Structures of pyruvate kinases display evolutionarily divergent allosteric strategies

The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (k(cat)/S(0.5)). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (k(cat)/S(0.5)) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors

Clinical significance of circulating tumor cells in predicating the outcomes of patients with colorectal cancer

Background: Relapse and metastasis of patients with Colorectal Cancer (CRC) is the major obstacle to the long-term life of patients. Its mechanisms remain defined. Methods: A total of 48 CRC patients were enrolled and 68 samples were obtained from the peripheral blood of patients before or after treatments in this study. Twenty non-cancer patients were also detected as a negative control. Circulating Tumor Cells (CTCs), including Epithelial CTCs (eCTCs), Mesenchymal (MCTCs), and epithelial/mesenchymal mixed phenotypes (mixed CTCs), were identified by CanPatrolTM CTC enrichment and RNA in situ hybridization. The relationship between CTCs number and Progression-Free Survival (PFS) or Overall Survival (OS) was evaluated. Results: Thirty-four of 48 patients (70.8%) were found to have positive CTCs. Total CTCs and MCTCs in the post-treatment had a significant correlation PFS and OS. When total CTCs or MCTCs in 5 mL blood of patients were more than 6 CTCs or 5 MCTCs, PFS of the patients was significantly shorter (p < 0.05) than that in patients with less than 6 CTCs or 5 MCTCs. The patients with > 5 CTCs count changes were found to exhibit poor PFS and OS rates (p < 0.05). Conclusion: Total CTCs and MCTCs number detection in patients with colorectal cancer was very useful biomarker for predicting the prognosis of patients. Higher CTCs or MCTCs had poorer PFS and OS rates

Pyruvate Kinase regulates the Pentose-Phosphate pathway in Response to Hypoxia in Mycobacterium tuberculosis

In response to the stress of infection, Mycobacterium tuberculosis (Mtb) reprograms its metabolism to accommodate nutrient and energetic demands in a changing environment. Pyruvate kinase (PYK) is an essential glycolytic enzyme in the phosphoenolpyruvate–pyruvate–oxaloacetate node that is a central switch point for carbon flux distribution. Here we show that the competitive binding of pentose monophosphate inhibitors or the activator glucose 6-phosphate (G6P) to MtbPYK tightly regulates the metabolic flux. Intriguingly, pentose monophosphates were found to share the same binding site with G6P. The determination of a crystal structure of MtbPYK with bound ribose 5-phosphate (R5P), combined with biochemical analyses and molecular dynamic simulations, revealed that the allosteric inhibitor pentose monophosphate increases PYK structural dynamics, weakens the structural network communication, and impairs substrate binding. G6P, on the other hand, primes and activates the tetramer by decreasing protein flexibility and strengthening allosteric coupling. Therefore, we propose that MtbPYK uses these differences in conformational dynamics to up- and down-regulate enzymic activity. Importantly, metabolome profiling in mycobacteria reveals a significant increase in the levels of pentose monophosphate during hypoxia, which provides insights into how PYK uses dynamics of the tetramer as a competitive allosteric mechanism to retard glycolysis and facilitate metabolic reprogramming toward the pentose-phosphate pathway for achieving redox balance and an anticipatory metabolic response in Mtb

Pyruvate kinases have an intrinsic and conserved decarboxylase activity

The phosphotransfer mechanism of pyruvate kinases (PYKs) has been studied in detail, but the mechanism of the intrinsic decarboxylase reaction catalysed by PYKs is still unknown. 1H NMR was used in this work to follow oxaloacetate (OAA) decarboxylation by trypanosomatid and human PYKs confirming that the decarboxylase activity is conserved across distantly related species. Crystal structures of Trypanosoma brucei PYK (TbPYK) complexed with the product of the decarboxylase reaction (pyruvate), and a series of substrate analogues (D-malate, α-ketoglutarate and oxalate) show that the OAA analogues bind to the kinase active site with similar binding modes, confirming that both decarboxylase and kinase activities share a common site for substrate binding and catalysis. Decarboxylation of OAA as monitored by NMR for TbPYK is relatively slow with turn-over values of 0.86 s-1 and 1.47 s-1 in the absence and presence of fructose 2,6-bisphosphate (F26BP), respectively. Human M1PYK has a measured turn-over value of 0.50 s-1. The X-ray structures explain why the decarboxylation activity is specific for OAA and is not general for α-keto acid analogues. Conservation of the decarboxylase reaction across divergent species is a consequence of piggybacking on the conserved kinase mechanism which requires a stabilised enol intermediate

