Rifampicin and its derivatives are at the forefront of the current standard chemotherapeutic regimen for active tuberculosis; they act by inhibiting the transcription activity of prokaryotic RNA polymerase. Rifampicin is believed to interact with the b subunit of RNA polymerase. However, it has been observed that protein-protein interactions with RNA polymerase core enzyme lead to its reduced susceptibility to rifampicin. This mechanism became more diversified with the discovery of RbpA, a novel RNA polymerase-binding protein, in Streptomyces coelicolor that could mitigate the effect of rifampicin on RNA polymerase activity. MsRbpA is a homologue of RbpA in Mycobacterium smegmatis. On deciphering the role of MsRbpA in M. smegmatis we found that it interacts with RNA polymerase and increases the rifampicin tolerance levels, both in vitro and in vivo. It interacts with the b subunit of RNA polymerase. However, it was found to be incapable of rescuing rifampicin-resistant RNA polymerases in the presence of rifampicin at the respective IC 50 .

The protein MsRbpA from Mycobacterium smegmatis rescues RNA polymerase (RNAP) from the inhibitory effect of rifampicin (Rif). We have reported previously that MsRbpA interacts with the b-subunit of RNAP and that the effect of MsRbpA on Rif-resistant (Rif R ) RNAP is minimal. Here we attempted to gain molecular insights into the mechanism of action of this protein with respect to its role in rescuing RNAP from Rif-mediated transcription inhibition. Our experimental approach comprised multiple-round transcription assays, fluorescence spectroscopy, MS and surface plasmon resonance in order to meet the above objective. Based on our molecular studies we propose here that Rif is released from its binding site in the RNAP-Rif complex in the presence of MsRbpA. Biophysical studies reveal that the location of MsRbpA on RNAP is at the junction of the b-and b9-subunits, close to the Rif-binding site and the (i+1) site on RNAP.

Sonic hedgehog (Shh) signaling is closely coupled with bioenergetics of medulloblastoma, the most common malignant pediatric brain tumor. Shh-associated medulloblastoma arises from cerebellar granule neuron precursors (CGNPs), a neural progenitor whose developmental expansion requires signaling by Shh, a ligand secreted by the neighboring Purkinje neurons. Previous observations show that Shh signaling inhibits fatty acid oxidation while driving increased fatty acid synthesis. Proliferating CGNPs and mouse Shh medulloblastomas feature high levels of glycolytic enzymes in vivo and in vitro. Since both of these metabolic processes are closely linked to mitochondrial bioenergetics, the role of Shh signaling in mitochondrial biogenesis was investigated. This report uncovers a surprising decrease in mitochondrial membrane potential (MMP) and overall ATP production in CGNPs exposed to Shh, consistent with increased glycolysis resulting in high intracellular acidity, leading to mitochondrial fragmentation. Ultrastructural examination of mitochondria revealed a spherical shape in Shh-treated cells, in contrast to the elongated appearance in vehicle-treated post-mitotic cells. Expression of Mitofusin 1 and 2 was reduced in these cells, while their ectopic expression restored the mitochondrial membrane potential to the non-proliferating state and the morphology to a fused, interconnected state. Mouse Shh medulloblastoma cells featured drastically impaired mitochondrial morphology, restoration of which by ectopic mitofusin expression was also associated with a decrease in the expression of Cyclin D2 protein, a marker for proliferation.

The sonic hedgehog subtype of medulloblastoma (SHH MB) is associated with treatment failure and poor outcome. Current strategies utilizing whole brain radiation therapy result in deleterious off-target effects on the normal developing childhood brain. Most conventional chemotherapies remain limited by ineffective blood–brain barrier (BBB) penetrance. These challenges signify an unmet need for drug carriers that can cross the BBB and deliver drugs to targeted sites with high drug-loading efficiency and long-term stability. We herein leverage the enhanced stability and targeting ability of engineered high-density lipoprotein-mimetic nanoparticles (eHNPs) to cross the BBB and deliver a SHH inhibitor effectively to the cancer stem-like cell population in SHH MB. Our microfluidic technology enabled highly reproducible production of multicomponent eHNPs incorporated with apolipoprotein A1, anti-CD15, and a SHH inhibitor (LDE225). We demonstrate the dual-targeted delivery and enhanced therapeutic effect of eHNP-A1-CD15-LDE225 via scavenger receptor class B type 1 (SR-B1) and CD15 on brain SHH MB cells in vitro, ex vivo, and in vivo. Moreover, we show that eHNP-A1 not only serves as a stable drug carrier, but also has a therapeutic effect itself through SR-B1-mediated intracellular cholesterol depletion in SHH MB cells. Through the facilitated and targeted cellular uptake of drugs and direct therapeutic role of this engineered biomimetic nanocarrier in SHH MB, our multifunctional nanoparticle provides intriguing therapeutic promise as an effective and potent nanomedicine for the treatment of SHH MB.

Post-natal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for the SHH-associated subgroup of medulloblastoma (MB), is driven by Sonic Hedgehog (Shh) and Insulin-like Growth Factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is up-regulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh-subgroup of MB in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and medulloblastoma cells and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas.