The mammalian COOH-terminal binding proteins (CtBPs) CtBP1 and CtBP2 are metabolically regulated transcriptional co-repressors that are degraded upon acute exposure to the alternative reading frame (ARF) tumor suppressor. We reported previously that CtBP stimulates cell migration in certain contexts via repression of PTEN transcription and activation of the phosphatidylinositol 3-kinase (PI3K) pathway. We have now identified an additional and direct mechanism for CtBP stimulation of cell migration via regulation of T-cell lymphoma invasion and metastasis 1 (Tiam1) protein. Tiam1 is a guanine nucleotide exchange factor (GEF) for Rac GTPase that plays a critical role in regulating cell adhesion, invasion, and migration and has been directly implicated in the promotion of cancer progression and metastasis. We noted a strict positive correlation between CtBP2 and Tiam1 expression levels and that CtBP promotion of cell migration required CtBP-dependent transcriptional activation of Tiam1. RNA interference (RNAi)-mediated knockdown of CtBP2 in human colon or lung carcinoma cells led to decreased Tiam1 protein and mRNA expression, while overexpression of CtBP2 increased Tiam1 expression levels. RNAi and overexpression studies also demonstrated that Tiam1 is a key downstream mediator of CtBP2-mediated cell migration. An analysis of the Tiam1 promoter revealed binding sites for the CtBP-interacting Kruppel-like factor 8 (KLF8), and a Tiam1 promoter luciferase reporter was induced in the presence of both KLF8 and CtBP2, consistent with KLF8-dependent CtBP transactivation of Tiam1. Chromatin immunoprecipitation analyses demonstrated CtBP2 occupancy of the Tiam1 promoter that was dependent on the presence of KLF8. Our results indicate that Tiam1 is a transcriptional activation target of CtBP2 and that this interaction promotes the pro-oncogenic function of CtBP2 leading to cancer cell migration. Transcriptional activation thus plays a role in CtBP pro-oncogenic functions along with the previously characterized CtBP co-repressor function.

Addressing the urgent need to develop novel drugs against drug-resistant Mycobacterium tuberculosis (M. tb) strains, ecumicin (ECU) and rufomycin I (RUFI) are being explored as promising new leads targeting cellular proteostasis via the caseinolytic protein ClpC1. Details of the binding topology and chemical mode of (inter)action of these cyclopeptides help drive further development of novel potency-optimized entities as tuberculosis drugs. ClpC1 M. tb protein constructs with mutations driving resistance to ECU and RUFI show reduced binding affinity by surface plasmon resonance (SPR). Despite certain structural similarities, ECU and RUFI resistant mutation sites did not overlap in their SPR binding patterns. SPR competition experiments show ECU prevents RUFI binding, whereas RUFI partially inhibits ECU binding. The X-ray structure of the ClpC1-NTD-RUFI complex reveals distinct differences compared to the previously reported ClpC1-NTD-cyclomarin A structure. Surprisingly, the complex structure revealed that the epoxide moiety of RUFI opened and covalently bound to ClpC1-NTD via the sulfur atom of Met1. Furthermore, RUFI analogues indicate that the epoxy group of RUFI is critical for binding and bactericidal activity. The outcomes demonstrate the significance of ClpC1 as a novel target and the importance of SAR analysis of identified macrocyclic peptides for drug discovery.

Menaquinone (vitamin K2) plays a vital role in energy generation and environmental adaptation in many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb). Although menaquinone levels are known to be tightly linked to the cellular redox/energy status of the cell, the regulatory mechanisms underpinning this phenomenon are unclear. The first committed step in menaquinone biosynthesis is catalyzed by MenD, a thiamine diphosphate–dependent enzyme comprising three domains. Domains I and III form the MenD active site, but no function has yet been ascribed to domain II. Here, we show that the last cytosolic metabolite in the menaquinone biosynthesis pathway, 1,4-dihydroxy-2-naphthoic acid (DHNA), binds to domain II of Mtb-MenD and inhibits its activity. Using X-ray crystallography of four apo- and cofactor-bound Mtb-MenD structures, along with several spectroscopy assays, we identified three arginine residues (Arg-97, Arg-277, and Arg-303) that are important for both enzyme activity and the feedback inhibition by DHNA. Among these residues, Arg-277 appeared to be particularly important for signal propagation from the allosteric site to the active site. This is the first evidence of feedback regulation of the menaquinone biosynthesis pathway in bacteria, identifying a protein-level regulatory mechanism that controls menaquinone levels within the cell and may therefore represent a good target for disrupting menaquinone biosynthesis in M. tuberculosis.

bChronic myeloid leukemia (CML) is derived from a stem cell, and it is widely accepted that the existence of leukemia stem cells (LSCs) is one of the major reasons for the relapse of CML treated with kinase inhibitors. Key to eradicating LSCs is to identify genes that play a critical role in survival regulation of these stem cells. Using BCR-ABL-induced CML mouse model, here we show that expression of the stearoyl-CoA desaturase 1 (Scd1) gene is downregulated in LSCs and that Scd1 plays a tumor-suppressive role in LSCs with no effect on the function of normal hematopoietic stem cells. Deletion of Scd1 causes acceleration of CML development and conversely overexpression of Scd1 delays CML development. In addition, using genetic approaches, we show that Pten, p53, and Bcl2 are regulated by Scd1 in LSCs. Furthermore, we find that induction of Scd1 expression by a PPAR␥ agonist suppresses LSCs and delays CML development. Our results demonstrate a critical role for Scd1 in functional regulation of LSCs, providing a new anti-LSC strategy through enhancing Scd1 activity.