The 'Not-So-Famous Five' in tumorigenesis: tRNAs, tRNA fragments, and tRNA epitranscriptome in concert with AARSs and AIMPs.

RNA profiling studies have revealed that ∼75% of the human genome is transcribed to RNA but only a meagre fraction of it is translated to proteins. Majority of transcribed RNA constitute a specialized pool of non-coding RNAs. Human genome contains approximately 506 genes encoding a set of 51 different tRNAs, constituting a unique class of non-coding RNAs that not only have essential housekeeping functions as translator molecules during protein synthesis, but have numerous uncharted regulatory functions. Intriguing findings regarding a variety of non-canonical functions of tRNAs, tRNA derived fragments (tRFs), esoteric epitranscriptomic modifications of tRNAs, along with aminoacyl-tRNA synthetases (AARSs) and ARS-interacting multifunctional proteins (AIMPs), envision a 'peripheral dogma' controlling the flow of genetic information in the backdrop of qualitative information wrung out of the long-live central dogma of molecular biology, to drive cells towards either proliferation or differentiation programs. Our review will substantiate intriguing peculiarities of tRNA gene clusters, atypical tRNA-transcription from internal promoters catalysed by another distinct RNA polymerase enzyme, dynamically diverse tRNA epitranscriptome, intricate mechanism of tRNA-charging by AARSs governing translation fidelity, epigenetic regulation of gene expression by tRNA fragments, and the role of tRNAs and tRNA derived/associated molecules as quantitative determinants of the functional proteome, covertly orchestrating the process of tumorigenesis, through a deregulated tRNA-ome mediating selective codon-biased translation of cancer related gene transcripts.

Biophysical and theoretical studies of the interaction between a bioactive compound 3,5-dimethoxy-4-hydroxycinnamic acid with calf thymus DNA.

3,5-Dimethoxy-4-hydroxycinnamic acid commonly known as Sinapic acid is a well-known derivative of hydroxycinnamic acids, is commonly present in human diet. Due to its wide variety of pharmacological activities like antioxidant, antimicrobial, anti-inflammatory, anticancer, and anti-anxiety, it has attracted much attention for the researchers. In our previous published work we have already analyzed the interaction between sinapic acid (SA) with a model transport protein. In this work our aim is to demonstrate a detailed investigation of the binding interaction between sinapic acid with another carrier of genetic information in a living cell, the DNA. Here we have used calf thymus DNA (ct-DNA) as a model. The binding characteristic of SA with ct-DNA was investigated by different spectroscopic and theoretical tools. The spectroscopic investigation revealed that quenching of intrinsic fluorescence of SA by ct-DNA occurs through dynamic quenching mechanism. The thermodynamic parameters established the involvement of hydrogen bonding and weak van der Waals forces in the interaction. Further, the circular dichroism, competitive binding experiment with ethidium bromide and potassium iodide quenching experiment suggested that SA possibly binds to the groove position of the ct-DNA. Finally, molecular docking analysis established the SA binds to minor groove position of ct-DNA in G-C rich region through hydrogen bonding interaction. Additionally, gel electrophoresis analysis has been performed to determine the protective efficacy of SA against UVB induced DNA damage and 50 µM of SA was found to protect the DNA from UVB induced damage. We hope that our study could provide the validation of SA on behalf of therapeutics and development of next generation therapeutic drug as well as designing new efficient drug molecule and methodology for the interaction study of the drug with DNA.

Differential proteomics study of platelets in asymptomatic constitutional macrothrombocytopenia: altered levels of cytoskeletal proteins.

OBJECTIVES: Harris platelet syndrome (HPS), also known as asymptomatic constitutional macrothrombocytopenia (ACMT), is an autosomal dominant platelet disorder characterized by mild-to-severe thrombocytopenia and giant platelets with normal platelet aggregation and absence of bleeding symptoms. We have attempted a comparative proteomics study for profiling of platelet proteins in healthy vs. pathological states to discover characteristic protein expression changes in macrothrombocytes and decipher the factors responsible for the functionally active yet morphologically distinct platelets. METHODS: We have used 2-D gel-based protein separation techniques coupled with MALDI-ToF/ToF-based mass spectrometric identification and characterization of the proteins to investigate the differential proteome profiling of platelet proteins isolated from the peripheral blood samples of patients and normal volunteers. RESULTS AND CONCLUSION: Our study revealed altered levels of actin-binding proteins such as myosin light chain, coactosin-like protein, actin-related protein 2/3 complex, and transgelin2 that hint toward the cytoskeletal changes necessary to maintain the structural and functional integrity of macrothrombocytes. We have also observed over expressed levels of peroxiredoxin2 that signifies the prevailing oxidative stress in these cells. Additionally, altered levels of protein disulfide isomerase and transthyretin provide insights into the measures adapted by the macrothrombocytes to maintain their normal functional activity. This first proteomics study of platelets from ACMT may provide an understanding of the structural stability and normal functioning of these platelets in spite of their large size.

