), representing higher substrate affinity for anserine-CN1 interactions. Molecular docking studies showed that anserine binds at the catalytic site of the carnosinases with an affinity similar to carnosine. Overall, the present study elucidated the inherent promiscuity of human carnosinases in hydrolyzing anserine using a sensitive LCMS/MS approach.

}, keywords = {Anserine, Carnosinase, Carnosine, Diabetes, LC-MS, MS, Multiple reaction monitoring (MRM), Promiscuity}, issn = {0304-4165}, doi = {10.1016/j.bbagen.2022.130290}, author = {Pandya, Vaibhav Kumar and Shankar, S. Shiva and Rajesh, S. and Rathore, Rajeshwari and Kumaran, Sangaralingam and Kulkarni, Mahesh J.} } @article {49603, title = {Simple freeze-thaw based method for efficient purification of recombinant human proinsulin from inclusion bodies}, journal = {Protein Expression and Purification}, volume = {227}, year = {2025}, month = {MAR}, pages = {106645}, type = {Article}, abstract = {

Insulin is a pivotal peptide hormone essential for regulating glucose homeostasis. It has been known for over 100 years, but its production and purification methods are still under improvement. Escherichia coli based bacterial expression system is primarily used for insulin production. The human insulin protein expressed in bacteria usually forms inclusion bodies, complicating the purification process. Traditionally, insulin purification is a timeconsuming process involving urea-based denaturation methods, and various refolding techniques, followed by extensive chromatographic methods. Here, we report an easy and efficient purification of human proinsulin involving freeze-thaw based solubilization method. The extracted proinsulin inclusion bodies are treated with different concentrations of urea, followed by a freeze-thaw based solubilization. The freezing was carried out at various temperatures, mainly -80 degrees C, -20 degrees C, and -196 degrees C to determine the optimum condition for solubilization. Highest solubilization of proinsulin from the inclusion body was achieved with 0.5M urea and -20 degrees C. Further Nickel NTA-based purification was performed, and the purified protein was characterized for disulfide mapping by high-resolution mass spectrometer (HRMS). We also performed glucose uptake assays to validate the functional properties of purified proinsulin. This freeze-thaw based mild solubilization approach is a fast and effective method for getting bioactive proinsulin, which will help further design better purification and processing strategies for insulin production.

}, keywords = {Diabetes, Disulfide mapping, glucose, insulin, Mass spectrometry}, issn = {1046-5928}, doi = {10.1016/j.pep.2024.106645}, author = {Rajesh, S. and Jathar, Swaraj and Banarjee, Reema and Sharma, Monika and Palkar, Shivani and Shankar, S. Shiva and Kulkarni, Mahesh J.} }