Identifying metalloproteins through X-ray fluorescence mapping and mass spectrometry

Metals are critical and dynamic components of biochemistry. To understand their roles, we greatly need tools to identify the ligands that bind them within the complexity of natural systems. This work describes the development of methods that not only detect and distinguish metals, but also characterize the proteins that bind them. We describe an approach to expose, identify and quantify metalloproteins in complex mixtures by sequential non-denaturing 2D-gel electrophoresis (2D GE)/X-ray Fluorescence (XRF) and tandem mass spectrometry (MS/MS) in the same spot of a sample. We first apply the development of 2D GE/XRF to Shewanella oneidensis MR-1, a well-studied system, and verify our electrophoretic approach. Then, we identified a novel periplasmic zinc protein in Pseudomonas aeruginosa PAO1 through 2D GE/XRF followed by MS/MS. The identity and function of this protein was verified through a gene mutation experiment.Fil: Raimunda, Daniel Cesar. Worcester Polytechnic Institute; Estados UnidosFil: Khare, Tripti. Argonne National Laboratory; Estados UnidosFil: Giometti, Carol. Argonne National Laboratory; Estados UnidosFil: Vogt, Stefan. Argonne National Laboratory; Estados UnidosFil: Argüello, José M.. Worcester Polytechnic Institute; Estados UnidosFil: Finney, Lydia. Argonne National Laboratory; Estados Unido

Transcriptional and Proteomic Analysis of a Ferric Uptake Regulator (Fur) Mutant of Shewanella oneidensis: Possible Involvement of Fur in Energy Metabolism, Transcriptional Regulation, and Oxidative Stress

The iron-directed, coordinate regulation of genes depends on the fur (ferric uptake regulator) gene product, which acts as an iron-responsive, transcriptional repressor protein. To investigate the biological function of a fur homolog in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1, a fur knockout strain (FUR1) was generated by suicide plasmid integration into this gene and characterized using phenotype assays, DNA microarrays containing 691 arrayed genes, and two-dimensional polyacrylamide gel electrophoresis. Physiological studies indicated that FUR1 was similar to the wild-type strain when they were compared for anaerobic growth and reduction of various electron acceptors. Transcription profiling, however, revealed that genes with predicted functions in electron transport, energy metabolism, transcriptional regulation, and oxidative stress protection were either repressed (ccoNQ, etrA, cytochrome b and c maturation-encoding genes, qor, yiaY, sodB, rpoH, phoB, and chvI) or induced (yggW, pdhC, prpC, aceE, fdhD, and ppc) in the fur mutant. Disruption of fur also resulted in derepression of genes (hxuC, alcC, fhuA, hemR, irgA, and ompW) putatively involved in iron uptake. This agreed with the finding that the fur mutant produced threefold-higher levels of siderophore than the wild-type strain under conditions of sufficient iron. Analysis of a subset of the FUR1 proteome (i.e., primarily soluble cytoplasmic and periplasmic proteins) indicated that 11 major protein species reproducibly showed significant (P < 0.05) differences in abundance relative to the wild type. Protein identification using mass spectrometry indicated that the expression of two of these proteins (SodB and AlcC) correlated with the microarray data. These results suggest a possible regulatory role of S. oneidensis MR-1 Fur in energy metabolism that extends the traditional model of Fur as a negative regulator of iron acquisition systems

Knock-out of SO1377 gene, which encodes the member of a conserved hypothetical bacterial protein family COG2268, results in alteration of iron metabolism, increased spontaneous mutation and hydrogen peroxide sensitivity in Shewanella oneidensis MR-1

BACKGROUND: Shewanella oneidensis MR-1 is a facultative, gram-negative bacterium capable of coupling the oxidation of organic carbon to a wide range of electron acceptors such as oxygen, nitrate and metals, and has potential for bioremediation of heavy metal contaminated sites. The complete 5-Mb genome of S. oneidensis MR-1 was sequenced and standard sequence-comparison methods revealed approximately 42% of the MR-1 genome encodes proteins of unknown function. Defining the functions of hypothetical proteins is a great challenge and may need a systems approach. In this study, by using integrated approaches including whole genomic microarray and proteomics, we examined knockout effects of the gene encoding SO1377 (gi24372955), a member of the conserved, hypothetical, bacterial protein family COG2268 (Clusters of Orthologous Group) in bacterium Shewanella oneidensis MR-1, under various physiological conditions. RESULTS: Compared with the wild-type strain, growth assays showed that the deletion mutant had a decreased growth rate when cultured aerobically, but not affected under anaerobic conditions. Whole-genome expression (RNA and protein) profiles revealed numerous gene and protein expression changes relative to the wild-type control, including some involved in iron metabolism, oxidative damage protection and respiratory electron transfer, e. g. complex IV of the respiration chain. Although total intracellular iron levels remained unchanged, whole-cell electron paramagnetic resonance (EPR) demonstrated that the level of free iron in mutant cells was 3 times less than that of the wild-type strain. Siderophore excretion in the mutant also decreased in iron-depleted medium. The mutant was more sensitive to hydrogen peroxide and gave rise to 100 times more colonies resistant to gentamicin or kanamycin. CONCLUSION: Our results showed that the knock-out of SO1377 gene had pleiotropic effects and suggested that SO1377 may play a role in iron homeostasis and oxidative damage protection in S. oneidensis MR-1

