Melanoma in advanced stages is one of the most aggressive tumors and the deadliest of skin cancers. To date, the histopathological staging focuses on tumor thickness, and clinical staging is a major estimate of the clinical behavior of primary melanoma. Here we report on an observational study with in-depth molecular profiling at the protein level including post-translational modifications (PTMs) on eleven primary tumors from melanoma patients. Global proteomics, phosphoproteomics, and acetylomics were performed on each sample. We observed an up-regulation of key mitochondrial functions, including the mitochondrial translation machinery and the down-regulation of structural proteins involved in cell adhesion, the cytoskeleton organization, and epidermis development, which dictates the progression of the disease. Additionally, the PTM level pathways related to RNA processing and transport, as well as chromatin organization, were dysregulated in relation to the progression of melanoma. Most of the pathways dysregulated in this cohort were enriched in genes differentially expressed at the transcript level when similar groups are compared or metastasis to primary melanomas. At the genome level, we found significant differences in the mutation profiles between metastatic and primary melanomas. Our findings also highlighted sex-related differences in the molecular profiles. Remarkably, primary melanomas in women showed higher levels of antigen processing and presentation, and activation of the immune system response. Our results provide novel insights, relevant for developing personalized precision treatments for melanoma patients.

Cryo-electron tomography (cryo-ET) enables the three-dimensional (3D) structural characterization of macromolecular complexes in their physiological environment. Thus, cryo-ET is uniquely suited to study the structural basis of biomolecular processes that are extremely difficult or even impossible to reconstitute using purified components. Translation of mitochondrial genes, which occurs in the secluded interior of mitochondria, falls into this category. Here, we describe the principles of cryo-ET in the context of mitochondrial translation and outline recent developments and challenges of the method. The 3D image of a frozen-hydrated biological sample is computed from its 2D projections, which are acquired using a transmission electron microscope. In conjunction with automated detection of different copies of the molecule of interest and averaging of the corresponding subtomograms, cryo-ET enables macromolecular structure determination in the native environment (i.e. in situ) at sub-nanometer resolution. The preservation of the native environment furthermore allows the extraction of contextual information about the molecules, including the location of specific molecules with respect to membranes, their relative positioning and the spatial organization with respect to other types of macromolecules. Recent preparative developments extend the field of application of cryo-ET from isolated organelles to cultured eukaryotic cells and even tissue, making the traditional borders between molecular and cellular structural biology disappear.

Mitochondrial (mt) tRNA gene mutations are an important cause of human morbidity and are associated with different syndromes. We have previously shown that the mitochondrial protein synthesis elongation factor EF-Tu and isolated sequences from the carboxy-terminal domain of yeast and human mt leucyl-tRNA synthetases (LeuRS), have a wide range of suppression capability among different yeast mt tRNA mutants having defective respiratory phenotype. Here we show that the rescuing capability can be restricted to a specific sequence of six amino acids from the carboxy-terminal domain of mt LeuRS. On the other hand by overexpressing a mutated version of mt EF-Tu in a yeast strain deleted for the endogenous nuclear gene we identified the specific region involved in suppression. Results support the possibility that a small peptide could correct defects associated with many mt tRNA mutations, suggesting a novel therapy for mitochondrial diseases treatment. The involvement of the mt EF-Tu in cellular heat stress response has also been suggested.