MiR-29b replacement inhibits proteasomes and disrupts aggresome+autophagosome formation to enhance the antimyeloma benefit of bortezomib

Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches. Although proteasome inhibitors (PIs) have transformed management of multiple myeloma (MM), drug resistance emerges through induction of the aggresome+autophagy pathway as a compensatory protein clearance mechanism. Genome-wide profiling identified microRNAs (miRs) differentially expressed in bortezomib-resistant myeloma cells compared with drug-naive cells. The effect of individual miRs on proteasomal degradation of short-lived fluorescent reporter proteins was then determined in live cells. MiR-29b was significantly reduced in bortezomib-resistant cells as well as in cells resistant to second-generation PIs carfilzomib and ixazomib. Luciferase reporter assays demonstrated that miR-29b targeted PSME4 that encodes the proteasome activator PA200. Synthetically engineered miR-29b replacements impaired the growth of myeloma cells, patient tumor cells and xenotransplants. MiR-29b replacements also decreased PA200 association with proteasomes, reduced the proteasome's peptidase activity and inhibited ornithine decarboxylase turnover, a proteasome substrate degraded through ubiquitin-independent mechanisms. Immunofluorescence studies revealed that miR-29b replacements enhanced the bortezomib-induced accumulation of ubiquitinated proteins but did not reveal aggresome or autophagosome formation. Taken together, our study identifies miR-29b replacements as the first-in-class miR-based PIs that also disrupt the autophagy pathway and highlight their potential to synergistically enhance the antimyeloma effect of bortezomib

NIK Stabilization in Osteoclasts Results in Osteoporosis and Enhanced Inflammatory Osteolysis

Maintenance of healthy bone requires the balanced activities of osteoclasts (OCs), which resorb bone, and osteoblasts, which build bone. Disproportionate action of OCs is responsible for the bone loss associated with postmenopausal osteoporosis and rheumatoid arthritis. NF-κB inducing kinase (NIK) controls activation of the alternative NF-κB pathway, a critical pathway for OC differentiation. Under basal conditions, TRAF3-mediated NIK degradation prevents downstream signaling, and disruption of the NIK:TRAF3 interaction stabilizes NIK leading to constitutive activation of the alternative NF-κB pathway.Using transgenic mice with OC-lineage expression of NIK lacking its TRAF3 binding domain (NT3), we now find that alternative NF-κB activation enhances not only OC differentiation but also OC function. Activating NT3 with either lysozyme M Cre or cathepsinK Cre causes high turnover osteoporosis with increased activity of OCs and osteoblasts. In vitro, NT3-expressing precursors form OCs more quickly and at lower doses of RANKL. When cultured on bone, they exhibit larger actin rings and increased resorptive activity. OC-specific NT3 transgenic mice also have an exaggerated osteolytic response to the serum transfer model of arthritis.Constitutive activation of NIK drives enhanced osteoclastogenesis and bone resorption, both in basal conditions and in response to inflammatory stimuli

Nod2 Downregulates TLR2/1 Mediated IL1β Gene Expression in Mouse Peritoneal Macrophages

Nod2 is a cytosolic pattern recognition receptor. It has been implicated in many inflammatory conditions. Its signaling has been suggested to modulate TLR responses in a variety of ways, yet little is known about the mechanistic details of the process. We show in this study that Nod2 knockdown mouse peritoneal macrophages secrete more IL1β than normal macrophages when stimulated with peptidoglycan (PGN). Muramyl dipeptide (MDP, a Nod2 ligand) + PGN co-stimulated macrophages have lower expression of IL1β than PGN (TLR2/1 ligand) stimulated macrophages. MDP co-stimulation have similar effects on Pam3CSK4 (synthetic TLR2/1 ligand) mediated IL1β expression suggesting that MDP mediated down regulating effects are receptor dependent and ligand independent. MDP mediated down regulation was specific for TLR2/1 signaling as MDP does not affect LPS (TLR4 ligand) or zymosan A (TLR2/6 ligand) mediated IL1β expression. Mechanistically, MDP exerts its down regulating effects by lowering PGN/Pam3CSK4 mediated nuclear cRel levels. Lower nuclear cRel level were observed to be because of enhanced transporting back rather than reduced nuclear translocation of cRel in MDP + PGN stimulated macrophages. These results demonstrate that Nod2 and TLR2/1 signaling pathways are independent and do not interact at the level of MAPK or NF-κB activation

A BAFF-R mutation associated with non-Hodgkin lymphoma alters TRAF recruitment and reveals new insights into BAFF-R signaling

