Galactinol synthase across evolutionary diverse taxa: Functional preference for higher plants?

Galactinol synthase (GolS), a GT8 family glycosyltransferase, synthesizes galactinol and raffinose series of oligosaccharides (RFOs). Identification and analysis of conserved domains in GTs among evolutionarily diverse taxa, structure prediction by homology modeling and determination of substrate binding pocket followed by phylogenetic analysis of GolS sequences establish presence of functional GolS predominantly in higher plants, fungi having the closest possible ancestral sequences. Evolutionary preference for a functional GolS expression in higher plants might have arisen in response to the need for galactinol and RFO synthesis to combat abiotic stress, in contrast to other organisms lacking functional GolS for such functions.     

The ends and means of artificially induced targeted protein degradation

Studies on knockout mutants and conditional mutants are invaluable to biological research and have been used extensively to probe the intricacies of biological systems through loss of function associated with attenuation of a particular protein. Besides, RNAi technology has been developed in recent years to further aid the process of scientific inquiry. Even though, the methods, dealing with DNA and RNA have met with great success, are not without their shortcomings. In order to overcome the inadequacies of existing methods, a host of new techniques, aimed at knockdowns at the protein rather than the nucleic acid level, have been devised. Essentially, these methods can achieve rapid degradation of cellular pools of a target protein in response to an inducible signal coupled with dose-dependent modulation and exquisite temporal control, features which are absent from techniques involving manipulations at the DNA or RNA level. This review aims to provide a broad overview of a gamut of these methods, while highlighting the strengths and weaknesses of each one. Last two decades of advances presented here in the field of targeted protein degradation serve as a beacon to further research and are likely to find applications in the areas of medicine and allied fields of biology.     

Identifying new therapeutic targets via modulation of protein corona formation by engineered nanoparticles

Background

We introduce a promising methodology to identify new therapeutic targets in cancer. Proteins bind to nanoparticles to form a protein corona. We modulate this corona by using surface-engineered nanoparticles, and identify protein composition to provide insight into disease development.

Methods/Principal Findings

Using a family of structurally homologous nanoparticles we have investigated the changes in the protein corona around surface-functionalized gold nanoparticles (AuNPs) from normal and malignant ovarian cell lysates. Proteomics analysis using mass spectrometry identified hepatoma-derived growth factor (HDGF) that is found exclusively on positively charged AuNPs (⁺AuNPs) after incubation with the lysates. We confirmed expression of HDGF in various ovarian cancer cells and validated binding selectivity to ⁺AuNPs by Western blot analysis. Silencing of HDGF by siRNA resulted s inhibition in proliferation of ovarian cancer cells.

Conclusion

We investigated the modulation of protein corona around surface-functionalized gold nanoparticles as a promising approach to identify new therapeutic targets. The potential of our method for identifying therapeutic targets was demonstrated through silencing of HDGF by siRNA, which inhibited proliferation of ovarian cancer cells. This integrated proteomics, bioinformatics, and nanotechnology strategy demonstrates that protein corona identification can be used to discover novel therapeutic targets in cancer.     

A Novel MMP-2 Inhibitor 3-azidowithaferin A (3-azidoWA) Abrogates Cancer Cell Invasion and Angiogenesis by Modulating Extracellular Par-4

BackgroundWithaferin A, which is a naturally derived steroidal lactone, has been found to prevent angiogenesis and metastasis in diverse tumor models. It has also been recognized by different groups for prominent anti-carcinogenic roles. However, in spite of these studies on withanolides, their detailed anti-metastatic mechanism of action remained unknown. The current study has poised to address the machinery involved in invasion regulation by stable derivative of Withaferin A, 3-azido Withaferin A (3-azidoWA) in human cervical HeLa and prostate PC-3 cells.Conclusion/SignificanceFor this report, we found that 3-azidoWA suppressed motility and invasion of HeLa and PC-3 cells in MMP-2 dependent manner. Our in vitro result strongly suggests that sub-toxic doses of 3-azidoWA enhanced the secretion of extracellular Par-4 that abolished secretory MMP-2 expression and activity. Depletion of secretory Par-4 restored MMP-2 expression and invasion capability of HeLa and PC-3 cells. Further, our findings implied that 3-azidoWA attenuated internal phospho-ERK and phospho-Akt expression in a dose dependent manner might play a key role in inhibition of mouse angiogenesis by 3-azidoWA.     

