Glucose restriction drives spatial reorganization of mevalonate metabolism

Eukaryotes compartmentalize metabolic pathways into sub-cellular domains, but the role of inter-organelle contacts in organizing metabolic reactions remains poorly understood. Here, we show that in response to acute glucose restriction (AGR) yeast undergo metabolic remodeling of their mevalonate pathway that is spatially coordinated at nucleus-vacuole junctions (NVJs). The NVJ serves as a metabolic platform by selectively retaining HMG-CoA Reductases (HMGCRs), driving mevalonate pathway flux in an Upc2-dependent manner. Both spatial retention of HMGCRs and increased mevalonate pathway flux during AGR is dependent on NVJ tether Nvj1. Furthermore, we demonstrate that HMGCRs associate into high molecular weight assemblies during AGR in an Nvj1-dependent manner. Loss of Nvj1-mediated HMGCR partitioning can be bypassed by artificially multimerizing HMGCRs, indicating NVJ compartmentalization enhances mevalonate pathway flux by promoting the association of HMGCRs in high molecular weight assemblies. Loss of HMGCR compartmentalization perturbs yeast growth following glucose starvation, indicating it promotes adaptive metabolic remodeling. Collectively we propose a non-canonical mechanism regulating mevalonate metabolism via the spatial compartmentalization of rate-limiting HMGCR enzymes at an inter-organelle contact site.

Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression

In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a ‘terminator catalog’ by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3′-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.

The Dysregulation of OGT/OGA Cycle Mediates Tau and APP Neuropathology in Down Syndrome

Protein O-GlcNAcylation is a nutrient-related post-translational modification that, since its discovery some 30 years ago, has been associated with the development of neurodegenerative diseases. As reported in Alzheimer’s disease (AD), flaws in the cerebral glucose uptake translate into reduced hexosamine biosynthetic pathway flux and subsequently lead to aberrant protein O-GlcNAcylation. Notably, the reduction of O-GlcNAcylated proteins involves also tau and APP, thus promoting their aberrant phosphorylation in AD brain and the onset of AD pathological markers. Down syndrome (DS) individuals are characterized by the early development of AD by the age of 60 and, although the two conditions present the same pathological hallmarks and share the alteration of many molecular mechanisms driving brain degeneration, no evidence has been sought on the implication of O-GlcNAcylation in DS pathology. Our study aimed to unravel for the first time the role of protein O-GlcNacylation in DS brain alterations positing the attention of potential trisomy-related mechanisms triggering the aberrant regulation of OGT/OGA cycle. We demonstrate the disruption of O-GlcNAcylation homeostasis, as an effect of altered OGT and OGA regulatory mechanism, and confirm the relevance of O-GlcNAcylation in the appearance of AD hallmarks in the brain of a murine model of DS. Furthermore, we provide evidence for the neuroprotective effects of brain-targeted OGA inhibition. Indeed, the rescue of OGA activity was able to restore protein O-GlcNAcylation, and reduce AD-related hallmarks and decreased protein nitration, possibly as effect of induced autophagy.

Glucose restriction drives spatial re-organization of mevalonate metabolism and liquid-crystalline lipid droplet biogenesis

Eukaryotes compartmentalize metabolic pathways into sub-cellular domains, but the role of inter-organelle contacts in organizing metabolic reactions remains poorly understood. Here, we show that in response to acute glucose restriction (AGR) yeast undergo metabolic remodeling of their mevalonate pathway that is spatially coordinated at nucleus-vacuole junctions (NVJs). The NVJ serves as a metabolic platform by selectively retaining HMG-CoA Reductases (HMGCRs), driving mevalonate pathway flux in an Upc2-dependent manner. AGR-induced HMGCR compartmentalization enhances mevalonate metabolism and sterol-ester biosynthesis that generates lipid droplets (LDs) with liquid-crystalline sub-architecture. Loss of NVJ-dependent HMGCR partitioning affects yeast growth, but can be bypassed by artificially multimerizing HMGCRs, indicating NVJ compartmentalization enhances mevalonate pathway flux by promoting HMGCR inter-enzyme associations. We propose a non-canonical mechanism regulating mevalonate metabolism via the spatial compartmentalization of rate-limiting HMGCR enzymes, and reveal that AGR creates LDs with remarkable phase transition properties.One Sentence SummarySpatial compartmentalization of HMG-CoA Reductases at ER-lysosome contacts modulates mevalonate pathway flux

Activity of fructose-1,6-bisphosphatase from Campylobacter jejuni

The glycolytic pathway of the enteric pathogen Campylobacter jejuni is incomplete; the absence of phosphofructokinase means that the suppression of futile cycling at this point in the glycolytic–gluconeogenic pathway might not be required, and therefore the mechanism for controlling pathway flux is likely to be quite different or absent. In this study, the characteristics of fructose-1,6-bisphosphatase (FBPase) of C. jejuni are described and the regulation of this enzyme is compared with the equivalent enzymes from organisms capable of glycolysis. The enzyme is insensitive to AMP inhibition, unlike other type I FBPases. Campylobacter jejuni FBPase also shows limited sensitivity to other glycolytic and gluconeogenic intermediates. The allosteric cooperative control of the enzyme’s activity found in type I FBPases appears to have been lost.

