Search Results (7)

Following the success of mRNA vaccines against COVID-19, mRNA-based therapeutics have now become a great interest and potential. The development of this approach has been preceded by studies of modifications found on mRNA ribonucleotides that influence the stability, translation and immunogenicity of this molecule. The 5′ cap of eukaryotic mRNA plays a critical role in these cellular functions and is thus the focus of intensive chemical modifications to affect the biological properties of in vitro-prepared mRNA. Enzymatic removal of the 5′ cap affects the stability of mRNA in vivo. The NUDIX hydrolase Dcp2 was identified as the first eukaryotic decapping enzyme and is routinely used to analyse the synthetic cap at the 5′ end of RNA. Here we highlight three additional NUDIX enzymes with known decapping activity, namely Nudt2, Nudt12 and Nudt16. These enzymes possess a different and some overlapping activity towards numerous 5′ RNA cap structures, including non-canonical and chemically modified ones. Therefore, they appear as potent tools for comprehensive in vitro characterisation of capped RNA transcripts, with special focus on synthetic RNAs with therapeutic activity. Full article

Intratumoral hypoxia is associated with tumor progression, aggressiveness, and therapeutic resistance in several cancers. Hypoxia causes cancer cells to experience replication stress, thereby activating DNA damage and repair pathways. MutT homologue-1 (MTH1, also known as NUDT1), a member of the Nudix family, maintains the genomic integrity and viability of tumor cells in the hypoxic tumor microenvironment. Although hypoxia is associated with poor prognosis and can cause therapeutic resistance by regulating the microenvironment, it has not been considered a treatable target in cancer. This study aimed to investigate whether hypoxia-induced MTH1 is a useful target for immunotherapy and whether hypoxic conditions influence the antitumor activity of immune cells. Our results showed that MTH1 expression was elevated under hypoxic conditions in head and neck cancer cell lines. Furthermore, we identified a novel MTH1-targeting epitope peptide that can activate peptide-specific CD4+ helper T cells with cytotoxic activity. The proliferation and cytotoxic activity of T cells were maintained under hypoxic conditions, and PD-1 blockade further augmented the cytotoxicity. These results indicate that MTH1-targeted immunotherapy combined with checkpoint blockade can be an effective strategy for the treatment of hypoxic tumors. Full article

Expression of microRNAs, such as miR-365, is known to be dysregulated in many tumors, including oral cancers, although little is known about their role or functions. The objective of this project is to evaluate the downstream targets of miR-365 to determine any potential pathways or effects. Downstream targets for miR-365 (miRdatabase target scores > 90) were used for qPCR screening of oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, CAL27). Each oral cancer cell line expressed miR-365 downstream targets molybdenum cofactor synthesis-2 (MOCS2), erythropoietin receptor (EPOR), IQ motif containing-K (IQCK), carboxypeptidase A3 (CPA3), solute carrier family 24 member-3 (SLC24A3), and coiled-coil domain containing 47 (CCDC47)—although the expression levels varied somewhat. However, differential results were observed with ubiquitin protein ligase E3 component n-recognin-3 (UBR3), nudix hydrolase-12 (NUDT12), zinc finger CCHC-type containing-14 (ZCCHC14), and homeobox and leucine zipper encoding (HOMEZ). These data suggest that many of the miR-365 targets are expressed in the oral cancers screened, with the differential expression of UBR3, ZCCHC14, HOMEZ, and NUDT12, which may be correlated with chemoresistance among two specific oral cancer cell lines (SCC25, SCC9). These results suggest this differential expression may signal potential targets for patient treatment with tumors exhibiting miR-365 and chemotherapeutic resistance. Full article

Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a nucleoside diphosphate linked to another moiety X. Several substrates for hNudt16 and various possible biological functions have been reported. However, some of these reports contradict each other and studies comparing the substrate specificity of the hNudt16 protein are limited. Therefore, we quantitatively compared the affinity of hNudt16 towards a set of previously published substrates, as well as identified novel potential substrates. Here, we show that hNudt16 has the highest affinity towards IDP and GppG, with K_d below 100 nM. Other tested ligands exhibited a weaker affinity of several orders of magnitude. Among the investigated compounds, only IDP, GppG, m⁷GppG, AppA, dpCoA, and NADH were hydrolyzed by hNudt16 with a strong substrate preference for inosine or guanosine containing compounds. A new identified substrate for hNudt16, GppG, which binds the enzyme with an affinity comparable to that of IDP, suggests another potential regulatory role of this protein. Molecular docking of hNudt16-ligand binding inside the hNudt16 pocket revealed two binding modes for representative substrates. Nucleobase stabilization by Π stacking interactions with His24 has been associated with strong binding of hNudt16 substrates. Full article

Twenty to thirty percent of the septating mycobacterial cells of the mid-log phase population showed highly deviated asymmetric constriction during division (ACD), while the remaining underwent symmetric constriction during division (SCD). The ACD produced short-sized cells (SCs) and normal/long-sized cells (NCs) as the sister–daughter cells, but with significant differential susceptibility to antibiotic/oxidative/nitrite stress. Here we report that, at 0.2% glycerol, formulated in the Middlebrook 7H9 medium, a significantly high proportion of the cells were divided by SCD. When the glycerol concentration decreased to 0.1% due to cell-growth/division, the ACD proportion gradually increased until the ACD:SCD ratio reached ~50:50. With further decrease in the glycerol levels, the SCD proportion increased with concomitant decrease in the ACD proportion. Maintenance of glycerol at 0.1%, through replenishment, held the ACD:SCD proportion at ~50:50. Transfer of the cells from one culture with a specific glycerol level to the supernatant from another culture, with a different glycerol level, made the cells change the ACD:SCD proportion to that of the culture from which the supernatant was taken. RT-qPCR data showed the possibility of diadenosine tetraphosphate phosphorylase (MSMEG_2932), phosphatidylinositol synthase (MSMEG_2933), and a Nudix family hydrolase (MSMEG_2936) involved in the ACD:SCD proportion-change in response to glycerol levels. We also discussed its physiological significance. Full article

Despite global research efforts, breast cancer remains the leading cause of cancer death in women worldwide. The majority of these deaths are due to metastasis occurring years after the initial treatment of the primary tumor and occurs at a higher frequency in hormone receptor-positive (Estrogen and Progesterone; HR+) breast cancers. We have previously described the role of NUDT5 (Nudix-linked to moiety X-5) in HR+ breast cancer progression, specifically with regards to the growth of breast cancer stem cells (BCSCs). BCSCs are known to be the initiators of epithelial-to-mesenchyme transition (EMT), metastatic colonization, and growth. Therefore, a greater understanding of the proteins and signaling pathways involved in the metastatic process may open the door for therapeutic opportunities. In this review, we discuss the role of NUDT5 and other members of the NUDT family of enzymes in breast and other cancer types. We highlight the use of global omics data based on our recent phosphoproteomic analysis of progestin signaling pathways in breast cancer cells and how this experimental approach provides insight into novel crosstalk mechanisms for stratification and drug discovery projects aiming to treat patients with aggressive cancer. Full article

Here, we present the characterisation of the so-far-unstudied NUDIX hydrolase family member NUDT22. We previously identified the unique hydrolase activity of NUDT22 towards UDP-glucose from a family-wide biochemical substrate screen. UDP-glucose hydrolysis was found to result in the production of uridine monophosphate (UMP) and glucose 1-phosphate (G-1-P). We furthermore solved the first crystal structure of NUDT22 in complex with its substrate UDP-glucose [¹]. Our mechanistic studies revealed increased replication stress in NUDT22-deficient cells that could be rescued by nucleoside supplementation. We therefore propose the discovery of a novel NUDT22-mediated pyrimidine salvage pathway. Increased replication rates resulting in replication stress is a hallmark of cancer cells, and NUDT22 gene expression alterations are present in several cancer tissues, which makes NUDT22 an interesting new target for the development of small molecule inhibitors for uses in cancer. We employed our NUDT22 crystal structure to perform an in silico docking screen on available small molecule libraries to identify starting points for the development of first-in-class NUDT22 inhibitors. Chemically optimised NUDT22 inhibitors are currently being validated in biochemical assays and cellular target engagement assays, and their cellular activity is being assessed in vitro. Full article