Martin Piskacek

Functionally Suppressive CD8 T Regulatory Cells Are Increased in Patients with Multiple Myeloma: A Cause for Immune Impairment

Background Multiple myeloma (MM) is a plasma cell malignancy frequently associated with impaired immune cell numbers and functions. In MM, several studies have previously shown that CD4 regulatory T (Treg) cells hamper effector T cell functions and enhance immune dysfunction. In this study, we aimed to prove the presence of functionally suppressive Treg cells expressing CD8 phenotype (CD8 Treg cells) in MM. To the best of our knowledge, this has not been reported previously in MM. Methods We analyzed CD8 Treg cells and their transcription factor FoxP3 from 64 newly diagnosed MM patients using flow cytometry and real time-polymerase chain reaction (RT-PCR). RNA profile of cytokines in CD8 Treg cells was also assessed using RT-PCR. CD8 Treg cells from 5 MM patients and 5 healthy donors were functionally evaluated using proliferation assays. Results CD8 Treg cells (CD8+CD25hi+) were significantly elevated in MM patients (P<0.0001), and their transcription factor FoxP3 expression was also higher in MM (P<0.0001) compared to healthy donors which was evidenced by flow cytometry and RT-PCR analyses. CD8 Treg cells negatively correlated with total lymphocyte count (P = 0.016). Functional studies revealed that CD8 Treg cells isolated from MM patients and healthy donors inhibited proliferation of CD4 T cells in a concentration dependent manner. In the presence of CD8 Treg cells in proliferation assays, level of IFN-γ was decreased but not IL-10. CD4 T cells from MM patients secreted abnormal level of IL-10 compared to healthy donors (P = 0.01) in proliferation assays without CD8 Treg cells. RNA profile of cytokines from CD8 Treg cells did not differ significantly between MM patients and healthy donors. Conclusions These findings show the presence of increased number of functionally suppressive CD8 Treg cells in MM patients. We believe that these suppressive CD8 Treg cells might enhance immune impairment and disease progression in MM.

The 9aaTAD Is Exclusive Activation Domain in Gal4.

The Gal4 protein is a well-known prototypic acidic activator that has multiple activation domains. We have previously identified a new activation domain called the nine amino acid transactivation domain (9aaTAD) in Gal4 protein. The family of the 9aaTAD activators currently comprises over 40 members including p53, MLL, E2A and other members of the Gal4 family; Oaf1, Pip2, Pdr1 and Pdr3. In this study, we revised function of all reported Gal4 activation domains. Surprisingly, we found that beside of the activation domain 9aaTAD none of the previously reported activation domains had considerable transactivation potential and were not involved in the activation of transcription. Our results demonstrated that the 9aaTAD domain is the only decisive activation domain in the Gal4 protein. We found that the artificial peptides included in the original Gal4 constructs were results of an unintended consequence of cloning that were responsible for the artificial transcriptional activity. Importantly, the activation domain 9aaTAD, which is the exclusive activation domain in Gal4, is also the central part of a conserved sequence recognized by the inhibitory protein Gal80. We propose a revision of the Gal4 regulation, in which the activation domain 9aaTAD is directly linked to both activation function and Gal80 mediated inhibition.

Do human B-lymphocytes avoid aging until 60 years?

Broad changes in human innate and adaptive immunity are associated with advanced age. The age-related alteration of gene expression was reported for both T and B lymphocytes. We analysed the genome-wide expression profiles (n=20) of naive and whole B cell populations from young and early aged healthy donors under 60 years. We revealed large homogeneity of all analysed genome-wide expression profiles but did not identified any significant gene deregulation between young (30-45 years) and early aged healthy donors (50-60 years). We argue that B cells avoid the aging program on molecular level until 60 years of age. Our results demonstrate the potential of hematopoietic stem cells to generate uncompromised lymphocytes in early elderly. These are very encouraging findings for the general health and the immunity maintenance would not need any intervention to naive B cells. Rather, a suitable immune stimulation in healthy body environment warrants further research into aging of older elderly.