Pathway-flux based biomarker for immunotherapy response in melanoma.

e22064 Background: Immune checkpoint therapy (ICT) refers to therapeutic interventions that specifically target immune evasion mechanisms to restore the host immunity with anti-tumor ability. The ICT has revolutionized the immune-based treatment across > 30 different cancers including solid tumors and hematopoietic malignancies, with an ORR of 30% and a 7%-12% grades 3-5 irAEs in average. However, a substantial unmet point is the development of a biomarker with which response of ICT can be predicted before treatment for individual patients. Methods: In order to face this challenge this study developed an advanced genome-scale pathway flux analysis (GPFA) to evaluate the strength of signaling transduction and metabolic flux in immune system. The input of GPFA is the gene expression profiles of individual objects and the output of GPFA can be summarized in a index system, IM.Index, with following definition: IM.Index = 1.78E-4 * Σ flux(P) + 2.37E-4 * Σ flux(P) p ∈ signaling transduction p ∈ energy metabolism. Subsequently, the IM.Index was applied to analyze genetic data of two independent cohorts of melanoma patients treated with anti-PD-1 therapy (nivolumab a. pembrolizumab). Results: The IM.Index predicted the response of anti-PD-1 therapy (nivolumab) in the first cohort with an odds ratio (OR) of 3.14 (95%CI: 1.16-8.45; p = 3.10E-3; AUC = 0.82) and with a sensitivity 89% and specificity 76%. The prediction on overall survival (OS) of this cohort achieved an hazard ratio (HR) of 1.53 (95%CI: 1.22-1.92; p = 7.8E-3). Subsequently, the prediction result for the anti-PD-1 therapy (pembrolizumab) in the second cohort achieved an OR of 2.12 (95%CI: 1.22-3.66; p = 4.50E-4; AUC = 0.87) and the OS prediction in this cohort reached an HR of 1.24 (95%CI: 1.04-1.47; p = 1.40E-2). Comparison with other four potential biomarkers (TMB, TNB, neo-peptide load and cytolytic score) related to immunotherapy showed a comparative outcome of the IM.Index regarding diagnosis and prognosis in melanoma. For instance, IM.Index showed a superior performance on objective response rate (ORR) of 70% and AUC of 0.83. Conclusions: In conclusion this study demonstrated that a pathway flux analysis at a genome-scale may be explorative in biomarker research in immunotherapy, since this type of analysis could reflect the strength or functional status of the immune system. The IM.Index developed in this study may also be applied to investigation the treatment response of immunotherapy in other types of cancer.

Genome-scale Pathway Flux Analysis Predicts Efficacy of anti-PD-1 Therapy in Melanoma

Background: Currently, predicting treatment efficacy to immunotherapy has been under extensive investigation. However, a putative biomarker for immunotherapy in melanoma remains to be found.Methods: Utilizing genetic data from two independent melanoma patient cohorts treated with anti-PD-1 therapy, the study described herein conducted a genome-scale pathway flux analysis (GPFA) and related statistical methods to determine whether specific pathways could be identified that are relevant to immunotherapy efficacy.Results: The analysis results highlighted three mechanisms responsible for the efficacy of immunotherapy in melanoma including 1) proper cellular functions in immune cells; 2) angiogenesis for the development and differentiation of immune cells; 3) energy metabolic remodeling to meet the activation of immune cells. Based on these discoveries, a pathway flux-based biomarker (IM.Index) was developed and assessed to validate its predictive ability with odds ratio (OR) of 3.14 (95%CI: 1.16-8.45; p=3.10E-3), sensitivity 76% and specificity 89%. The IM.Index achieved an objective response rate (ORR) of 70%. Comparison to other four putative biomarkers (TMB, NAL, neo-peptide load and cytolytic score) showed a comparative outcome with an hazard ratio (HR) of 1.83 (95%CI: 1.26-2.67; p=1.62E-3) and area under curve (AUC) of 0.82.Conclusion: These results indicate a translational potential of IM.Index, as a biomarker, for anti-PD-1 therapy in melanoma and the GPFA might pave a new path for biomarker discovery in immunotherapy.

Niche-selective inhibition of pathogenic Th17 cells by targeting metabolic redundancy

SummaryTargeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.