Pressure-Temperature Analysis of the Stability of the CTL9 Domain Reveals Hidden Intermediates

The observation of two-state unfolding for many small single-domain proteins by denaturants has led to speculation that protein sequences may have evolved to limit the population of partially folded states that could be detrimental to fitness. How such strong cooperativity arises from a multitude of individual interactions is not well understood. Here, we investigate the stability and folding cooperativity of the C-terminal domain of the ribosomal protein L9 in the pressure-temperature plane using site-specific NMR. In contrast to apparent cooperative unfolding detected with denaturant-induced and thermal-induced unfolding experiments and stopped-flow refolding studies at ambient pressure, NMR-detected pressure unfolding revealed significant deviation from two-state behavior, with a core region that was selectively destabilized by increasing temperature. Comparison of pressure-dependent NMR signals from both the folded and unfolded states revealed the population of at least one invisible excited state at atmospheric pressure. The core destabilizing cavity-creating I98A mutation apparently increased the cooperativity of the loss of folded-state peak intensity while also increasing the population of this invisible excited state present at atmospheric pressure. These observations highlight how local stability is subtly modulated by sequence to tune protein conformational landscapes and illustrate the ability of pressure- and temperature-dependent studies to reveal otherwise hidden states.     

LytR-CpsA-Psr proteins in Staphylococcus aureus display partial functional redundancy and the deletion of all three severely impairs septum placement and cell separation

Staphylococcus aureus contains three members of the LytR-CpsA-Psr (LCP) family of membrane proteins: MsrR, SA0908 and SA2103. The characterization of single-, double- and triple-deletion mutants revealed distinct phenotypes for each of the three proteins. MsrR was involved in cell separation and septum formation and influenced β-lactam resistance; SA0908 protected cells from autolysis; and SA2103, although displaying no apparent phenotype by itself, enhanced the properties of msrR and sa0908 mutants when deleted. The deletion of sa0908 and sa2103 also further attenuated the virulence of msrR mutants in a nematode-killing assay. The severely defective growth phenotype of the triple mutant revealed that LytR-CpsA-Psr proteins are essential for optimal cell division in S. aureus. Growth could be rescued to varying degrees by any one of the three proteins, indicating some functional redundancy within members of this protein family. However, differing phenotypic characteristics of all single and double mutants and complemented triple mutants indicated that each protein played a distinct role(s) and contributed differently to phenotypes influencing cell separation, autolysis, cell surface properties and virulence.     

MicroRNAs 130a/b are regulated by BCR-ABL and downregulate expression of CCN3 in CML

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterized by the expression of the oncoprotein, Bcr-Abl kinase. CCN3 normally functions as a negative growth regulator, but it is downregulated in CML, the mechanism of which is not known. MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate protein translation by binding to the complimentary sequences of the 3′ UTR of messenger RNAs. Deregulated miRNA expression has emerged as a hallmark of cancer. In CML, BCR-ABL upregulates oncogenic miRNAs and downregulates tumour suppressor miRNAs favouring leukaemic transformation. We report here that the downregulation of CCN3 in CML is mediated by BCR-ABL dependent miRNAs. Using the CML cell line K562, we profiled miRNAs, which are BCR-ABL dependent by transfecting K562 cells with anti-BCR-ABL siRNA. MiRNA expression levels were quantified using the Taqman Low Density miRNA array platform. From the miRNA target prediction databases we identified miRNAs that could potentially bind to CCN3 mRNA and reduce expression. Of these, miR-130a, miR-130b, miR-148a, miR-212 and miR-425-5p were significantly reduced on BCR-ABL knockdown, with both miR-130a and miR-130b decreasing the most within 24 h of siRNA treatment. Transfection of mature sequences of miR-130a and miR-130b individually into BCR-ABL negative HL60 cells resulted in a decrease of both CCN3 mRNA and protein. The reduction in CCN3 was greatest with overexpression of miR-130a whereas miR-130b overexpression resulted only in marginal repression of CCN3. This study shows that miRNAs modulate CCN3 expression. Deregulated miRNA expression initiated by BCR-ABL may be one mechanism of downregulating CCN3 whereby leukaemic cells evade negative growth regulation.     

CCN3 suppresses mitogenic signalling and reinstates growth control mechanisms in Chronic Myeloid Leukaemia

CCN3, a tumour suppressor gene, is down-regulated as a result of BCR-ABL tyrosine kinase activity in Chronic Myeloid Leukaemia (CML). We have established a stable CCN3 expression model in the human K562 CML cell line and have further validated the role for CCN3 in the leukaemogenic process. K562 cells stably transfected with CCN3 (K562/CCN3; 2.25 × 10⁶ copies per 50 ng cDNA) demonstrated over 50% reduction in cell growth in comparison to cells stably transfected with empty vector (K562/control; p = 0.005). K562/CCN3 cells had reduced colony formation capacity (reduced by 29.7%, p = 0.03) and reduced mitogenic signalling in comparison to K562/control cells (reduced by 29.5% (p = 0.002) and 37.4% (p = 0.017) for phosphorylation levels of ERK and AKT respectively). K562/CCN3 cells showed an accumulation of events within the subG₀ phase of the cell cycle and increased apoptosis was confirmed by a three-fold increase in annexin V binding (p < 0.05). K562/CCN3 cells exposed to Imatinib (1 μM and 5 μM) showed an increase in events within the subG₀ phase of cell cycle over 96 h and mirrored the enhanced cell kill demonstrated by Annexin staining. Wild type K562 cells treated with recombinant human Ccn3 (10 nM) in combination with Imatinib (5 μM) also displayed enhanced cell kill (p = 0.008). K562/CCN3 cells displayed increased adhesion to matrigel™ (2.92 ± 0.52 fold increase compared to K562/control) which was commensurate with increased expression of the alpha 6 and beta 4 integrins (6.53 ± 0.47 and 1.94 ± 0.07 fold increase in gene expression respectively (n = 3, p < 0.05)). CCN3 restores cellular growth regulatory properties that are absent in CML and sensitises CML cells to imatinib induced apoptosis. CCN3 may provide novel avenues for the development of alternate therapeutic strategies.