Natural Product-Based Nanomedicine in Treatment of Inflammatory Bowel Disease

Many synthetic drugs and monoclonal antibodies are currently in use to treat Inflammatory Bowel Disease (IBD). However, they all are implicated in causing severe side effects and long-term use results in many complications. Numerous in vitro and in vivo experiments demonstrate that phytochemicals and natural macromolecules from plants and animals reduce IBD-related complications with encouraging results. Additionally, many of them modify enzymatic activity, alleviate oxidative stress, and downregulate pro-inflammatory transcriptional factors and cytokine secretion. Translational significance of natural nanomedicine and strategies to investigate future natural product-based nanomedicine is discussed. Our focus in this review is to summarize the use of phytochemicals and macromolecules encapsulated in nanoparticles for the treatment of IBD and IBD-associated colorectal cancer

CXCL12-CXCR4/CXCR7 Axis in Colorectal Cancer: Therapeutic Target in Preclinical and Clinical Studies

Chemokines are chemotactic cytokines that promote cancer growth, metastasis, and regulate resistance to chemotherapy. Stromal cell-derived factor 1 (SDF1) also known as C-X-C motif chemokine 12 (CXCL12), a prognostic factor, is an extracellular homeostatic chemokine that is the natural ligand for chemokine receptors C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or cluster of differentiation 184 (CD184) and chemokine receptor type 7 (CXCR7). CXCR4 is the most widely expressed rhodopsin-like G protein coupled chemokine receptor (GPCR). The CXCL12–CXCR4 axis is involved in tumor growth, invasion, angiogenesis, and metastasis in colorectal cancer (CRC). CXCR7, recently termed as atypical chemokine receptor 3 (ACKR3), is amongst the G protein coupled cell surface receptor family that is also commonly expressed in a large variety of cancer cells. CXCR7, like CXCR4, regulates immunity, angiogenesis, stem cell trafficking, cell growth and organ-specific metastases. CXCR4 and CXCR7 are expressed individually or together, depending on the tumor type. When expressed together, CXCR4 and CXCR7 can form homo- or hetero-dimers. Homo- and hetero-dimerization of CXCL12 and its receptors CXCR4 and CXCR7 alter their signaling activity. Only few drugs have been approved for clinical use targeting CXCL12-CXCR4/CXCR7 axis. Several CXCR4 inhibitors are in clinical trials for solid tumor treatment with limited success whereas CXCR7-specific inhibitors are still in preclinical studies for CRC. This review focuses on current knowledge of chemokine CXCL12 and its receptors CXCR4 and CXCR7, with emphasis on targeting the CXCL12–CXCR4/CXCR7 axis as a treatment strategy for CRC

Aberrant regulation of CXCR4 in cancer via deviant microRNA-targeted interactions

CXCR4 is involved in many facets of cancer, including being a major player in establishing metastasis. This is in part due to the deregulation of CXCR4, which can be attributed to many genetic and epigenetic mechanisms, including aberrant microRNA–CXCR4 interaction. MicroRNAs (miRNAs) are a type of small non-coding RNA that primarily targets the 3’ UTR of mRNA transcripts, which in turn suppresses mRNA and subsequent protein expression. In this review, we reported and characterized the many aberrant miRNA–CXCR4 interactions that occur throughout human cancers. In particular, we reported known target sequences located on the 3’ UTR of CXCR4 transcripts that tumour suppressor miRNAs bind and therefore regulate expression by. From these aberrant interactions, we also documented affected downstream genes/pathways and whether a particular tumour suppressor miRNA was reported as a prognostic marker in its respected cancer type. In addition, a limited number of cancer-causing miRNAs coined ‘oncomirs’ were reported and described in relation to CXCR4 regulation. Moreover, the mechanisms underlying both tumour suppressor and oncomir deregulations concerning CXCR4 expression were also explored. Furthermore, the miR-146a-CXCR4 axis was delineated in oncoviral infected endothelial cells in the context of virus-causing cancers. Lastly, miRNA-driven therapies and CXCR4 antagonist drugs were discussed as potential future treatment options in reported cancers pertaining to deregulated miRNA–CXCR4 interactions

Hepatocellular Carcinoma: The Role of MicroRNAs

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. HCC is diagnosed in its advanced stage when limited treatment options are available. Substantial morphologic, genetic and epigenetic heterogeneity has been reported in HCC, which poses a challenge for the development of a targeted therapy. In this review, we discuss the role and involvement of several microRNAs (miRs) in the heterogeneity and metastasis of hepatocellular carcinoma with a special emphasis on their possible role as a diagnostic and prognostic tool in the risk prediction, early detection, and treatment of hepatocellular carcinoma