How to overcome the ATRA resistance with the 9aaTAD activation domains in retinoic acid receptors

In higher metazoa, the nuclear hormone receptors activate transcription trough their specific adaptors, nuclear hormone receptor cofactors NCoA, which are surprisingly absent in lower metazoa. In this study, we demonstrated that the 9aaTAD from NHR-49 receptor activates transcription as a small peptide. We showed, that the 9aaTAD domains are conserved in the human nuclear hormone receptors including HNF4, RARa, VDR and PPARg. The small 9aaTAD peptides derived from these nuclear hormone receptors also effectively activated transcription and that in absence of the NCoA adaptors. We identified adjacent inhibitory domains in the human HNF4 and RARa, which hindered their activation function. In acute promyelocytic leukaemia (PML-RARa), the receptor mutations often caused all-trans retinoic acid (ATRA) resistance. The fact that almost the entire receptor is needed for ATRA mediated receptor activation, this activation pathway is highly susceptible for loss of function when mutated. Nevertheless in the most of the reported mutants, the activation domains 9aaTAD are still intact. The release of activation 9aaTAD from its dormancy by a new drug might be the sound strategy in combat the ATRA resistance in PML leukaemia. Graphical Abstract

The inositol phosphatase SHIP2 enables sustained ERK activation downstream of FGF receptors by recruiting Src kinases

The scaffold function of the inositol phosphatase SHIP2 mediates sustained ERK signaling downstream of FGF receptors. Converting transient to sustained signaling Activation of fibroblast growth factor receptors (FGFRs) stimulates downstream signaling transiently because the receptors are endocytosed and degraded after activation. Nevertheless, FGFRs stimulate both sustained and transient ERK signaling. Fafilek et al. found that the inositol phosphatase SHIP2 was required for converting transient FGFR activation into sustained ERK signaling. The catalytic activity of SHIP2 was not required. Instead, SHIP2 acted as a scaffold that recruited Src family kinases to FGFR complexes, thus enhancing the phosphorylation of adaptor proteins that mediated signal relay from FGFRs to ERK. Because sustained ERK activation due to aberrant FGFR signaling is associated with oncogenesis and developmental disorders, SHIP2 may be a potential therapeutic target for these pathologies. Sustained activation of extracellular signal–regulated kinase (ERK) drives pathologies caused by mutations in fibroblast growth factor receptors (FGFRs). We previously identified the inositol phosphatase SHIP2 (also known as INPPL1) as an FGFR-interacting protein and a target of the tyrosine kinase activities of FGFR1, FGFR3, and FGFR4. We report that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued cell phenotypes triggered by pathologic FGFR-ERK signaling. Mutant forms of SHIP2 lacking phosphoinositide phosphatase activity still associated with FGFRs and did not prevent FGF-induced sustained ERK activation, demonstrating that the adaptor rather than the catalytic activity of SHIP2 was required. SHIP2 recruited Src family kinases to the FGFRs, which promoted FGFR-mediated phosphorylation and assembly of protein complexes that relayed signaling to ERK. SHIP2 interacted with FGFRs, was phosphorylated by active FGFRs, and promoted FGFR-ERK signaling at the level of phosphorylation of the adaptor FRS2 and recruitment of the tyrosine phosphatase PTPN11. Thus, SHIP2 is an essential component of canonical FGF-FGFR signal transduction and a potential therapeutic target in FGFR-related disorders.

Gal4 activation domain 9aaTAD could be inactivated by adjacent mini-inhibitory domain and reactivated by distal re-activation domain

The characterisation of the activation domains started three decades ago with Gcn4 and Gal4 activators. The amorphous character of the activation domains strongly hindered their definition. Moreover, during the attempts to localise the Gal4 activation domain, the artificial peptides, an unintended consequence of cloning, were responsible for artificial transcriptional activity of the several Gal4 constructs. These artefacts produced enormous experimental bias and misconception. The presence of inhibitory domains in some Gal4 constructs made the misperception even worse. Previously, we reported that the nine amino acid transactivation domain, 9aaTAD, is the exclusive activation domain in the Gal4 protein. The activation domain 9aaTAD could be identified in Gal4 paralogs Oaf1, Pip2, Pdr1, Pdr3 and other activators p53, E2A and MLL. Surprisingly, the activation domain 9aaTAD was reported as misconception for Gal4 activator. Here we demonstrated that small region of 10 amino acids adjacent to the Gal4 activation domain 9aaTAD is an inhibitory domain, which the authors included in their constructs. Moreover, we identified Gal4 region, which was able to the reverse the inhibitory effect. The 9aaTAD re-activation domain was localized to the 13 amino acid long region. In this report we clarified the numerous confusions and rebutted supposed 9aaTAD misconception. Summary The activation domain 9aaTAD has decisive function in Gal4 activation. Gal4 activation domain 9aaTAD could be inhibited by adjacent region of 10 amino acids. The inhibited Gal4 activation domain 9aaTAD could be reactivated by 13 amino acid long Gal4 region. The activation domains 9aaTAD could be identified by our 9aaTAD prediction algorithm, especially in the Gal4 family.