Malgorzata Palczewska

Day-Night Changes in Downstream Regulatory Element Antagonist Modulator/Potassium Channel Interacting Protein Activity Contribute to Circadian Gene Expression in Pineal Gland

The molecular mechanisms controlling the oscillatory synthesis of melatonin in rat pineal gland involve the rhythmic expression of several genes including arylalkylamine N-acetyltransferase (AA-NAT), inducible cAMP early repressor (ICER), and Fos-related antigen-2 (fra-2). Here we show that the calcium sensors downstream regulatory element antagonist modulator/potassium channel interacting protein (DREAM/KChIP)-3 and KChIP-1, -2 and -4 bind to downstream regulatory element (DRE) sites located in the regulatory regions of these genes and repress basal and induced transcription from ICER, fra-2 or AA-NAT promoters. Importantly, we demonstrate that the endogenous binding activity to DRE sites shows day-night oscillations in rat pineal gland and retina but not in the cerebellum. The peak of DRE binding activity occurs during the day period of the circadian cycle, coinciding with the lowest levels of fra-2, ICER, and AA-NAT transcripts. We show that a rapid clearance of DRE binding activity during the entry in the night period is related to changes at the posttranscriptional level of DREAM/KChIP. The circadian pattern of DREAM/KChIP activity is maintained under constant darkness, indicating that an endogenous clock controls DREAM/KChIP function. Our data suggest involvement of the family of DREAM repressors in the regulation of rhythmically expressed genes engaged in circadian rhythms.

Transcriptional repressor DREAM regulates T-lymphocyte proliferation and cytokine gene expression

Downstream Regulatory Element Antagonist Modulator (DREAM) is a Ca2+‐dependent transcriptional repressor expressed in the brain, thyroid gland and thymus. Here, we analyzed the function of DREAM and the related protein KChIP‐2 in the immune system using transgenic (tg) mice expressing a cross‐dominant active mutant (EFmDREAM) for DREAM and KChIPs Ca2+‐dependent transcriptional derepression. EFmDREAM tg mice showed reduced T‐cell proliferation. Tg T cells exhibited decreased interleukin (IL)‐2, ‐4 and interferon (IFN)γ production after polyclonal activation and following antigen‐specific response. Chromatin immunoprecipitation and transfection assays showed that DREAM binds to and represses transcription from these cytokine promoters. Importantly, specific transient knockdown of DREAM or KChIP‐2 induced basal expression of IL‐2 and IFNγ in wild‐type splenocytes. These data propose DREAM and KChIP‐2 as Ca2+‐dependent repressors of the immune response.

Sumoylation regulates nuclear localization of repressor DREAM

DREAM is a Ca(2+)-binding protein with specific functions in different cell compartments. In the nucleus, DREAM acts as a transcriptional repressor, although the mechanism that controls its nuclear localization is unknown. Yeast two-hybrid assay revealed the interaction between DREAM and the SUMO-conjugating enzyme Ubc9 and bioinformatic analysis identified four sumoylation-susceptible sites in the DREAM sequence. Single K-to-R mutations at positions K26 and K90 prevented in vitro sumoylation of recombinant DREAM. DREAM sumoylation mutants retained the ability to bind to the DRE sequence but showed reduced nuclear localization and failed to regulate DRE-dependent transcription. In PC12 cells, sumoylated DREAM is present exclusively in the nucleus and neuronal differentiation induced nuclear accumulation of sumoylated DREAM. In fully differentiated trigeminal neurons, DREAM and SUMO-1 colocalized in nuclear domains associated with transcription. Our results show that sumoylation regulates the nuclear localization of DREAM in differentiated neurons. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Matrix vesicles isolated from mineralization-competent Saos-2 cells are selectively enriched with annexins and S100 proteins.

Matrix vesicles (MVs) are cell-derived membranous entities crucial for mineral formation in the extracellular matrix. One of the dominant groups of constitutive proteins present in MVs, recognised as regulators of mineralization in norm and pathology, are annexins. In this report, besides the annexins already described (AnxA2 and AnxA6), we identified AnxA1 and AnxA7, but not AnxA4, to become selectively enriched in MVs of Saos-2 cells upon stimulation for mineralization. Among them, AnxA6 was found to be almost EGTA-non extractable from matrix vesicles. Moreover, our report provides the first evidence of annexin-binding S100 proteins to be present in MVs of mineralizing cells. We observed that S100A10 and S100A6, but not S100A11, were selectively translocated to the MVs of Saos-2 cells upon mineralization. This observation provides the rationale for more detailed studies on the role of annexin-S100 interactions in MV-mediated mineralization.

Diffusion nuclear magnetic resonance spectroscopy detects substoichiometric concentrations of small molecules in protein samples

Small molecules are difficult to detect in protein solutions, whether they originate from elution during affinity chromatography (e.g., imidazole, lactose), buffer exchange (Tris), stabilizers (e.g., beta-mercaptoethanol, glycerol), or excess labeling reagents (fluorescent reagents). Mass spectrometry and high-pressure liquid chromatography (HPLC) often require substantial efforts in optimization and sample manipulation to provide sufficient sensitivity and reliability for their detection. One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) could, in principle, detect residual amounts of small molecules in protein solutions down to equimolecular concentrations with the protein. However, at lower concentrations, the NMR signals of the contaminants can be hidden in the background spectrum of the protein. As an alternative, the 1D diffusion difference protocol used here is feasible. It even improves the detection level, picking up NMR signals from small-molecule contaminants at lower concentrations than the protein itself. We successfully observed 30 microM imidazole in the presence of four different proteins (1-1.5 mg/ml, 6-66 kDa, 25-250 microM) by 1D diffusion-ordered spectroscopy (DOSY) difference and 1-h total acquisition time. Of note, imidazole was not detected in the corresponding 1D (1)H NMR spectra. This protocol can be adapted to different sample preparation procedures and NMR acquisition methods with minimal manipulation in either deuterated or nondeuterated buffers.

