Possible Role of Correlation Coefficients and Network Analysis of Multiple Intracellular Proteins in Blood Cells of Patients with Bipolar Disorder in Studying the Mechanism of Lithium Responsiveness: A Proof-Concept Study.

Background: The mechanism of lithium treatment responsiveness in bipolar disorder (BD) remains unclear. The aim of this study was to explore the utility of correlation coefficients and protein-to-protein interaction (PPI) network analyses of intracellular proteins in monocytes and CD4+ lymphocytes of patients with BD in studying the potential mechanism of lithium treatment responsiveness. Methods: Patients with bipolar I or II disorder who were diagnosed with the MINI for DSM-5 and at any phase of the illness with at least mild symptom severity and received lithium (serum level ≥ 0.6 mEq/L) for 16 weeks were divided into two groups, responders (≥50% improvement in Montgomery-Asberg Depression Rating Scale and/or Young Mania Rating Scale scores from baseline) and non-responders. Twenty-eight intracellular proteins/analytes in CD4+ lymphocytes and monocytes were analyzed with a tyramine-based signal-amplified flow cytometry procedure. Correlation coefficients between analytes at baseline were estimated in both responders and non-responders and before and after lithium treatment in responders. PPI network, subnetwork, and pathway analyses were generated based on fold change/difference in studied proteins/analytes between responders and non-responders. Results: Of the 28 analytes from 12 lithium-responders and 11 lithium-non-responders, there were more significant correlations between analytes in responders than in non-responders at baseline. Of the nine lithium responders with pre- and post-lithium blood samples available, the correlations between most analytes were weakened after lithium treatment with cell-type specific patterns in CD4+ lymphocytes and monocytes. PPI network/subnetwork and pathway analyses showed that lithium response was involved in four pathways, including prolactin, leptin, neurotrophin, and brain-derived neurotrophic factor pathways. Glycogen synthase kinase 3 beta and nuclear factor NF-kappa-B p65 subunit genes were found in all four pathways. Conclusions: Using correlation coefficients, PPI network/subnetwork, and pathway analysis with multiple intracellular proteins appears to be a workable concept for studying the mechanism of lithium responsiveness in BD. Larger sample size studies are necessary to determine its utility.

Differences in intracellular protein levels in monocytes and CD4+ lymphocytes between bipolar depressed patients and healthy controls: A pilot study with tyramine-based signal-amplified flow cytometry.

BACKGROUND: Molecular biomarkers for bipolar disorder (BD) that distinguish it from other manifestations of depressive symptoms remain unknown. The aim of this study was to determine if a very sensitive tyramine-based signal-amplification technology for flow cytometry (CellPrint™) could facilitate the identification of cell-specific analyte expression profiles of peripheral blood cells for bipolar depression (BPD) versus healthy controls (HCs). METHODS: The diagnosis of psychiatric disorders was ascertained with Mini International Neuropsychiatric Interview for DSM-5. Expression levels for eighteen protein analytes previously shown to be related to bipolar disorder were assessed with CellPrint™ in CD4+ T cells and monocytes of bipolar patients and HCs. Implementation of protein-protein interaction (PPI) network and pathway analysis was subsequently used to identify new analytes and pathways for subsequent interrogations. RESULTS: Fourteen drug-naïve or -free patients with bipolar I or II depression and 17 healthy controls (HCs) were enrolled. The most distinguishable changes in analyte expression based on t-tests included GSK3ß, HMGB1, IRS2, phospho-GSK3αß, phospho-RELA, and TSPO in CD4+ T cells and calmodulin, GSK3ß, IRS2, and phospho-HS1 in monocytes. Subsequent PPI and pathway analysis indicated that prolactin, leptin, BDNF, and interleukin-3 signal pathways were significantly different between bipolar patients and HCs. LIMITATION: The sample size of the study was small and 2 patients were on medications. CONCLUSION: In this pilot study, CellPrint™ was able to detect differences in cell-specific protein levels between BPD patients and HCs. A subsequent study including samples from patients with BPD, major depressive disorder, and HCs is warranted.

Divergent Directionality of Immune Cell-Specific Protein Expression between Bipolar Lithium Responders and Non-Responders Revealed by Enhanced Flow Cytometry.

