Transketolase drives the development of aortic dissection by impairing mitochondrial bioenergetics.

AIM: Aortic dissection (AD) is a disease with rapid onset but with no effective therapeutic drugs yet. Previous studies have suggested that glucose metabolism plays a critical role in the progression of AD. Transketolase (TKT) is an essential bridge between glycolysis and the pentose phosphate pathway. However, its role in the development of AD has not yet been elucidated. In this study, we aimed to explore the role of TKT in AD. METHODS: We collected AD patients' aortic tissues and used high-throughput proteome sequencing to analyze the main factors influencing AD development. We generated an AD model using BAPN in combination with angiotensin II (Ang II) and pharmacological inhibitors to reduce TKT expression. The effects of TKT and its downstream mediators on AD were elucidated using human aortic vascular smooth muscle cells (HAVSMCs). RESULTS: We found that glucose metabolism plays an important role in the development of AD and that TKT is upregulated in patients with AD. Western blot and immunohistochemistry confirmed that TKT expression was upregulated in mice with AD. Reduced TKT expression attenuated AD incidence and mortality, maintained the structural integrity of the aorta, aligned elastic fibers, and reduced collagen deposition. Mechanistically, TKT was positively associated with impaired mitochondrial bioenergetics by upregulating AKT/MDM2 expression, ultimately contributing to NDUFS1 downregulation. CONCLUSION: Our results provide new insights into the role of TKT in mitochondrial bioenergetics and AD progression. These findings provide new intervention options for the treatment of AD.

Neocortical neurogenesis in development and evolution-Human-specific features.

In this review, we focus on human-specific features of neocortical neurogenesis in development and evolution. Two distinct topics will be addressed. In the first section, we discuss the expansion of the neocortex during human evolution and concentrate on the human-specific gene ARHGAP11B. We review the ability of ARHGAP11B to amplify basal progenitors and to expand a primate neocortex. We discuss the contribution of ARHGAP11B to neocortex expansion during human evolution and its potential implications for neurodevelopmental disorders and brain tumors. We then review the action of ARHGAP11B in mitochondria as a regulator of basal progenitor metabolism, and how it promotes glutaminolysis and basal progenitor proliferation. Finally, we discuss the increase in cognitive performance due to the ARHGAP11B-induced neocortical expansion. In the second section, we focus on neocortical development in modern humans versus Neanderthals. Specifically, we discuss two recent findings pointing to differences in neocortical neurogenesis between these two hominins that are due to a small number of amino acid substitutions in certain key proteins. One set of such proteins are the kinetochore-associated proteins KIF18a and KNL1, where three modern human-specific amino acid substitutions underlie the prolongation of metaphase during apical progenitor mitosis. This prolongation in turn is associated with an increased fidelity of chromosome segregation to the apical progenitor progeny during modern human neocortical development, with implications for the proper formation of radial units. Another such key protein is transketolase-like 1 (TKTL1), where a single modern human-specific amino acid substitution endows TKTL1 with the ability to amplify basal radial glia, resulting in an increase in upper-layer neuron generation. TKTL1's ability is based on its action in the pentose phosphate pathway, resulting in increased fatty acid synthesis. The data imply greater neurogenesis during neocortical development in modern humans than Neanderthals due to TKTL1, in particular in the developing frontal lobe.

Posttranslational Acylations of the Rat Brain Transketolase Discriminate the Enzyme Responses to Inhibitors of ThDP-Dependent Enzymes or Thiamine Transport.

Transketolase (TKT) is an essential thiamine diphosphate (ThDP)-dependent enzyme of the non-oxidative branch of the pentose phosphate pathway, with the glucose-6P flux through the pathway regulated in various medically important conditions. Here, we characterize the brain TKT regulation by acylation in rats with perturbed thiamine-dependent metabolism, known to occur in neurodegenerative diseases. The perturbations are modeled by the administration of oxythiamine inhibiting ThDP-dependent enzymes in vivo or by reduced thiamine availability in the presence of metformin and amprolium, inhibiting intracellular thiamine transporters. Compared to control rats, chronic administration of oxythiamine does not significantly change the modification level of the two detected TKT acetylation sites (K6 and K102) but doubles malonylation of TKT K499, concomitantly decreasing 1.7-fold the level of demalonylase sirtuin 5. The inhibitors of thiamine transporters do not change average levels of TKT acylation or sirtuin 5. TKT structures indicate that the acylated residues are distant from the active sites. The acylations-perturbed electrostatic interactions may be involved in conformational shifts and/or the formation of TKT complexes with other proteins or nucleic acids. Acetylation of K102 may affect the active site entrance/exit and subunit interactions. Correlation analysis reveals that the action of oxythiamine is characterized by significant negative correlations of K499 malonylation or K6 acetylation with TKT activity, not observed upon the action of the inhibitors of thiamine transport. However, the transport inhibitors induce significant negative correlations between the TKT activity and K102 acetylation or TKT expression, absent in the oxythiamine group. Thus, perturbations in the ThDP-dependent catalysis or thiamine transport manifest in the insult-specific patterns of the brain TKT malonylation and acetylations.