Comparative proteomics and glycoproteomics of plasma proteins in Indian visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) is a deadly parasitic diseases caused by Leishmania donovani; it is a major health problem in many countries. A lack of proper understanding of the disease biology, poor diagnostic methods and increasing drug resistance are the main reasons for the growing burden of VL infection. Comparative plasma proteomics are a relatively useful technique that can be used to investigate disease-associated alterations that can help in understanding host responses against pathogens, and might be useful in disease management and diagnosis. RESULT: In this study, a comparative proteomics and glycoproteomics approach using 2DE and 2D-DIGE was employed between early diagnosed VL patients of all age groups and healthy endemic and non-endemic controls in order to aid the recognition of disease-associated alterations in host plasma. Comparative proteomics was performed by the depletion of seven highly abundant plasma proteins. Comparative glycoproteomics was performed by the depletion of albumin and IgG, followed by purification of plasma glycoproteins using a multi lectin affinity column. From these two approaches, 39 differentially expressed protein spots were identified and sequenced using MALDI-TOF/TOF mass spectrometry. This revealed ten distinct proteins that appeared in multiple spots, suggesting micro-heterogeneity. Among these proteins, alpha-1-antitrypsin, alpha-1-B glycoprotein and amyloid-A1 precursor were up-regulated, whereas vitamin-D binding protein, apolipoprotein-A-I and transthyretin were down-regulated in VL. Alterations in the levels of these proteins in VL-infected plasma were further confirmed by western blot and ELISA. CONCLUSIONS: These proteins may be involved in the survival of parasites, resisting neutrophil elastase, and in their multiplication in macrophages, potentially maintaining endogenous anti-inflammatory and immunosuppressive conditions. Consequently, the results of this study may help in understanding the host response against L.donovani, which could help in the discovery of new drugs and disease management. Finally, these alterations on protein levels might be beneficial in improving early diagnosis considering those as biomarkers in Indian VL.

2D DIGE based proteomics study of erythrocyte cytosol in sickle cell disease: altered proteostasis and oxidative stress.

Sickle cell disease (SCD) is a hemolytic disorder caused by a mutation in beta-globin gene and affects millions of people worldwide. Though clinical manifestations of the disease are quite heterogeneous, many of them occur due to erythrocyte sickling at reduced oxygen concentration and vascular occlusion mediated via blood cell adhesion to the vessel wall. We have followed proteomic approach to resolve the differentially regulated proteins of erythrocyte cytosol. The deregulated proteins mainly fall in the group of chaperone proteins such as heat shock protein 70, alpha hemoglobin stabilizing protein, and redox regulators such as aldehyde dehydrogenase and peroxiredoxin-2 proteoforms. Proteasomal subunits are found to be upregulated and phospho-catalase level also got altered. Severe oxidative stress inside erythrocyte is evident from the ROS analysis and Oxyblot(TM) experiments. Peroxiredoxin-2 shows significant dimerization in the SCD patients, a hallmark of oxidative stress inside erythrocytes. One interesting fact is that most of the differentially regulated proteins are also common for hemoglobinopathies such as Eß thalassemia. These could provide important clues in understanding the pathophysiology of SCD and lead us to better patient management in the future.

Elevated levels of redox regulators, membrane-bound globin chains, and cytoskeletal protein fragments in hereditary spherocytosis erythrocyte proteome.

OBJECTIVE: Hereditary spherocytosis (HS), a common inherited hemolytic anemia characterized by decreased deformability, reduced surface to volume ratio, and increased osmotic fragility of the spheroidal erythrocytes, is associated with several mutations of α- and ß-spectrin, ankyrin, band 3, band 4.2. HS manifests itself with high degrees of clinical heterogeneity and the molecular events leading to premature hemolysis of the spherocytes are unclear. We have employed proteomic techniques to identify differentially regulated proteins in the membrane and hemoglobin-depleted cytosol of HS erythrocytes. METHODS: We have employed 2-D gel electrophoresis and tandem matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry to investigate the differential proteome profiling of membrane and hemoglobin-depleted cytosol of erythrocytes isolated from the peripheral blood samples of HS patients and normal volunteers. RESULTS: Our study showed that redox regulators are up-regulated; while a co-chaperone and a nucleotide kinase are down-regulated in HS erythrocyte cytosol. We observed elevated levels of membrane-associated globin chains and low-molecular weight fragments of several major cytoskeletal proteins. CONCLUSION: The observed changes in the erythrocyte proteomes indicate altered redox regulation, nucleotide metabolism, protein aggregation and/or degradation, cytoskeletal disorganization, and severe oxidative stress in HS. Taken together, this study could enlighten upon disease progression and pathophysiology of HS.