Background and Objectives: There is no biomarker to predict lithium response. This study used CellPrint™ enhanced flow cytometry to study 28 proteins representing a spectrum of cellular pathways in monocytes and CD4+ lymphocytes before and after lithium treatment in patients with bipolar disorder (BD). Materials and Methods: Symptomatic patients with BD type I or II received lithium (serum level ≥ 0.6 mEq/L) for 16 weeks. Patients were assessed with standard rating scales and divided into two groups, responders (≥50% improvement from baseline) and non-responders. Twenty-eight intracellular proteins in CD4+ lymphocytes and monocytes were analyzed with CellPrint™, an enhanced flow cytometry procedure. Data were analyzed for differences in protein expression levels. Results: The intent-to-treat sample included 13 lithium-responders (12 blood samples before treatment and 9 after treatment) and 11 lithium-non-responders (11 blood samples before treatment and 4 after treatment). No significant differences in expression between the groups was observed prior to lithium treatment. After treatment, the majority of analytes increased expression in responders and decreased expression in non-responders. Significant increases were seen for PDEB4 and NR3C1 in responders. A significant decrease was seen for NR3C1 in non-responders. Conclusions: Lithium induced divergent directionality of protein expression depending on the whether the patient was a responder or non-responder, elucidating molecular characteristics of lithium responsiveness. A subsequent study with a larger sample size is warranted.

Protein Biomarkers in Monocytes and CD4+ Lymphocytes for Predicting Lithium Treatment Response of Bipolar Disorder: a Feasibility Study with Tyramine-Based Signal-Amplified Flow Cytometry.

Purpose: To determine if enhanced flow cytometry (CellPrint™) can identify intracellular proteins of lithium responsiveness in monocytes and CD4+ lymphocytes from patients with bipolar disorder. Methods: Eligible bipolar I or II patients were openly treated with lithium for 16-weeks. Baseline levels of Bcl2, BDNF, calmodulin, Fyn, phospho-Fyn/phospho-Yes, GSK3ß, phospho-GSK3αß, HMGB1, iNOS, IRS2, mTor, NLPR3, PGM1, PKA C-α, PPAR-γ, phospho-RelA, and TPH1 in monocytes and CD4+ lymphocytes of lithium responders and non-responders were measured with CellPrint™. Their utility of discriminating responders from non-responders was explored. Protein-protein network and pathway enrichment analyses were conducted. Results: Of the 24 intent-to-treat patients, 12 patients completed the 16-week study. Eleven of 13 responders and 8 of 11 non-responders were available for this analysis. The levels of the majority of analytes in lithium responders were lower than non-responders in both cell types, but only the level of GSK3ß in monocytes was significantly different (p = 0.034). The combination of GSK3ß and phospho-GSK3αß levels in monocytes correctly classified 11/11 responders and 5/8 non-responders. Combination of GSK3ß, phospho-RelA, TPH1 and PGM1 correctly classified 10/11 responders and 6/7 non-responders, both with a likelihood of ≥ 85%. Prolactin, leptin, BDNF, neurotrophin, and epidermal growth factor/epidermal growth factor receptor signaling pathways are involved in the lithium treatment response. GSK3ß and RelA genes are involved in 4 of 5 these pathways. Conclusion: CellPrint™ flow cytometry was able to detect differences in multiple proteins in monocytes and CD4+ lymphocytes between lithium responders and non-responders. A large study is warranted to confirm or refute these findings.

Prognostic information and biological insights in chronic lymphocytic leukemia by high-resolution immunophenotypic analysis of ZAP70.

The E2997 Phase III trial included preservation of valuable chronic lymphocytic leukemia (CLL) patient specimens and relevant clinical outcome data. Using a novel high-resolution technology on a flow cytometry platform, we assessed 79 E2997 samples for the expression of 27 analytes that reflected the activity of signaling pathways and apoptosis. We found that the expression levels of ZAP70 segregated the samples into two subpopulations with the distribution showing a peak-trough-peak configuration. Although prior assessment of ZAP70 by standard procedures did not reveal any prognostic information, we found by using the trough in the distribution as a cutpoint that ZAP70 expression levels were significantly correlated with both progression-free survival and overall survival. Additionally, the cells expressing high versus low levels of ZAP70 demonstrated distinct molecular organization as indicated by the other analytes assessed. Our analysis demonstrates the value of ZAP70 expression as a prognostic indicator and suggests that the different clinical results may be due to the distinct molecular biology of the ZAP70-low versus the ZAP70-high CLL samples.

Intrinsic RNA binding by the eukaryotic initiation factor 4F depends on a minimal RNA length but not on the m7G cap.