A BAFF receptor mutation associated with non-Hodgkin lymphoma provides new insight into the proximal players of normal BAFF-R signaling

Features of teaching Russian as a foreign language on the basis of local history texts

The paper deals with topical issues of teaching Russian as a foreign language (RAFL) and the peculiarities of teaching, taking into account the Linguistic-cultural component. Linguistic-cultural component is considered as materials on regional studies, local history, history, culture and the basics of legislation. Mastering this material will allow students of RAFL courses to master a wide range of background knowledge about the country, traditions, etc. A typology of textbooks on RAFL is given. The most frequently used textbooks on RAFL are examined from the point of view of the material containing a linguistic-cultural component and features that must be taken into account when working with local history material

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death’ (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death’ (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death

A Reporter Screen in a Human Haploid Cell Line Identifies CYLD as a Constitutive Inhibitor of NF-κB

The development of forward genetic screens in human haploid cells has the potential to transform our understanding of the genetic basis of cellular processes unique to man. So far, this approach has been limited mostly to the identification of genes that mediate cell death in response to a lethal agent, likely due to the ease with which this phenotype can be observed. Here, we perform the first reporter screen in the near-haploid KBM7 cell line to identify constitutive inhibitors of NF-κB. CYLD was the only currently known negative regulator of NF-κB to be identified, thus uniquely distinguishing this gene. Also identified were three genes with no previous known connection to NF-κB. Our results demonstrate that reporter screens in haploid human cells can be applied to investigate the many complex signaling pathways that converge upon transcription factors

A Study of Nuclear Transcription Factor-Kappa B in Childhood Autism

BACKGROUND: Several children with autism show regression in language and social development while maintaining normal motor milestones. A clear period of normal development followed by regression and subsequent improvement with treatment, suggests a multifactorial etiology. The role of inflammation in autism is now a major area of study. Viral and bacterial infections, hypoxia, or medication could affect both foetus and infant. These stressors could upregulate transcription factors like nuclear factor kappa B (NF-κB), a master switch for many genes including some implicated in autism like tumor necrosis factor (TNF). On this hypothesis, it was proposed to determine NF-κB in children with autism. METHODS: Peripheral blood samples of 67 children with autism and 29 control children were evaluated for NF-κB using electrophoretic mobility shift assay (EMSA). A phosphor imaging technique was used to quantify values. The fold increase over the control sample was calculated and statistical analysis was carried out using SPSS 15. RESULTS: We have noted significant increase in NF-κB DNA binding activity in peripheral blood samples of children with autism. When the fold increase of NF-κB in cases (n = 67) was compared with that of controls (n = 29), there was a significant difference (3.14 vs. 1.40, respectively; p<0.02). CONCLUSION: This finding has immense value in understanding many of the known biochemical changes reported in autism. As NF-κB is a response to stressors of several kinds and a master switch for many genes, autism may then arise at least in part from an NF-κB pathway gone awry

HOIL-1L Interacting Protein (HOIP) as an NF-κB Regulating Component of the CD40 Signaling Complex

The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-κB activation through the ubiquitin-dependent activation of IKKγ. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-κB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling

Inhibition of 26S Protease Regulatory Subunit 7 (MSS1) Suppresses Neuroinflammation

Recently, researchers have focused on immunosuppression induced by rifampicin. Our previous investigation found that rifampicin was neuroprotective by inhibiting the production of pro-inflammatory mediators, thereby suppressing microglial activation. In this study, using 2-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we discovered that 26S protease regulatory subunit 7 (MSS1) was decreased in rifampicin-treated microglia. Western blot analysis verified the downregulation of MSS1 expression by rifampicin. As it is indicated that the modulation of the ubiquitin-26S proteasome system (UPS) with proteasome inhibitors is efficacious for the treatment of neuro-inflammatory disorders, we next hypothesized that silencing MSS1 gene expression might inhibit microglial inflammation. Using RNA interference (RNAi), we showed significant reduction of IkBα degradation and NF-kB activation. The production of lipopolysaccharides-induced pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide, cyclooxygenase-2, and prostaglandin E2 were also reduced by MSS1 gene knockdown. Taken together, our findings suggested that rifampicin inhibited microglial inflammation by suppressing MSS1 protein production. Silencing MSS1 gene expression decreased neuroinflammation. We concluded that MSS1 inhibition, in addition to anti-inflammatory rifampicin, might represent a novel mechanism for the treatment of neuroinflammatory disorders