Differential expression of red cell proteins in hemoglobinopathy.

Red blood cell proteome has not been studied well until recently, as the large abundance of hemoglobin posed challenge to the detection of other cytosolic proteins in the linear dynamic range. However, in the last couple of years, due to emergence of various novel hemoglobin depletion strategies and more state-of-the-art detection techniques, a number of works on erythrocyte proteome have appeared in the literature. As a result, we now have much deeper information about both the membrane as well as the cytosolic proteins of erythrocytes. In this review, we have discussed the role of red cell proteome on the two most well-studied hemoglobin disorders, sickle cell disease and thalassemia, emphasizing on the differential expression of the redox regulator proteins and chaperones, in particular. We have also touched upon the importance of the association of the varying levels of hemoglobin variants, particularly HbE on the clinical manifestation of composite diseases like HbEß thalassemia.

Differential regulation of redox proteins and chaperones in HbEß-thalassemia erythrocyte proteome.

PURPOSE: In (hemoglobin, Hb) HbEß-thalassemia, HbE (ß-26 Glu→Lys) interacts with ß-thalassemia to produce clinical manifestation of varying severity. This is the first proteomic effort to study changes in protein levels of erythrocytes isolated from HbEß-thalassemic patients compared to normal. EXPERIMENTAL DESIGN: We have used 2-DE and MALDI-MS/MS-based techniques to investigate the differential proteome profiling of membrane and Hb-depleted fraction of cytosolic proteins of erythrocytes isolated from the peripheral blood samples of HbEß-thalassemia patients and normal volunteers. RESULTS: Our study showed that redox regulators such as peroxiredoxin 2, Cu-Zn superoxide dismutase and thioredoxin and chaperones such as α-hemoglobin stabilizing protein and HSP-70 were upregulated in HbEß-thalassemia. We have also observed larger amounts of membrane associated globin chains and indications of disruption of spectrin-based junctional complex in the membrane skeleton of HbEß-thalassemic erythrocytes upon detection of low molecular weight fragments of ß-spectrin and decrease in ß-actin and dematin content. CONCLUSION AND CLINICAL RELEVANCE: We have observed interesting changes in the proteomic levels of redox regulators and chaperons in the thalassemic hemolysates and have observed strong correlation or association of the extent of such proteomic changes with HbE levels. This could be important in understanding the role of HbE in disease progression and pathophysiology.

Oxidative crosslinking, spectrin and membrane interactions of hemoglobin mixtures in HbEbeta-thalassemia.

The authors have studied the interactions of intact hemoglobin mixtures of HbE and HbA, with the major erythroid membrane skeletal protein, spectrin and tailor-made phospholipids membranes containing aminophospholipids to understand the role of spectrin and phospholipids of erythrocytes in the overall pathophysiology of the hemoglobin disorders. Hemoglobin mixtures were isolated and purified from the peripheral blood samples of HbE carriers and different HbEbeta thalassemia patients, taken for diagnosis. Spectrin binding was studied by fluorescence and oxidative crosslinking, by SDS-PAGE. Membrane perturbation experiments were carried out to study the leakage of the self-quenching fluorophore, carboxyfluorescein, entrapped in the phospholipid vesicles. Hemoglobin mixtures with elevated levels of HbE showed stronger interactions with spectrin reflected in the decrease in binding dissociation constant from 17 to 5 muM upon increase in HbE% from about 30 to 90% in the hemolysates. The yield of the spectrin crosslinked complexes of such hemoglobin mixtures also increased with increase in HbE levels. Presence of ATP/Mg and DPG were found to decrease the overall yield of such complexes and the binding affinity of hemoglobins to spectrin. HbE rich hemolysates also induced greater leakage of entrapped carboxyfluorescein (CF) from phospholipid membranes containing aminophospholipids. Results from this study indicate the roles of skeletal proteins and aminophospholipids, particularly under oxidative stress conditions to be important in the premature destruction of erythrocytes in hemoglobin disorders, e.g. HbEbeta-thalassaemia.

Porous red cell ultrastructure and loss of membrane asymmetry in a novel case of hemolytic anemia.

Transmission electron microscopic study revealed large pores on the erythrocyte ghost membranes, disrupted cytoskeleton and microcytosis of circulating erythrocytes in a novel case of hemolytic anemia. Greater loss of phosphatidylserine (PS) asymmetry was observed in younger erythrocytes compared with the aged ones in contrast to the normal red cells. Levels of sialylated glycoconjugates, such as glycophorin, measured by the binding of wheat germ agglutinin, showed greater loss upon aging. Such drastic loss of PS asymmetry leads to faster eryptosis, mediated by shedding of glycophorin-containing microvesicles leaving highly PS-exposed erythrocytes accessible to the phagocytes.