The eukaryotic initiation factor 4F (eIF4F) is thought to be the first factor to bind mRNA during 7-methylguanosine (m7G) cap-dependent translation initiation. The multipartite eIF4F contains the cap-binding protein eIF4E, and it is assumed that eIF4F binds mRNAs primarily at the 5' m7G cap structure. We have analyzed equilibrium binding of rabbit eIF4F to a series of diverse RNAs and found no impact of the 5'-cap on the stability of eIF4F-RNA complexes. However, eIF4F preferentially and cooperatively binds to RNAs with a minimum length of approximately 60 nucleotides in vitro. Furthermore, translation activity in rabbit reticulocyte lysate is strongly inhibited by RNAs exceeding this length, but not by shorter ones, consistent with the notion that eIF4F in its physiological environment preferentially binds longer RNAs, too. Collectively, our results indicate that intrinsic RNA binding by eIF4F depends on a minimal RNA length, rather than on cap recognition. The nonetheless essential m7G cap may either function at steps subsequent to eIF4F-RNA binding, or other factors facilitate preferential binding of eIF4F to the m7G cap.

Analysis of substrate recognition by the ribonucleoprotein endonuclease RNase P.

Ribonuclease P (RNase P), is a ribonucleoprotein complex that catalyzes the site-specific cleavage of pre-tRNA and a wide variety of other substrates. Although RNase P RNA is the catalytic subunit of the holoenzyme, the protein subunit plays a critical role in substrate binding. Thus, RNase P is an excellent model system for studying ribonucleoprotein function. In this review we describe methods applied to the in vitro study of substrate recognition by bacterial RNase P, covering general considerations of reaction conditions, quantitative measurement of substrate binding equilibria, enzymatic and chemical protection, cross-linking, modification interference, and analysis of site-specific substitutions. We describe application of these methods to substrate binding by RNase P RNA alone and experimental considerations for examining the holoenzyme. The combined use of these approaches has shown that the RNA and protein subunits cooperate to bind different portions of the substrate structure, with the RNA subunit predominantly interacting with the mature domain of tRNA and the protein interacting with the 5(') leader sequence. However, important questions concerning the interface between the two subunits and the coordination of RNA and protein subunits in binding and catalysis remain.

Conservation of helical structure contributes to functional metal ion interactions in the catalytic domain of ribonuclease P RNA.

Like protein enzymes, catalytic RNAs contain conserved structure motifs important for function. A universal feature of the catalytic domain of ribonuclease P RNA is a bulged-helix motif within the P1-P4 helix junction. Here, we show that changes in bulged nucleotide identity and position within helix P4 affect both catalysis and substrate binding, while a subset of the mutations resulted only in catalytic defects. We find that the proximity of the bulge to sites of metal ion coordination in P4 is important for catalysis; moving the bulge distal to these sites and deleting it had similarly large effects, while moving it proximal to these sites had only a moderate effect on catalysis. To test whether the effects of the mutations are linked to metal ion interactions, we used terbium-dependent cleavage of the phosphate backbone to probe metal ion-binding sites in the wild-type and mutant ribozymes. We detect cleavages at specific sites within the catalytic domain, including helix P4 and J3/4, which have previously been shown to participate directly in metal ion interactions. Mutations introduced into P4 cause local changes in the terbium cleavage pattern due to alternate metal ion-binding configurations with the helix. In addition, a bulge deletion mutation results in a 100-fold decrease in the single turnover cleavage rate constant at saturating magnesium levels, and a reduced affinity for magnesium ions important for catalysis. In light of the alternate terbium cleavage pattern in P4 caused by bulge deletion, this decreased ability to utilize magnesium ions for catalysis appears to be due to localized structural changes in the ribozyme's catalytic core that weaken metal ion interactions in P4 and J3/4. The information reported here, therefore, provides evidence that the universal conservation of the P4 structure is based in part on optimization of metal ion interactions important for catalysis.

Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA.

Interactions with divalent metal ions are essential for the folding and function of the catalytic RNA component of the tRNA processing enzyme ribonuclease P (RNase P RNA). However, the number and location of specific metal ion interactions in this large, highly structured RNA are poorly understood. Using atomic mutagenesis and quantitative analysis of thiophilic metal ion rescue we provide evidence for metal ion interactions at the pro-R(P) and pro-S(P) non-bridging phosphate oxygens at nucleotide A67 in the universally conserved helix P4. Moreover, second-site modifications within helix P4 and the adjacent single stranded region (J3/4) provide the first evidence for metal ion interactions with nucleotide base functional groups in RNase P RNA and reveal the presence of an additional metal ion important for catalytic function. Together, these data are consistent with a cluster of metal ion interactions in the P1-P4 multi-helix junction that defines the catalytic core of the RNase P ribozyme.

NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis.

The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1-P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+ concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly important for substrate cleavage and demonstrate a close association between catalytic function and metal ion-dependent structure in the highly conserved P1-P4 multihelix junction.