Human papillomavirus type 16 E6 promotes cervical cancer proliferation by upregulating transketolase enzymatic activity through the activation of protein kinase B.

Over 99% of precancerous cervical lesions are associated with human papillomavirus (HPV) infection, with HPV types 16 and 18 (especially type 16) found in over 70% of cervical cancer cases globally. E6, a critical HPV gene, triggers malignant proliferation by degrading p53; however, this mechanism alone cannot fully explain the oncogenic effects of HPV16 E6. Therefore, we aimed to investigate new targets of HPV oncogenic mechanisms. Our results revealed significant changes in nonoxidative pentose phosphate pathway (PPP) metabolites in HPV16-positive cells. However, the role of nonoxidative PPP in HPV-associated cell transformation and tumor development remained unexplored. In this study, we investigated the impact and mechanisms of HPV16 E6 on cervical cancer proliferation using the HPV-negative cervical cancer cell line (C33A). HPV16 E6 was found to promote cervical cancer cell proliferation both in vitro and in vivo, activating the nonoxidative PPP. Transketolase (TKT), a key enzyme in the nonoxidative PPP, is highly expressed in cervical cancer tissues and associated with poor prognosis. HPV16 E6 promotes cervical cancer cell proliferation by upregulating TKT activity through the activation of AKT. In addition, oxythiamine (OT), a TKT inhibitor, hindered tumor growth, with enhanced effects when combined with cisplatin (DDP). In conclusion, HPV16 E6 promotes cervical cancer proliferation by upregulating TKT activity through the activation of AKT. OT demonstrates the potential to inhibit HPV16-positive cervical cancer growth, and when combined with DDP, could further enhance the tumor-suppressive effect of DDP.

Enrichment of grape berries and tomato fruit with health-promoting tartaric acid by expression of the Vitis vinifera transketolase VvTK2 gene.

Tartaric acid (TA) is a major non-fermentable plant soluble acid that abundantly occur in grapes and wines, imparting low pH and tart flavour to berries thereby regulating numerous quality attributes of wine, such as flavour, microbial stability, and aging potential. Evaluation of acidity in mature fruits of 21 wine grape (Vitis vinifera) varieties revealed significant variation between 'Beichun' and 'Gewürztraminer', which was correlated with TA content. RNA-seq analysis of fruits from the two cultivars at different developmental stages revealed that a transketolase gene, VvTK2, was significantly dominantly expressed in the high TA phenotype 'Beichun' variety. Subcellular localization assay showed that VvTK2 protein was located in the chloroplast. Virus-induced VvTK2 gene silencing significantly decreased the expression of 2-keto-L-gulonic acid reductase (Vv2-KGR) as well as L-idonate dehydrogenase (VvL-IdnDH3) and inhibited TA accumulation, while its transient over-expression in grape showed the opposite results. Heterologous VvTK2 over-expression in tomato demonstrated its obvious capacity to induce TA synthesis. Overall, these results highlights a novel role of VvTK2 in modulating TA biosynthesis, which could be an excellent strategy for future genetic improvement of grape flavour.

Structural determination and kinetic analysis of the transketolase from Vibrio vulnificus reveal unexpected cooperative behavior.

