Current Proteomics - Volume 14, Issue 2, 2017

Diabetic neuropathy is the most common complication of diabetes mellitus and a major factor associated with foot ulcers and amputations in patients with diabetes. Diagnosing diabetic neuropathy in early stages may reduce the risk for comorbidities. However, it still represents a major challenge for the healthcare providers, despite the complexity of available techniques. Discovering new serum biomarkers, useful for early diagnosis of diabetic neuropathy and monitoring the disease progression is of growing interest for the scientific research. In this regard, the field of proteomics is extremely promising. Mass spectrometry technology and bioinformatic analysis of proteomic data accelerated the detection of biomarkers related to peripheral nervous system pathology. Herein, we analyze what role proteomics approaches play in the modern diagnostic strategy of diabetic neuropathy.

Background: Melanoma is an aggressive malignancy and its increasing incidence worldwide has boosted interest in biomarker discovery in blood to monitor melanoma development, therapy efficacy, and to find new therapy targets. Our study aimed to discover candidate protein biomarkers that could have a double role, differentiate between melanoma patients and controls and distinguish patients with good clinical outcomes from patients with unfavorable one. Method: Melanoma patients in stages I-IV were followed-up for 5 years in specialized dermatology centers in southern Romania, from the years 2007 to 2015. Blood samples were drawn at diagnosis and at the time when the clinical outcome could be scored by the clinician as unfavorable or good. In order to obtain the best peptidic pattern of plasma, samples were fractionated using ProteoMinerTM Bead library and after fractionation subjected to SELDI-TOF-MS (surface-enhanced laser desorption/ ionization (SELDI) technology used with time-of-flight (TOF) mass spectrometers) analysis. Results: Nine peaks (p-values <0.01) in the peptide (mass) range were found up-regulated in melanoma patients. We found 3 peaks whose apparent relative abundances decreased in > 80% of patients with good outcomes, which was consistent with reverting to a proteomic profile more similar to controls. Conclusion: Mass spectrometry analysis of plasma can provide validated markers in development and treatment of cutaneous melanoma.

Background: Secretory production of heterologous proteins in bacterial hosts has been a topic of interest due to its various advantages. However, it is difficult to achieve because of process complexity and the need for selection of an appropriate signal peptide for each protein and host. IL- 11, marketed as Neumega®, is a multifunctional cytokine approved for platelet recovery in chemotherapy- induced thrombocytopenia. Objective: The in silico evaluation of 30 signal peptides for finding the best theoretical candidates for the secretory production of recombinant IL-11 in Escherichia coli. Methods: The prediction of signal peptide presence and location of cleavage sites were done by SignalP 4.1. Five signal peptides (ompT, npr, TorA, caf1M, and phoA) were excluded from further evaluations due to cleavage issues. Different physicochemical properties of signal peptides, which may affect protein secretion, were studied by ProtParam and PROSO II servers. Results/Conclusion: Computational analysis of the influencing factors identified TorT, sfmC, and ompC, respectively, as the best theoretical candidates for the secretory production of IL-11 in E. coli. However, in the experimental investigation, other influencing factors and a system biology approach should be considered.

Background: The chemokine receptors are an important subfamily of rhodopsin-like G protein-coupled receptors (GPCRs) and have fundamental roles in many physiological and pathological processes. Therefore, they are among the drug targets in pharmaceutical development. Objective: The binding of these receptors with their chemokines is complex. The present work aims to contribute to a better understanding of this complexity. Material and Methods: Several tools and statistical analysis were used on the composition of DNA and protein sequences of GPCRs including chemokine receptors. Results: Our study reveals that these receptors have an uneven chromosomal distribution where the majority are located on chromosome 3 and are encoded by multiexonic genes. The principal component analysis, followed by hierarchical clustering, demonstrates that the composition in small amino acids, the extracellular region and the gene size have the highest ability to explain variability in the dataset. Besides this, XCR1, CCR1, CCR10 and CCR7 are the elements that best express the groups. Finding Charge Clusters in Protein tool shows that negative clusters are localized only in the NTerminal segment then the positive clusters are localized in the C-Terminal domain, in the fifth and sixth transmembrane regions, more precisely at positions 208-209, 225-228. ScanProsite analysis detects two intra-domain biological signatures frequently found with only few exceptions: glycosylation and sulfation domains localized especially in the N-Terminal tail, containing specific amino acids such as Ser, Thr, Asn and Tyr. Conclusion: The identification of characteristics and classification of chemokine receptors are powerful approaches for discovering new features of their high-affinity interaction with chemokines.

Objective: Osteoporotic fracture is one of the most common health risks and aggravates the quality of life among postmenopausal women worldwide. In this study, osteoporosis-associated protein biomarkers were identified from urine of osteoporotic female Sprague-Dawley rats developed by ovariectomy. Method: Four months after the operation, the bone mineral density of the femur of ovariectomized rats was significantly lowered in comparison with that of the sham operated rats. The protein profiles of the urine samples collected from the sham, ovariectomized (OVX) and 2 month-old non-operated (Young) rats were compared by 2-D gel and MS spectrometry. Results: Proteins consistently expressed between Young and sham but differentially expressed in OVX rats were selected and identified. One down-regulated 21 kDa protein, superoxide dismutase (SOD), and 1 up-regulated 53-54 kDa protein, alph-1-antitrypsin (A1AT), were selected from urine of the ovariectomized rats by 2-D gel analysis. Further, a total of 30 with 19 up-regulated and 11- down-regulated proteins were selected by LC-MS analysis with more than 2-fold differences in spectral counts. The fact that SOD and A1AT are also listed in the 30 differential proteins suggests that our biomarker isolation procedure suitably represents osteoporosis-associated proteins in urine. Conclusion: Supporting the facts, the differential expressions of SOD and A1AT in urine could be validated by Western blotting. These urinary osteoporosis-associated proteins have high potentials to become candidates for non-invasive diagnosis of osteoporosis from urine.

