Differential Scanning Fluorometry Signatures as Indicators of Enzyme Inhibitor Mode of Action: Case Study of Glutathione S-Transferase

Differential scanning fluorometry (DSF), also referred to as fluorescence thermal shift, is emerging as a convenient method to evaluate the stabilizing effect of small molecules on proteins of interest. However, its use in the mechanism of action studies has received far less attention. Herein, the ability of DSF to report on inhibitor mode of action was evaluated using glutathione S-transferase (GST) as a model enzyme that utilizes two distinct substrates and is known to be subject to a range of inhibition modes. Detailed investigation of the propensity of small molecule inhibitors to protect GST from thermal denaturation revealed that compounds with different inhibition modes displayed distinct thermal shift signatures when tested in the presence or absence of the enzyme's native co-substrate glutathione (GSH). Glutathione-competitive inhibitors produced dose-dependent thermal shift trendlines that converged at high compound concentrations. Inhibitors acting via the formation of glutathione conjugates induced a very pronounced stabilizing effect toward the protein only when GSH was present. Lastly, compounds known to act as noncompetitive inhibitors exhibited parallel concentration-dependent trends. Similar effects were observed with human GST isozymes A1-1 and M1-1. The results illustrate the potential of DSF as a tool to differentiate diverse classes of inhibitors based on simple analysis of co-substrate dependency of protein stabilization

Telomere length dynamics in normal individuals and in patients with hematopoietic stem cell-associated disorders

The telomere length in nucleated peripheral blood (PB) cells indirectly reflects the mitotic history of their precursors: the hematopoietic stem cells (HSCs). The average length of telomeres in PB leukocytes can be measured using fluorescence in situ hybridization and flow cytometry (flow FISH). We previously used flow FISH to characterize the age-related turnover of HSCs in healthy individuals. In this review, we describe results of recent flow FISH studies in patients with selected hematopoietic stem cell-associated disorders: chronic myelogeneous leukemia (CML) and several bone marrow failure syndromes. CML is characterized by a marked expansion of myeloid Philadelphia chromosome positive (Ph+) cells. Nevertheless, nonmalignant (Ph-) HSCs typically coexist in the bone marrow of CML patients. We analyzed the telomere length in > 150 peripheral blood leukocytes (PBLs) and bone marrow samples of patients with CML as well as samples of Ph- T-lymphocytes. Compared to normal controls, the overall telomere fluorescence in PBLs of patients with CML was significantly reduced. However, no telomere shortening was observed in Ph- T-lymphocytes. Patients in late chronic phase (CP) had significantly shorter telomeres than those assessed earlier in CP. Our data suggest that progressive telomere shortening is correlated with disease progression in CML. Within the group of patients with bone marrow failure syndromes, we only found significantly shortened telomeres (compared to age-adjusted controls) in granulocytes from patients with aplastic anemia (AA). Strikingly, the telomere length in granulocytes from AA patients who had recovered after immunosuppressive therapy (recAA) did not differ significantly from controls, whereas untreated patients and nonresponders with persistent severe pancytopenia (sAANR) showed marked and significant telomere shortening compared to healthy donors and patients with recAA. Furthermore, an inverse correlation between age-adjusted telomere length and peripheral blood counts was found in support of a model in which the degree of cytopenia and the amount of telomere shortening are correlated. These results support the concept of extensive proliferation of HSCs in subgroups of AA patients and suggest a potential use of telomere-length measurements as a prognostic tool in this group of disorders as well

Telomere length change plateaus at 4 years of age in Latino children: associations with baseline length and maternal change

Telomeres are the protective complexes at the end of chromosomes, required for genomic stability. Little is known about predictors of attrition in young children or the relationship between parental and child patterns of telomere change. Telomere length was assessed twice over one year, at 4 and at 5 years of age, in Latino preschool children (n = 77) and their mothers (n = 70) in whole blood leukocytes. Maternal and child rates of attrition during the same time period were compared in 70 mother-child pairs. More children showed lengthened telomeres over one year compared to their mothers and very few children showed attrition (2.6 %). Approximately 31 % of children and 16 % of mothers displayed lengthening over one year while 66 % of children showed maintenance in contrast with 74 % of mothers. The strongest predictor for child telomere length change was child's baseline telomere length (r = -0.61, p < 0.01). Maternal rate of change was associated with child rate of change (r = 0.33, p < 0.01). After controlling for child baseline telomere length, the relationship between child and maternal rate of change trended towards significance (Coeff = 0.20, 95 % CI -0.03 to 0.43; p = 0.08). We found primarily maintenance and lengthening from 4 to 5 years of age in children, with minimal telomere attrition, indicating that most of the telomere loss happens in the first 4 years, plateauing by age 4. Lastly, we found close to 10 % of the variance in rate of change in children shared by mothers. While some of this shared variance is genetic, there are likely environmental factors that need to be further identified that impact rate of telomere length change

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous con-ditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this re-ceptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent context. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across ap-plications, species and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease

TGF-beta receptor mediated telomerase inhibition, telomere shortening and breast cancer cell senescence

ABSTRACT Human telomerase reverse transcriptase (hTERT) plays a central role in telomere lengthening for continuous cell proliferation, but it remains unclear how extracellular cues regulate telomerase lengthening of telomeres. Here we report that the cytokine bone morphogenetic protein-7 (BMP7) induces the hTERT gene repression in a BMPRII receptor- and Smad3-dependent manner in human breast cancer cells. Chonic exposure of human breast cancer cells to BMP7 results in short telomeres, cell senescence and apoptosis. Mutation of the BMPRII receptor, but not TGFbRII, ACTRIIA or ACTRIIB receptor, inhibits BMP7-induced repression of the hTERT gene promoter activity, leading to increased telomerase activity, lengthened telomeres and continued cell proliferation. Expression of hTERT prevents BMP7-induced breast cancer cell senescence and apoptosis. Thus, our data suggest that BMP7 induces breast cancer cell aging by a mechanism involving BMPRII receptor- and Smad3-mediated repression of the hTERT gene