Biochemical and structural studies on trypanosomatid pyruvate kinases

Glycolytic enzymes have been indicated as potential drug targets in trypanosomatid parasites such as Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania spp. Pyruvate kinase (PYK) catalyses the final reaction in the glycolytic pathway to produce ATP and pyruvate from ADP and phosphoenolpyruvate (PEP), and has been validated by RNAi experiments as a suitable drug target in T. brucei. This thesis describes biochemical and structural studies of PYKs from T. cruzi (TcPYK) and T. brucei (TbPYK), providing not only a foundation but also new clues for PYK-specific inhibitor screening and structure-based drug design. Soluble TcPYK and TbPYK (81% sequence identity) have been expressed and purified from E. coli, and their kinetics have been fully characterised. X-ray crystal structures of apoenzyme TcPYK (apo TcPYK), and of TbPYK in complex with fructose 2,6-bisphosphate (F26BP) (TbPYK/F26BP/Mg) have been determined, and each possesses a tetrameric architecture composed of four identical protein chains. Each chain contains four domains which are A-domain, B-domain, C-domain and N-terminal domain. The active site is located in the cleft between the A- and B-domains, while the F26BP-bound effector site is within the C-domain. The conformational transition between inactive T-state and active R-state for both enzymes requires a concerted 8o rigid-body rotation of each of the four AC-cores (Aand C-domains) in the tetramer. During the T- to R-state transition induced by F26BP binding, the side chain of Arg311 is re-orientated to stabilise the short Aα6′ helix at the active site, and the flexible loop at the effector site is stabilised by F26BP. In this active conformation additional salt bridges form across the C-C interface to lock the enzyme in a more stable R-state. TbPYK/F26BP/Mg is the first ‘effector only’ PYK structure and identifies a third Mg2+ binding site (Mg-3) which is distinct from the two canonical Mg2+ binding sites. The substrate PEP was soaked into crystals of TbPYK/F26BP/Mg resulting in an ‘in crystallo’ 23° B-domain rotation forming a partially closed active site. This is accompanied by active site side-chain reorientations, and the movement of Mg2+ from its ‘priming’ position Mg-3 to its canonical position Mg-1. It is plausible that Mg2+ is retained in its ‘priming’ position after product release to act as a co-activator with F26BP to maintain the enzyme in its R-state conformation, as long as F26BP is present. The inherent oxaloacetate decarboxylase activity of PYK was reported over 30 years ago and has been further characterised by 1H NMR studies in this thesis. In addition, a series of TbPYK structures in complex with product (pyruvate), with analogues of the decarboxylase substrate oxaloacetate (D-malate and α-ketoglutarate), or with the competitive inhibitor oxalate have been determined by crystal soaking, and indicate that both decarboxylase activity and kinase activity share a common active site. A proposed mechanism explains the conserved decarboxylase activity of PYK where the active-site Mg2+ and Lys239 in TbPYK (which is conserved between species) play essential roles in the decarboxylation reaction. Three strategies for designing novel inhibitors against trypanosomatid PYKs have been proposed in this thesis. (1) Develop selective modulators to increase the binding affinity of inhibitors. As an example, F16BP has been shown to regulate the inhibitory effect of PEP analogues (oxalate, D-malate, α-ketoglutarate, malonate and L-tartrate) on TbPYK activity. (2) Develop allosteric inhibitors in order to lock trypanosomatid PYKs in an inactive state where the enzyme has low affinity for substrate binding. (3) A third strategy is to combine multiple modulators and inhibitors to increase the inhibition efficiency and selectivity

High-Precision Detection Algorithm for Metal Workpiece Defects Based on Deep Learning

Computer vision technology is increasingly being widely applied in automated industrial production. However, the accuracy of workpiece detection is the bottleneck in the field of computer vision detection technology. Herein, a new object detection and classification deep learning algorithm called CSW-Yolov7 is proposed based on the improvement of the Yolov7 deep learning network. Firstly, the CotNet Transformer structure was combined to guide the learning of dynamic attention matrices and enhance visual representation capabilities. Afterwards, the parameter-free attention mechanism SimAM was introduced, effectively enhancing the detection accuracy without increasing computational complexity. Finally, using WIoUv3 as the loss function effectively mitigated many negative influences during training, thereby improving the model’s accuracy faster. The experimental results manifested that the [email protected] of CSW-Yolov7 reached 93.3%, outperforming other models. Further, this study also designed a polyhedral metal workpiece detection system. A large number of experiments were conducted in this system to verify the effectiveness and robustness of the proposed algorithm

Application of Treatment for Deep Hole Drilling Debris in Gas-Rich Soft Coal Seams

Hole accidents occurring in the drilling process and borehole collapse encountered in gas production are obstacles standing in the way of high efficiency of gas drainage through the outburst-prone soft coal seams in China. The fundamental testing data from existing suspending agents were combined to prepare a suspension in a specific density for testing debris disposal technology considering several adverse effects. Those effects include the sizeable residual volume of drilling debris, difficult debris disposal, the low effective utilization rate of drill holes, and the unideal extraction effect in drill holes with long-distance downward drill holes. The testing outcomes indicated that the suspension-aided debris disposal could contribute to the following aspects: discharging residual drilling debris out of the drill hole, rinsing the drill hole, preventing drilling debris from filling the coal seam, providing the adequate drill hole depth, and improving the effects of gas extraction. As a result, the gas production in shallow drill holes was increased by 40%, with gas concentration elevated by 21%. In addition, the scalar quantity and extraction concentration in the gas concentration of deep drill holes were increased by 84% and 260%, respectively. This study was undertaken to provide a specific reference for debris disposal work of deep drill holes for gas-rich soft coal seams

Synthesis and Cytotoxicity Evaluation of Naphthalimide Derived N-Mustards

A series of N-mustards, which was conjugated to mono- or bis-naphthalimides with a flexible amine link, were synthesized and evaluated for cytotoxicity against five cancer cell lines (HCT-116, PC-3, U87 MG, Hep G2 and SK-OV-3). Several compounds displayed better activities than the control compound amonafide. Further evaluations by fluorescence spectroscopy studies and DNA-interstrand cross-linking assays revealed that the derivatives showed both alkylating and intercalating properties. Among the derivatives, the bis-naphthalimide N-mustard derivative 11b was found to exhibit the highest cytotoxic activity and DNA cross-linking ability. Both 11b and 7b induce HCT-116 cell apoptosis by S phase arrest