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The Major Histocompatibility Complex class I (MHC-I) molecules present antigenic peptides (AP) to CD8+T cells for self versus non-self recognition. Loading of AP on MHC-I takes place in the endoplasmic reticulum (ER), upon shuttling of cytoplasmic AP substrates to the ER. Understanding of this process has been influenced by the view that MHC-I antigens are produced from the proteasomal degradation of cellular proteins. Recent observations on the intronic and untranslated region-derived peptides as well as on the non-AUG translation products presented on the MHC-I open the possibility that antigenic peptides can derive from pre-spliced mRNAs translated in the nuclear compartment. In this brief report, we show that a fraction of human MHC-I molecules (human leukocyte antigens type A, HLA-A) is present in the nuclei of cells, in the proximity of histone H2B. With this finding, we hope to initiate a new direction of research on the nuclear role of MHC-I and ask whether the loading of antigens can take place in the nuclear compartment.

RNA editing is one of the most prevalent and abundant forms of post-transcriptional RNA modification observed in normal physiological processes and often aberrant in diseases including cancer. RNA editing changes the sequences of mRNAs, making them different from the source DNA sequence. Edited mRNAs can produce editing-recoded protein isoforms that are functionally different from the corresponding genome-encoded protein isoforms. The major type of RNA editing in mammals occurs by enzymatic deamination of adenosine to inosine (A-to-I) within double-stranded RNAs (dsRNAs) or hairpins in pre-mRNA transcripts. Enzymes that catalyse these processes belong to the adenosine deaminase acting on RNA (ADAR) family. The vast majority of knowledge on the RNA editing landscape relevant to human disease has been acquired using in vitro cancer cell culture models. The limitation of such in vitro models, however, is that the physiological or disease relevance of results obtained is not necessarily obvious. In this review we focus on discussing in vivo occurring RNA editing events that have been identified in human cancer tissue using samples surgically resected or clinically retrieved from patients. We discuss how RNA editing events occurring in tumours in vivo can identify pathological signalling mechanisms relevant to human cancer physiology which is linked to the different stages of cancer progression including initiation, promotion, survival, proliferation, immune escape and metastasis.

Natural killer cells are innate lymphocytes with the ability to lyse tumour cells depending on the balance of their activating and inhibiting receptors. Growing numbers of clinical trials show promising results of NK cell-based immunotherapies. Unlike T cells, NK cells can lyse tumour cells independent of antigen presentation, based simply on their activation and inhibition receptors. Various strategies to improve NK cell-based therapies are being developed, all with one goal: to shift the balance to activation. In this review, we discuss the current understanding of ways NK cells can lyse tumour cells and all the inhibitory signals stopping their cytotoxic potential.

In recent years, much research has been focused on the field of adoptive cell therapies (ACT) that use native or genetically modified T cells as therapeutic tools. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) demonstrated great success in the treatment of haematologic malignancies, whereas adoptive transfer of autologous tumour infiltrating lymphocytes (TILs) proved to be highly effective in metastatic melanoma. These encouraging results initiated many studies where ACT was tested as a treatment for various solid tumours. In this review, we provide an overview of the challenges of T cell-based immunotherapies of solid tumours. We describe alternative approaches for choosing the most efficient T cells for cancer treatment in terms of their tumour-specificity and phenotype. Finally, we present strategies for improvement of anti-tumour potential of T cells, including combination therapies.

Proteomics of human tissues and isolated cellular subpopulations create new opportunities for therapy and monitoring of a patients’ treatment in the clinic. Important considerations in such analysis include recovery of adequate amounts of protein for analysis and reproducibility in sample collection. In this study we compared several protocols for proteomic sample preparation: i) filter-aided sample preparation (FASP), ii) in-solution digestion (ISD) and iii) a pressure-assisted digestion (PCT) method. PCT method is known for already a decade [1], however it is not widely used in proteomic research. We assessed protocols for proteome profiling of isolated immune cell subsets and formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that the ISD method has very good efficiency of protein and peptide identification from the whole proteome, while the FASP method is particularly effective in identification of membrane proteins. Pressure-assisted digestion methods generally provide lower numbers of protein/peptide identifications, but have gained in popularity due to their shorter digestion time making them considerably faster than for ISD or FASP. Furthermore, PCT does not result in substantial sample loss when applied to samples of 50 000 cells. Analysis of FFPE tissues shows comparable results. ISD method similarly yields the highest number of identifications. Furthermore, proteins isolated from FFPE samples show a significant reduction of cleavages at lysine sites due to chemical modifications with formaldehyde-such as methylation (+14 Da) being among the most common. The data we present will be helpful for making decisions about the robust preparation of clinical samples for biomarker discovery and studies on pathomechanisms of various diseases.

Small heat shock proteins are ubiquitous molecular chaperones that, during cellular stress, bind to misfolded proteins and maintain them in a refolding competent state. Two members of the small heat shock protein family, IbpA and IbpB, are present in Escherichia coli. Despite 48% sequence identity, the proteins have distinct activities in promoting protein disaggregation. Cooperation between IbpA and IbpB is crucial for prevention of the irreversible aggregation of proteins. In this study, we investigated the importance of the N- and C-terminal regions of IbpA for self-oligomerization and chaperone functions. Deletion of either the N- or C-terminal region of IbpA resulted in a defect in the IbpA fibril formation process. The deletions also impaired IbpA chaperone function, defined as the ability to stabilize, in cooperation with IbpB, protein aggregates in a disaggregation-competent state. Our results show that the defect in chaperone function, observed in truncated versions of IbpA, is due to the inability of these proteins to interact with substrate proteins and consequently to change the properties of aggregates. At the same time, these versions of IbpA interact with IbpB similarly to the wild type protein. Competition experiments performed with the pC peptide, which corresponds to the IbpA C terminus, suggested the importance of IbpA intermolecular interactions in the stabilization of aggregates in a state competent for disaggregation. Our results suggest that these interactions are not only dependent on the universally conserved IEI motif but also on arginine 133 neighboring the IEI motif. IbpA mutated at arginine 133 to alanine lacked chaperone activity.

Hsp100 chaperones cooperate with the Hsp70 chaperone system to disaggregate and reactivate heat-denatured aggregated proteins to promote cell survival after heat stress. The homology models of Hsp100 disaggregases suggest the presence of a conserved network of ionic interactions between the first nucleotide binding domain (NBD1) and the coiled-coil middle subdomain, the signature domain of disaggregating chaperones. Mutations intended to disrupt the putative ionic interactions in yeast Hsp104 and bacterial ClpB disaggregases resulted in remarkable changes of their biochemical properties. These included an increase in ATPase activity, a significant increase in the rate of in vitro substrate renaturation, and partial independence from the Hsp70 chaperone in disaggregation. Paradoxically, the increased activities resulted in serious growth impediments in yeast and bacterial cells instead of improvement of their thermotolerance. Our results suggest that this toxic activity is due to the ability of the mutated disaggregases to unfold independently from Hsp70, native folded proteins. Complementary changes that restore particular salt bridges within the suggested network suppressed the toxic effects. We propose a novel structural aspect of Hsp100 chaperones crucial for specificity and efficiency of the disaggregation reaction.