Vibrio vulnificus (vv) is a multidrug-resistant human bacterial pathogen whose prevalence is expected to increase over the years. Transketolases (TK), transferases catalyzing two reactions of the nonoxidative branch of the pentose-phosphate pathway and therefore linked to several crucial metabolic pathways, are potential targets for new drugs against this pathogen. Here, the vvTK is crystallized and its structure is solved at 2.1 Å. A crown of 6 histidyl residues is observed in the active site and expected to participate in the thiamine pyrophosphate (cofactor) activation. Docking of fructose-6-phosphate and ferricyanide used in the activity assay, suggests that both substrates can bind vvTK simultaneously. This is confirmed by steady-state kinetics showing a sequential mechanism, on the contrary to the natural transferase reaction which follows a substituted mechanism. Inhibition by the I38-49 inhibitor (2-(4-ethoxyphenyl)-1-(pyrimidin-2-yl)-1H-pyrrolo[2,3-b]pyridine) reveals for the first time a cooperative behavior of a TK and docking experiments suggest a previously undescribed binding site at the interface between the pyrophosphate and pyridinium domains.

Elevated FBXL6 expression in hepatocytes activates VRK2-transketolase-ROS-mTOR-mediated immune evasion and liver cancer metastasis in mice.

Metastatic hepatocellular carcinoma (HCC) is the most lethal malignancy and lacks effective treatment. FBXL6 is overexpressed in human hepatocellular carcinoma (HCC), but whether this change drives liver tumorigenesis and lung metastasis in vivo remains unknown. In this study, we aimed to identify FBXL6 (F-Box and Leucine Rich Repeat Protein 6) as a key driver of HCC metastasis and to provide a new paradigm for HCC therapy. We found that elevated FBXL6 expression in hepatocytes drove HCC lung metastasis and was a much stronger driver than Kras mutation (KrasG12D/+;Alb-Cre), p53 haploinsufficiency (p53+/-) or Tsc1 loss (Tsc1fl/fl;Alb-Cre). Mechanistically, VRK2 promoted Thr287 phosphorylation of TKT and then recruited FBXL6 to promote TKT ubiquitination and activation. Activated TKT further increased PD-L1 and VRK2 expression via the ROS-mTOR axis, leading to immune evasion and HCC metastasis. Targeting or knockdown of TKT significantly blocked FBXL6-driven immune evasion and HCC metastasis in vitro and in vivo. Notably, the level of active TKT (p-Thr287 TKT) was increased and was positively correlated with the FBXL6 and VRK2 expression levels in HCC patients. Our work provides novel mechanistic insights into FBXL6-driven HCC metastasis and suggests that targeting the TKT-ROS-mTOR-PD-L1/VRK2 axis is a new paradigm for treating patients with metastatic HCC with high FBXL6 expression.

Expression of cancer-testis antigens in the immune microenvironment of non-small cell lung cancer.

The antigenic repertoire of tumors is critical for successful anti-cancer immune response and the efficacy of immunotherapy. Cancer-testis antigens (CTAs) are targets of humoral and cellular immune reactions. We aimed to characterize CTA expression in non-small cell lung cancer (NSCLC) in the context of the immune microenvironment. Of 90 CTAs validated by RNA sequencing, eight CTAs (DPEP3, EZHIP, MAGEA4, MAGEB2, MAGEC2, PAGE1, PRAME, and TKTL1) were selected for immunohistochemical profiling in cancer tissues from 328 NSCLC patients. CTA expression was compared with immune cell densities in the tumor environment and with genomic, transcriptomic, and clinical data. Most NSCLC cases (79%) expressed at least one of the analyzed CTAs, and CTA protein expression correlated generally with RNA expression. CTA profiles were associated with immune profiles: high MAGEA4 expression was related to M2 macrophages (CD163) and regulatory T cells (FOXP3), low MAGEA4 was associated with T cells (CD3), and high EZHIP was associated with plasma cell infiltration (adj. P-value < 0.05). None of the CTAs correlated with clinical outcomes. The current study provides a comprehensive evaluation of CTAs and suggests that their association with immune cells may indicate in situ immunogenic effects. The findings support the rationale to harness CTAs as targets for immunotherapy.

Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography.