Transcriptional repressor DREAM regulates trigeminal noxious perception.

Expression of the downstream regulatory element antagonist modulator (DREAM) protein in dorsal root ganglia and spinal cord is related to endogenous control mechanisms of acute and chronic pain. In primary sensory trigeminal neurons, high levels of endogenous DREAM protein are preferentially localized in the nucleus, suggesting a major transcriptional role. Here, we show that transgenic mice expressing a dominant active mutant of DREAM in trigeminal neurons show increased responses following orofacial sensory stimulation, which correlates with a decreased expression of prodynorphin and brain‐derived neurotrophic factor in trigeminal ganglia. Genome‐wide analysis of trigeminal neurons in daDREAM transgenic mice identified cathepsin L and the monoglyceride lipase as two new DREAM transcriptional targets related to pain. Our results suggest a role for DREAM in the regulation of trigeminal nociception.

Stimulators of mineralization limit the invasive phenotype of human osteosarcoma cells by a mechanism involving impaired invadopodia formation.

Background Osteosarcoma (OS) is a highly aggressive bone cancer affecting children and young adults. Growing evidence connects the invasive potential of OS cells with their ability to form invadopodia (structures specialized in extracellular matrix proteolysis). Results In this study, we tested the hypothesis that commonly used in vitro stimulators of mineralization limit the invadopodia formation in OS cells. Here we examined the invasive potential of human osteoblast-like cells (Saos-2) and osteolytic-like (143B) OS cells treated with the stimulators of mineralization (ascorbic acid and B-glycerophosphate) and observed a significant difference in response of the tested cells to the treatment. In contrast to 143B cells, osteoblast-like cells developed a mineralization phenotype that was accompanied by a decreased proliferation rate, prolongation of the cell cycle progression and apoptosis. On the other hand, stimulators of mineralization limited osteolytic-like OS cell invasiveness into collagen matrix. We are the first to evidence the ability of 143B cells to degrade extracellular matrix to be driven by invadopodia. Herein, we show that this ability of osteolytic-like cells in vitro is limited by stimulators of mineralization. Conclusions Our study demonstrates that mineralization competency determines the invasive potential of cancer cells. A better understanding of the molecular mechanisms by which stimulators of mineralization regulate and execute invadopodia formation would reveal novel clinical targets for treating osteosarcoma.

Characterization of calretinin I–II as an EF-hand, Ca2+, H+-sensing domain

Calretinin, a neuronal protein with well‐defined calcium‐binding properties, has a poorly defined function. The pH dependent properties of calretinin (CR), the N‐terminal (CR I–II), and C‐terminal (CR III–VI) domains were investigated. A drop in pH within the intracellular range (from pH 7.5 to pH 6.5) leads to an increased hydrophobicity of calcium‐bound CR and its domains as reported by fluorescence spectroscopy with the hydrophobic probe 2‐(p‐toluidino)‐6‐naphthalenesulfonic acid (TNS). The TNS data for the N‐ and C‐terminal domains of CR are additive, providing further support for their independence within the full‐length protein. Our work concentrated on CR I–II, which was found to have hydrophobic properties similar to calmodulin at lower pH. The elution of CR I–II from a phenyl‐Sepharose column was consistent with the TNS data. The pH‐dependent structural changes were further localized to residues 13–28 and 44–51 using nuclear magnetic resonance spectroscopy chemical shift analysis, and there appear to be no large changes in secondary structure. Protonation of His 12 and/or His 27 side chains, coupled with calcium chelation, appears to lead to the organization of a hydrophobic pocket in the N‐terminal domain. CR may sense and respond to calcium, proton, and other signals, contributing to conflicting data on the proteins role as a calcium sensor or calcium buffer.

NMR Investigations of Lectin—Carbohydrate Interactions

Publisher Summary Lectins are proteins that interact with carbohydrate ligands and often occur in oligomeric forms. Lectins are rarely suitable subjects for NMR structural studies due to their size. Carbohydrate ligands also provide challenges to the NMR spectroscopist. The poor dispersion of signals, compared to similar sized molecules, and the resulting overlap of resonances results in an ambiguity in data assignment. In other words, lectin—carbohydrate complexes present significant challenges to 3D structure determination for which NMR is a well-known tool. Although highly detailed 3D lectin—carbohydrate structures still cannot be routinely obtained, a wide range of other useful information about the binding site and binding processes are available by NMR methods. This chapter is aimed at enlightening researchers without a specialized knowledge of the NMR spectroscopy field. It introduces basic principles that govern NMR, and describes methods to obtain recombinant proteins suitable for NMR studies. The simplest NMR experiments that allow the measurement of chemical shifts of lectin and/or ligand during a titration to yield binding data are discussed. The assignment of the chemical shift data can provide an insight into structural changes, or the location of the binding site, although this is best done after obtaining a fuller 3D picture of the complex. Under the right conditions, it is possible to obtain data that specifically detail the bound conformation of the ligand.