Translocator protein (TSPO) in breast cancer

Several molecular and cellular markers are currently used as prognostic indicators for diagnosis and therapeutic intervention of breast cancer. Although some of these markers have helped clinicians provide an earlier diagnosis (or prognosis), they have failed to provide adequate information about the mechanisms responsible for different stages of tumor malignancy so that more effective anticancer therapies can be developed. Recently translocator protein (TSPO), formerly known as the peripheral benzodiazepine receptor (PBR), has received attention as a potential target for anticancer drug development. It is a well-conserved protein, located at outer-inner mitochondrial membrane contact sites, and is expressed in almost all tissues, although the level of expression varies. TSPO is closely associated with the 32 kDa voltage-dependent anion channel (VDAC) and the 30 kDa adenine nucleotide translocase (ANT), considered to form the core of a mitochondria multiprotein complex [named the mitochondrial permeability transition pore (MPTP)] and plays a role in apoptotic cell death. As the major role of TSPO is steroid biosynthesis, TSPO expression is particularly high in organs involved in steroidogenesis such as the adrenals, testes, ovaries, placenta, prostate, colon, kidney, and cardiovascular system. It is well known that TSPO is over-expressed in highly aggressive tumors, especially those of the breast, and that expression correlates with advancing stages of this malignancy. TSPO expression, nuclear localization, and TSPO-mediated cholesterol transport into the nucleus are involved in breast cancer cell proliferation and aggressive phenotype expression. Hence, it can be used as a biomarker in the stage-dependent diagnosis of this cancer. In addition, cell proliferation, invasion and migration appears to be decreased when treated with high doses of TSPO ligand PK-11195, a compound that may represent a therapeutic agent for the control of breast cancer progression. Control of breast cancer development by consumption of dietary soy protein has been linked to down-regulation of the expression of TSPO-mediated angiogenic signaling molecules. This chapter provides insight into the potential of TSPO as a rational target for the development of novel therapeutics for breast cancer.     

Proteomics and mass spectrometric studies reveal planktonic growth of Mycobacterium smegmatis in biofilm cultures in the absence of rpoZ

Mycobacterium smegmatis is known to form biofilms and many cell surface molecules like core glycopeptidolipids and short-chain mycolates appear to play important role in the process. However, the involvement of the cell surface molecules in mycobacteria towards complete maturation of biofilms is still not clear. This work demonstrates the importance of the glycopeptidolipid species with hydroxylated alkyl chain and the epoxylated mycolic acids, during the process of biofilm development. In our previous study, we reported the impairment of biofilm formation in rpoZ-deleted M. smegmatis, where rpoZ codes for the omega subunit of RNA polymerase (R. Mathew, R. Mukherjee, R. Balachandar, D. Chatterji, Microbiology 152 (2006) 1741). Here we report the occurrence of planktonic growth in a mc(2)155 strain which is devoid of rpoZ gene. This strain is deficient in selective incorporation of the hydroxylated glycopeptidolipids and the epoxy mycolates to their respective locations in the cell wall. Hence it forms a mutant biofilm defective in maturation, wherein the cells undertake various alternative metabolic pathways to survive in an environment where oxygen, the terminal electron acceptor, is limiting.     

Development and application of a method for analysis of lufenuron in wheat flour by gas chromatography-mass spectrometry and confirmation of bio-efficacy against Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

A new analytical method using gas chromatography with mass spectrometry (GC-MS) for the quantitative determination of lufenuron, a benzoylphenylurea (BPU) class of insecticide, from wheat flour has been developed and applied for time-dependent residue monitoring in treated wheat flour. The analyte was extracted from wheat flour by a single step solid-liquid extraction by using ethyl acetate and subsequently cleaned up using the Primary Secondary Amine as a sorbent prior to GC-MS analysis. The present method provides sufficient sensitivity as reflected by the values of limit of detection (LOD) and limit of quantification (LOQ), 5 ng/mL (S/N approximately 3) and 50 ng/mL (the lowest validation point on the calibration curve), respectively. The calibration curve showed an excellent linearity in the concentration range of 50-1000 ng/mL (r2=0.998). The average recovery for spiked samples at three concentrations (150, 300, and 450 ng/g) was 98.23+/-2.52% R.S.D. The method was applied for the determination of lufenuron residues in treated wheat flour samples. Simultaneous determination of bio-efficacy of lufenuron residues was also carried out against the red flour beetle, Tribolium castaneum to correlate the actual residual effect of lufenuron as detected by the analytical method, over a period of 3 months. The findings revealed that the residual concentration of lufenuron were neither uniform nor in descending order over a period of 3 months in wheat flour, possibly because of an uneven dispersal in the treated wheat which was subsequently milled into flour, as confirmed by GC-MS analysis. However, the residues of lufenuron were sufficient to produce 100% mortality of T. castaneum larvae up to 3 months. The results have been discussed in view of the potential of lufenuron as a candidate molecule for the control of stored product pests.