Phosphoketolase and transketolase are thiamine diphosphate-dependent enzymes and play a central role in the primary metabolism of bifidobacteria: the bifid shunt. The enzymes both catalyze phosphorolytic cleavage of xylulose 5-phosphate or fructose 6-phosphate in the first reaction step, but possess different substrate specificity in the second reaction step, where phosphoketolase and transketolase utilize inorganic phosphate (Pi) and D-ribose 5-phosphate, respectively, as the acceptor substrate. Structures of Bifidobacterium longum phosphoketolase holoenzyme and its complex with a putative inhibitor, phosphoenolpyruvate, were determined at 2.5 Šresolution by serial femtosecond crystallography using an X-ray free-electron laser. In the complex structure, phosphoenolpyruvate was present at the entrance to the active-site pocket and plugged the channel to thiamine diphosphate. The phosphate-group position of phosphoenolpyruvate coincided well with those of xylulose 5-phosphate and fructose 6-phosphate in the structures of their complexes with transketolase. The most striking structural change was observed in a loop consisting of Gln546-Asp547-His548-Asn549 (the QN-loop) at the entrance to the active-site pocket. Contrary to the conformation of the QN-loop that partially covers the entrance to the active-site pocket (`closed form') in the known crystal structures, including the phosphoketolase holoenzyme and its complexes with reaction intermediates, the QN-loop in the current ambient structures showed a more compact conformation with a widened entrance to the active-site pocket (`open form'). In the phosphoketolase reaction, the `open form' QN-loop may play a role in providing the binding site for xylulose 5-phosphate or fructose 6-phosphate in the first step, and the `closed form' QN-loop may help confer specificity for Pi in the second step.

New Role of Water in Transketolase Catalysis.

Transketolase catalyzes the interconversion of keto and aldo sugars. Its coenzyme is thiamine diphosphate. The binding of keto sugar with thiamine diphosphate is possible only after C2 deprotonation of its thiazole ring. It is believed that deprotonation occurs due to the direct transfer of a proton to the amino group of its aminopyrimidine ring. Using mass spectrometry, it is shown that a water molecule is directly involved in the deprotonation process. After the binding of thiamine diphosphate with transketolase and its subsequent cleavage, a thiamine diphosphate molecule is formed with a mass increased by one oxygen molecule. After fragmentation, a thiamine diphosphate molecule is formed with a mass reduced by one and two hydrogen atoms, that is, HO and H2O are split off. Based on these data, it is assumed that after the formation of holotransketolase, water is covalently bound to thiamine diphosphate, and carbanion is formed as a result of its elimination. This may be a common mechanism for other thiamine enzymes. The participation of a water molecule in the catalysis of the one-substrate transketolase reaction and a possible reason for the effect of the acceptor substrate on the affinity of the donor substrate for active sites are also shown.

Overexpression of a predicted transketolase gene and disruption of an α-1,3-glucan synthase gene in Aspergillus oryzae DGLA3 strain enhances the yield of free dihomo-γ-linolenic acid.

Free dihomo-γ-linolenic acid (DGLA), a polyunsaturated free fatty acid (FFA), can potentially be used to produce eicosanoid pharmaceuticals, such as prostaglandin E1. Previously, we constructed an Aspergillus oryzae mutant strain, named DGLA3, which produced free DGLA at an increased yield by faaA gene disruption and cooverexpression of one elongase and two desaturase genes. In this study, we achieved a further increase. Since FFA production is increased by enhancing the pentose phosphate pathway, we overexpressed a predicted transketolase gene composing the pathway in DGLA3, which consequently increased the free DGLA yield by 1.9-fold to 403 mg/L. Additionally, we disrupted the α-1,3-glucan synthase gene agsB involved in cell-wall biosynthesis, which further increased it by 1.3-fold to 533 mg/L. Overall, the yield increased by 2.5-fold. Free DGLA productivity and biomass increased similarly, but residual glucose concentration decreased. Increased hyphal dispersion appeared to cause additional glucose consumption, resulting in an increase in biomass and yield.

Structural and biochemical characterizations of Thermus thermophilus HB8 transketolase producing a heptulose.

Transketolase is a key enzyme in the pentose phosphate pathway in all organisms, recognizing sugar phosphates as substrates. Transketolase with a cofactor of thiamine pyrophosphate catalyzes the transfer of a 2-carbon unit from D-xylulose-5-phosphate to D-ribose-5-phosphate (5-carbon aldose), giving D-sedoheptulose-7-phosphate (7-carbon ketose). Transketolases can also recognize non-phosphorylated monosaccharides as substrates, and catalyze the formation of non-phosphorylated 7-carbon ketose (heptulose), which has attracted pharmaceutical attention as an inhibitor of sugar metabolism. Here, we report the structural and biochemical characterizations of transketolase from Thermus thermophilus HB8 (TtTK), a well-characterized thermophilic Gram-negative bacterium. TtTK showed marked thermostability with maximum enzyme activity at 85 °C, and efficiently catalyzed the formation of heptuloses from lithium hydroxypyruvate and four aldopentoses: D-ribose, L-lyxose, L-arabinose, and D-xylose. The X-ray structure showed that TtTK tightly forms a homodimer with more interactions between subunits compared with transketolase from other organisms, contributing to its thermal stability. A modeling study based on X-ray structures suggested that D-ribose and L-lyxose could bind to the catalytic site of TtTK to form favorable hydrogen bonds with the enzyme, explaining the high conversion rates of 41% (D-ribose) and 43% (L-lyxose) to heptulose. These results demonstrate the potential of TtTK as an enzyme producing a rare sugar of heptulose. KEY POINTS: • Transketolase catalyzes the formation of a 7-carbon sugar phosphate • Structural and biochemical characterizations of thermophilic transketolase were done • The enzyme could produce non-phosphorylated 7-carbon ketoses from sugars.

Loss of transketolase promotes the anti-diabetic role of brown adipose tissues.

Transketolase (TKT), an enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), bi-directionally regulates the carbon flux between the PPP and glycolysis. Loss of TKT in adipose tissues decreased glycolysis and increased lipolysis and uncoupling protein-1 (UCP1) expression, protecting mice from high-fat diet-induced obesity. However, the role of TKT in brown adipose tissue (BAT)-dependent glucose homeostasis under normal chow diet remains to be elucidated. We found that TKT ablation increased levels of glucose transporter 4 (GLUT4), promoting glucose uptake and glycogen accumulation in BAT. Using the streptozotocin (STZ)-induced diabetic mouse model, we discovered that enhanced glucose uptake due to TKT deficiency in BAT contributed to decreasing blood glucose and weight loss, protecting mice from STZ-induced diabetes. Mechanistically, TKT deficiency decreased the level of thioredoxin-interacting protein, a known inhibitor for GLUT4, by decreasing NADPH and glutathione levels and inducing oxidative stress in BAT. Therefore, our data reveal a new role of TKT in regulating the anti-diabetic function of BAT as well as glucose homeostasis.

Novel transketolase inhibitor oroxylin A suppresses the non-oxidative pentose phosphate pathway and hepatocellular carcinoma tumour growth in mice and patient-derived organoids.

BACKGROUND: Transketolase (TKT), a key rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), provides more than 85% of the ribose required for de novo nucleotide biosynthesis and promotes the development of hepatocellular carcinoma (HCC). Pharmacologic inhibition of TKT could impede HCC development and enhance treatment efficacy. However, no safe and effective TKT inhibitor has been approved. METHODS: An online two-dimensional TKT protein immobilised biochromatographic system was established for high-throughput screening of TKT ligands. Oroxylin A was found to specifically bind TKT. Drug affinity responsive target stability, cellular thermal shift assay, surface plasmon resonance, molecular docking, competitive displacement assay, and site mutation were performed to identify the binding of oroxylin A with TKT. Antitumour effects of oroxylin A were evaluated in vitro, in human xenograft mice, diethylnitrosamine (DEN)-induced HCC mice, and patient-derived organoids (PDOs). Metabolomic analysis was applied to detect the enzyme activity. Transcriptome profiling was conducted to illustrate the anti-HCC mechanism of oroxylin A. TKT knocking-down HCC cell lines and PDOs were established to evaluate the role of TKT in oroxylin A-induced HCC suppression. RESULTS: By targeting TKT, oroxylin A stabilised the protein to proteases and temperature extremes, decreased its activity and expression, resulted in accumulation of non-oxidative PPP substrates, and activated p53 signalling. In addition, oroxylin A suppressed cell proliferation, induced apoptosis and cell-cycle arrest, and inhibited the growth of human xenograft tumours and DEN-induced HCC in mice. Crucially, TKT depletion exerted identical effects to oroxylin A, and the promising inhibitor also exhibited excellent therapeutic efficacy against clinically relevant HCC PDOs. CONCLUSIONS: These results uncover a unique role for oroxylin A in TKT inhibition, which directly targets TKT and suppresses the non-oxidative PPP. Our findings will facilitate the development of small-molecule inhibitors of TKT and novel therapeutics for HCC.

New mechanisms for bacterial degradation of sulfoquinovose.

Sulfoquinovose (SQ, 6-deoxy-6-sulfo-D-glucose) is a sulfo-sugar with a ubiquitous distribution in the environment due to its production by plants and other photosynthetic organisms. Bacteria play an important role in degradation of SQ and recycling of its constituent sulfur and carbon. Since its discovery in 1963, SQ was noted to have a structural resemblance to glucose-6-phosphate and proposed to be degraded through a pathway analogous to glycolysis, termed sulfoglycolysis. Studies in recent years have uncovered an unexpectedly diverse array of sulfoglycolytic pathways in different bacteria, including one analogous to the Embden-Meyerhof-Parnas pathway (sulfo-EMP), one analogous to the Entner-Doudoroff pathway (sulfo-ED), and two involving sulfo-sugar cleavage by a transaldolase (sulfo-TAL) and transketolase (sulfo-TK), respectively, analogous to reactions in the pentose phosphate (PP) pathway. In addition, a non-sulfoglycolytic SQ degradation pathway was also reported, involving oxygenolytic C-S cleavage catalyzed by a homolog of alkanesulfonate monooxygenase (sulfo-ASMO). Here, we review the discovery of these new mechanisms of SQ degradation and lessons learnt in the study of new catabolic enzymes and pathways in bacteria.

Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals.

Neanderthal brains were similar in size to those of modern humans. We sought to investigate potential differences in neurogenesis during neocortex development. Modern human transketolase-like 1 (TKTL1) differs from Neanderthal TKTL1 by a lysine-to-arginine amino acid substitution. Using overexpression in developing mouse and ferret neocortex, knockout in fetal human neocortical tissue, and genome-edited cerebral organoids, we found that the modern human variant, hTKTL1, but not the Neanderthal variant, increases the abundance of basal radial glia (bRG) but not that of intermediate progenitors (bIPs). bRG generate more neocortical neurons than bIPs. The hTKTL1 effect requires the pentose phosphate pathway and fatty acid synthesis. Inhibition of these metabolic pathways reduces bRG abundance in fetal human neocortical tissue. Our data suggest that neocortical neurogenesis in modern humans differs from that in Neanderthals.

Complete Biosynthetic Pathway of the Phosphonate Phosphonothrixin: Two Distinct Thiamine Diphosphate-Dependent Enzymes Divide the Work to Form a C-C Bond.

Phosphonates often exhibit biological activities by mimicking the phosphates and carboxylates of biological molecules. The phosphonate phosphonothrixin (PTX), produced by the soil-dwelling bacterium Saccharothrix sp. ST-888, exhibits herbicidal activity. In this study, we propose a complete biosynthetic pathway for PTX by reconstituting its biosynthesis in vitro. Our intensive analysis demonstrated that two dehydrogenases together reduce phosphonopyruvate (PnPy) to 2-hydroxy-3-phosphonopropanoic acid (HPPA) to accelerate the thermodynamically unfavorable rearrangement of phosphoenolpyruvate (PEP) to PnPy. The next four enzymes convert HPPA to (3-hydroxy-2-oxopropyl)phosphonic acid (HOPA). In the final stage of PTX biosynthesis, the "split-gene" transketolase homologue, PtxB5/6, catalyzes the transfer of a two-carbon unit attached to the thiamine diphosphate (TPP) cofactor (provided by the acetohydroxyacid synthase homologue, PtxB7) to HOPA to produce PTX. This study reveals a unique C-C bond formation in which two distinct TPP-dependent enzymes, PtxB5/6 and PtxB7, divide the work to transfer an acetyl group, highlighting an unprecedented biosynthetic strategy for natural products.

Uridylation and the SKI complex orchestrate the Calvin cycle of photosynthesis through RNA surveillance of TKL1 in Arabidopsis.

RNA uridylation, catalyzed by terminal uridylyl transferases (TUTases), represents a conserved and widespread posttranscriptional RNA modification in eukaryotes that affects RNA metabolism. In plants, several TUTases, including HEN1 SUPPRESSOR 1 (HESO1) and UTP: RNA URIDYLYLTRANSFERASE (URT1), have been characterized through genetic and biochemical approaches. However, little is known about their physiological significance during plant development. Here, we show that HESO1 and URT1 act cooperatively with the cytoplasmic 3'-5' exoribonucleolytic machinery component SUPERKILLER 2 (SKI2) to regulate photosynthesis through RNA surveillance of the Calvin cycle gene TRANSKETOLASE 1 (TKL1) in Arabidopsis. Simultaneous dysfunction of HESO1, URT1, and SKI2 resulted in leaf etiolation and reduced photosynthetic efficiency. In addition, we detected massive illegitimate short interfering RNAs (siRNAs) from the TKL1 locus in heso1 urt1 ski2, accompanied by reduced TKL1/2 expression and attenuated TKL activities. Consequently, the metabolic analysis revealed that the abundance of many Calvin cycle intermediates is dramatically disturbed in heso1 urt1 ski2. Importantly, all these molecular and physiological defects were largely rescued by the loss-of-function mutation in RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), demonstrating illegitimate siRNA-mediated TKL silencing. Taken together, our results suggest that HESO1- and URT1-mediated RNA uridylation connects to the cytoplasmic RNA degradation pathway for RNA surveillance, which is crucial for TKL expression and photosynthesis in Arabidopsis.

Comparative proteomics analysis of human FFPE testicular tissues reveals new candidate biomarkers for distinction among azoospermia types and subtypes.

Understanding molecular mechanisms that underpin azoospermia and discovery of biomarkers that could enable reliable, non-invasive diagnosis are highly needed. Using label-free data-independent LC-MS/MS acquisition coupled with ion mobility, we compared the FFPE testicular proteome of patients with obstructive (OA) and non-obstructive azoospermia (NOA) subtypes hypospermatogenesis (Hyp) and Sertoli cell-only syndrome (SCO). Out of 2044 proteins identified based on ≥2 peptides, 61 proteins had the power to quantitatively discriminate OA from NOA and 30 to quantitatively discriminate SCO from Hyp and OA. Among these, H1-6, RANBP1 and TKTL2 showed superior potential for quantitative discrimination among OA, Hyp and SCO. Integrin signaling pathway, adherens junction, planar cell polarity/convergent extension pathway and Dectin-1 mediated noncanonical NF-kB signaling were significantly associated with the proteins that could discriminate OA from NOA. Comparison with 2 transcriptome datasets revealed 278 and 55 co-differentially expressed proteins/genes with statistically significant positive correlation. Gene expression analysis by qPCR of 6 genes (H1-6, RANBP1, TKTL2, TKTL1, H2BC1, and ACTL7B) with the highest discriminatory power on protein level and the same regulation trend with transcriptomic datasets, confirmed the proteomics results. In summary, our results suggest some underlying pathways in azoospermia and broaden the range of potential novel candidates for diagnosis. SIGNIFICANCE: Using a comparative proteomics approach on testicular tissue we have identified several pathways associated with azoospermia and a number of testis-specific and germ cell-specific proteins that have the potential to pinpoint the type of spermatogenesis failure. Furthermore, comparison with transcriptomics datasets based on genome-wide gene expression analyses of human testis specimens from azoospermia patients identified proteins that could discriminate between obstructive and non-obstructive azoospermia subtypes on both protein and mRNA levels. Up to our knowledge, this is the first integrated comparative analysis of proteomics and transcriptomics data from testicular tissues. We believe that the data from our study contributes significantly to increase the knowledge of molecular mechanisms of azoospermia and pave the way for new investigations in regards to non-invasive diagnosis.

Epitope detection in monocytes (EDIM) for liquid biopsy including identification of GD2 in childhood neuroblastoma-a pilot study.

BACKGROUND: Neuroblastoma (NB) is the most common paediatric extracranial solid malignancy. We analysed the role of the epitope detection in monocytes (EDIM) technique for liquid biopsy in NB patients. METHODS: Tumour epitopes transketolase-like 1 (TKTL1), Apo10 (DNaseX) and GD2 were assessed: expression levels in seven NB tumour samples and five NB cell lines were analysed using RT-PCR and flow cytometry. LAN-1 cells were co-cultured with blood and assessed using EDIM. Peripheral blood macrophages of patients with neuroblastoma (n = 38) and healthy individuals (control group, n = 37) were labelled (CD14+/CD16+) and assessed for TKTL1, Apo10 and GD2 using the EDIM technology. RESULTS: mRNA expression of TKTL1 and DNaseX/Apo10 was elevated in 6/7 NB samples. Spike experiments showed upregulation of TKTL1, Apo10 and GD2 in LAN-1 cells following co-culturing with blood. TKTL1 and Apo10 were present in macrophages of 36/38 patients, and GD2 in 15/19 patients. The 37 control samples were all negative. EDIM expression scores of the three epitopes allowed differentiation between NB patients and healthy individuals. CONCLUSIONS: The EDIM test might serve as a non-invasive tool for liquid biopsy in children suffering from NB. Future studies are necessary for assessing risk stratification, tumour biology, treatment monitoring, and early detection of tumour relapses.