Objectives: We have analyzed the influence of Amorphophallus konjac tuber (KONJAC) extracts on protein expression of mitochondrial in cell line SGC-7901, identified those differentially expressed by mass spectrometry, and built a network of differentially expressed proteins using software. This provided the basis for a study of the molecular mechanisms of the anti-gastric cancer effects. Method: The cell viability was evaluated by cell counting kit-8. Cell apoptosis assay was used to detect cell apoptosis. Two-dimensional gel electrophoresis and MALDI-TOF-MS analysis was applied to identify the differentially-expressed mitochondrial protein treatment with or without KONJAC extracts. Ingenuity Pathways Analysis (IPA) software was to analysis an interacting protein. Result: Proliferation of SGC-7901 cells was reduced and early apoptosis increased after treatment with KONJAC extracts. Two-dimensional gel electrophoresis and MALDI-TOF-MS analysis showed that 682 protein spots in control group and 643 protein spots in the KONJAC extract treated group. Twenty proteins were identified that showed 10 up-regulated and 10 down-regulated proteins in these arrays. Bioinformatic IPA network analysis showed that ubiquitin-conjugating enzyme 2(E2) was located in the center of the network. Thus, the inhibition of cell proliferation and promotion of apoptosis by KONJAC extracts may be through ubiquitin conjugating enzyme-mediated mitochondrial apoptotic pathways. Conclusion: These results indicate that KONJAC extract-induced apoptosis and inhibit proliferation in SGC-7901 cells is mediated by pathways involving in mitochondrial apoptotic, gene transcription, cell cycle, oxidative stress response and energy metabolism. KONJAC extracts could be promising as a treatment for gastric cancer.

Objectives: Due to the complexity of DNA-Repair Mechanisms in Plants, this work will give more insights to better understanding the dark DNA-repair mechanism through the interactome analysis of DDBs proteins in plants. Method: Bioinformatics tools had been used in this work to analyses DNA Damage-Binding proteins; MSA, Phylogenetic tree construction, 3-D structure prediction, Domain analysis, subcellular localization prediction, Interactome analysis and docking sites. Result: DDB1a and DDB1b are closely related, while DDB2 protein is on the other branch, which further confirms our previous results about the homology present between proteins DDB1a and DDB1b. We found two domains of AtDDB1a and AtDDB1b. These are MMS1_N and CPSF_A; 464 and 314 amino acids long, respectively. AtDDB2 was identified with ten domains – two zinc fingers and five WD40 domains with three simple regions interspersed between them. DDB1a and DDB1b interact with much more proteins when compared to DDB2. In fact, DDB2 shows only 3 high confidence interactions, two of which are with DDB1a and DDB1b. Interesting result is that the only additional protein DDB2 interacts with is CUL4, which is a protein involved in ubiquitination. Conclusion: This study shed new light to that puzzle through development and analysis of the interactome of DDB proteins. Most importantly, the interaction of E3-ligase-forming complex (AtDDB1 – CUL4 – CSA) was confirmed. This is extremely important since E3-ligase is a key player in DNA repair mechanism. In addition, beside the legendary role of DDBs proteins in DNA-repair mechanism, they play other important roles in negative regulation of abscisic acid ABA and ABA-mediated developmental responses, including inhibition of seed germination, seedling establishment, and root growth.

Background: Estrogen receptor-α positive breast cancer is the most common dreadful disease and leading cause of death among women. In majority of human breast cancer, the interactions between kinases and ERα are considered to be critical in signaling pathway. Many kinases are known to regulate ERα activity. Recently Lemur tyrosine kinase-3 was identified as predictive oncogenic ERα regulator with a vital role in endocrine resistance. The role of LMTK3 in ERα regulation can be known by studying the interactions between them. Objectives: To understand the transient interactions between ER-α and LMTK3 using computational technique. Method: ERα-LMTK3 complex structure was obtained using PatchDock server. The interacting residues and interface area between ERα and LMTK3 were identified using PDBsum. Molecular dynamics simulation was used to study the conformational dynamics of ERα-LMTK3 complex. Result: The approximate interface area of ERα-LMTK3 was found to be 3175 Å2 with atomic contact energy of 191.77 kcal/mol. PDBsum results revealed that some of the residues in C-terminal region of LMTK3 displayed non-bonding interactions with the residues in the phosphorylation sites (Ser104 and Ser106) of ERα. We noticed the total number of interface residues in ERα-LMTK3 complex to be 50 and the interface area for ERα as well as LMTK3 chain involved in interaction to be more than 2380 Å2. From conformational dynamics study, ERα-LMTK3 complex structure was found to be stable. Conclusion: The outcomes of the current study enhance the understanding of interactions between ERα and LMTK3 which are thought to be critical in signaling pathway in majority of human breast cancers.