Learning from Embryogenesis-A Comparative Expression Analysis in Melanoblast Differentiation and Tumorigenesis Reveals miRNAs Driving Melanoma Development.

Malignant melanoma is one of the most dangerous tumor types due to its high metastasis rates and a steadily increasing incidence. During tumorigenesis, the molecular processes of embryonic development, exemplified by epithelial-mesenchymal transition (EMT), are often reactivated. For melanoma development, the exact molecular differences between melanoblasts, melanocytes, and melanoma cells are not completely understood. In this study, we aimed to identify microRNAs (miRNAs) that promote melanoma tumorigenesis and progression, based on an in vitro model of normal human epidermal melanocyte (NHEM) de-differentiation into melanoblast-like cells (MBrCs). Using miRNA-sequencing and differential expression analysis, we demonstrated in this study that a majority of miRNAs have an almost equal expression level in NHEMs and MBrCs but are significantly differentially regulated in primary tumor- and metastasis-derived melanoma cell lines. Further, a target gene analysis of strongly regulated but functionally unknown miRNAs yielded the implication of those miRNAs in many important cellular pathways driving malignancy. We hypothesize that many of the miRNAs discovered in our study are key drivers of melanoma development as they account for the tumorigenic potential that differentiates melanoma cells from proliferating or migrating embryonic cells.

MicroRNA-sequencing data analyzing melanoma development and progression.

MicroRNAs (miRNAs) deregulated in melanoma are of growing importance in cancer research. We aimed to define the miRNAome of melanoma cell lines and primary melanocytes by RNA-Seq using identical cell lines as in a published miRNA expression study based on cDNA arrays. We identified 79 miRNAs, which are significantly deregulated during melanoma development. In addition, we could also determine 29 miRNAs being involved in melanoma progression. Interestingly, not all characterized miRNAs derived from cDNA array analyses of our and other groups could be found to be differentially expressed using RNA-Seq analyses, however, new miRNAs, formerly not associated with melanoma, were found to be strongly regulated.

Argonaute Family Protein Expression in Normal Tissue and Cancer Entities.

The members of the Argonaute (AGO) protein family are key players in miRNA-guided gene silencing. They enable the interaction between small RNAs and their respective target mRNA(s) and support the catalytic destruction of the gene transcript or recruit additional proteins for downstream gene silencing. The human AGO family consists of four AGO proteins (AGO1-AGO4), but only AGO2 harbors nuclease activity. In this study, we characterized the expression of the four AGO proteins in cancer cell lines and normal tissues with a new mass spectrometry approach called AGO-APP (AGO Affinity Purification by Peptides). In all analyzed normal tissues, AGO1 and AGO2 were most prominent, but marked tissue-specific differences were identified. Furthermore, considerable changes during development were observed by comparing fetal and adult tissues. We also identified decreased overall AGO expression in melanoma derived cell lines compared to other tumor cell lines and normal tissues, with the largest differences in AGO2 expression. The experiments described in this study suggest that reduced amounts of AGO proteins, as key players in miRNA processing, have impact on several cellular processes. Deregulated miRNA expression has been attributed to chromosomal aberrations, promoter regulation and it is known to have a major impact on tumor development and progression. Our findings will further increase our basic understanding of the molecular basis of miRNA processing and its relevance for disease.

Testing Suitability of Cell Cultures for SILAC-Experiments Using SWATH-Mass Spectrometry.

Precise quantification is a major issue in contemporary proteomics. Both stable-isotope-labeling and label-free methods have been established for differential protein quantification and both approaches have different advantages and disadvantages. The present protocol uses the superior precision of label-free SWATH-mass spectrometry to test for suitability of cell lines for a SILAC-labeling approach as systematic regulations may be introduced upon incorporation of the "heavy" amino acids. The SILAC-labeled cell cultures can afterwards be used for further analyses where stable-isotope-labeling is mandatory or has substantial advantages over label-free approaches such as pulse-chase-experiments and differential protein interaction analyses based on co-immunoprecipitation. As SWATH-mass spectrometry avoids the missing-value-problem typically caused by undersampling in highly complex samples and shows superior precision for the quantification, it is better suited for the detection of systematic changes caused by the SILAC-labeling and thus, can serve as a useful tool to test cell lines for changes upon SILAC-labeling.

MicroRNAs in malignant melanoma.

Melanoma is the most aggressive form of skin cancer, and the incidence of melanoma has been increasing faster than that of most other cancers. While the survival rate following surgical resection of early-stage primary tumors is nearly 100%, the survival of patients with metastasized tumors is strongly reduced, likely due to resistance to conventional therapies. Therefore, it is important to use new molecular approaches to develop new biomarkers to better prevent and diagnose melanoma. MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate gene expression via repression of translation or direct degradation of their complementary mRNA. In this review, we summarize our current understanding of the involvement of miRNAs and their corresponding targets in melanomagenesis as well as the potential use of miRNAs as biomarkers.

Structural insights into functional modes of proteins involved in ubiquitin family pathways.

The conjugation of ubiquitin and related modifiers to selected proteins represents a general mechanism to alter the function of these protein targets, thereby increasing the complexity of the cellular proteome. Ubiquitylation is catalyzed by a hierarchical enzyme cascade consisting of ubiquitin activating, ubiquitin conjugating, and ubiquitin ligating enzymes, and their combined action results in a diverse topology of ubiquitin-linkages on the modified proteins. Counteracting this machinery are various deubiquitylating enzymes while ubiquitin recognition in all its facets is accomplished by numerous ubiquitin-binding elements. In the following chapter, we attempt to provide an overview on enzymes involved in ubiquitylation as well as the removal of ubiquitin and proteins involved in the recognition and binding of ubiquitin from a structural biologist's perspective.

And yet it moves: active site remodeling in the SUMO E1.

The activation of ubiquitin and ubiquin-like proteins is catalyzed by E1 enzymes via consecutive adenylation and thioesterification reactions involving two apparently distant active sites. Olsen et al. (2010) uncover dramatic conformational changes in the SUMO E1 that spatially merge the adenylation and thioesterification active sites, including a > 30 A movement of the active site cysteine.