Biology subjects

Bates, P. D.

Publications and source records attributed to Bates, P. D..

7 recordsLinked to original sources

bioRxiv2024

PII interactions with BADC and BCCP proteins co-regulate lipid and nitrogen metabolism in Arabidopsis

In plants the initiation of fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACCase) which produces malonyl-CoA. The heteromeric form of ACCase (htACCase) is a holoenzyme consisting of biotin carboxylase and carboxyltransferase sub-complexes, both of which are subject to extensive regulation. Biotin carboxylase activity is controlled in part by the presence of the catalytic biotin carboxyl carrier proteins (BCCP1/2) and/or the non-catalytic, non-biotinylated, biotin/lipoyl attachment domain-containing proteins (BADC1/2/3) that associate with backbone biotin carboxylase (BC) protein. However, the mechanisms regulating BADC and BCCP interaction with BC and thus ACCase activity in planta are not clear. Here we demonstrate the Arabidopsis thaliana regulatory protein PII modulates htACCase activity through independent interactions with BADC and BCCP proteins in a selective manner. Analysis of badc1/2 and badc1/3 mutant lines and the respective pii triple mutants reveal that changes in seed oil and protein accumulation of badc double mutants are PII/nitrogen dependent. Absolute quantification of htACCase subunits and PII in developing seeds suggests that Arabidopsis exerts tight regulation over individual protein stoichiometry to balance oil and protein accumulation. The effects on vegetative and seed development indicate PII and BADC proteins have distinct but overlapping roles in the regulation of plant metabolism.

plant biology↗

bioRxiv2024

TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant

BackgroundHigh-risk neuroblastoma (HR-NBL) is an aggressive tumor of the sympathetic nervous system with high risk of relapse and poor overall survival. Allogeneic hematopoietic cell transplant (allo-HCT) has been used previously in HR-NBL patients; however, graft-versus-host-disease (GVHD) and disease progression have limited clinical application. Ex-vivo stimulated allogeneic natural killer (NK) cells represent a potential approach to enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD but have not shown efficacy in NBL. MethodsEx-vivo stimulated NK cells from C57BL/6NCr (B6) mice were expanded with soluble IL-15/IL-15R alone or with irradiated CD137L/CD54+ AgN2a-4P (15-4P) at a 1:1 ratio for 10-12 days. Allogeneic NK cells were then analyzed for activation, proliferation, cytokine production, and cytotoxicity against two murine NBL cell lines, Neuro2a and NXS2, in the absence or presence of anti-TIM-3. Lethally irradiated B6AJF1 Mice received allo-HCT from B6 donors followed by NBL challenge after 7 days to mimic tumor relapse. Select groups received anti-TIM-3 starting on day 9 for every 4 days with/without infusions of 15-4P B6 NK cells on days 14, 21, and 28. In select experiments, T cell and NK cells were selectively depleted to establish their contribution to the GVT effect. All groups were analyzed for tumor growth, GVHD and overall survival. ResultsCo-culturing NK cells with 15-4P results in 78-fold expansion with increased expression of Ki-67 and NKG2D, NKp46, TRAIL and TIM-3. 15-4P stimulated allogeneic NK cells showed enhanced cytotoxicity against NBL compared to IL-15 NK cells alone but was limited in part due to high expression of TIM-3 ligands on Neuro-2a compared to NXS2. The addition of TIM-3 blockade further enhanced NK cytotoxicity versus Neuro-2a, with enhanced 15-4P NK cell degranulation, Eomes, TRAIL and FasL expression observed. Analysis of RNA from 15-4P NK cells exposed to TIM-3 blockade showed gene expression of chemokines, NKG2D/DAP12 signaling, non-canonical NF-{kappa}b pathway and TRAIL signaling. Blockade of NKG2D, TRAIL or FasL on 15-4P NK cells abrogated cytotoxicity. In vivo, the combination of 15-4P stimulated allogeneic NK cells and TIM-3 blockade after allo-HCT resulted in prolonged survival against NBL with decreased tumor burden compared to NK cells or anti-TIM-3 alone, without inducing GVHD. Depletion of NK cells, but not T cells, abrogated the GVT effect. ConclusionAllo-HCT can be a platform for treating NBL using combination ex-vivo stimulated allogeneic NK cell therapy with TIM-3 blockade to enhance the GVT effect without inducing GVHD. Ethics StatementThe animal study M005915 was reviewed and approved by University of Wisconsin-Madison IACUC. WHAT IS ALREADY KNOWN ON THIS TOPICO_LIT cell depleted (TCD) allogeneic hematopoietic cells transplant (allo-HCT) has potential to be a salvage therapy for relapsed/refractory neuroblastoma (NBL) through the graft-versus-tumor (GVT) effect, however, the high incidence of graft-vs-host disease (GVHD) and disease progression has hindered widespread clinical application. Allogeneic NK cell therapy can be a safe and feasible treatment but has had limited efficacy in NBL. TIM-3 blockade has shown encouraging results for a variety of tumors but has not been explored for NBL nor used to enhance the GVT effect. C_LI WHAT THIS STUDY ADDSO_LIEx vivo stimulated allogeneic NK cells demonstrate robust expansion, proliferation, activation and cytotoxicity against NBL which is further augmented by the addition of TIM-3 checkpoint blockade. In an allo-HCT model of NBL relapse, mice treated with IL-15/IL-15R/AgN2a-4P (15-4P) stimulated NK cells and anti-TIM-3 exhibited prolonged overall survival and reduced tumor growth without exacerbating GVHD. C_LI HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYO_LIRelapse of NBL after TCD allo-HCT can be successfully treated with the combination of adoptively transferred 15-4P stimulated allogeneic NK cells and TIM-3 checkpoint blockade, representing a novel therapeutic approach that can be translated to the clinic or used as a platform to treat other solid tumors with high TIM-3 ligand expression. C_LI

immunology↗

bioRxiv2024

Self-incompatibility based functional genomics for rapid phenotypic characterization of seed metabolism genes

Reverse-genetic characterization of plant gene function through technologies such as CRISPR/Cas, RNAi, or gene overexpression requires the ability to efficiently transform the plant species of interest. However, efficient transformation systems are not available for most plant species. Physaria fendleri is an oilseed plant valued for its unusual hydroxylated fatty acids (HFA, e.g. lesquerolic acid) that accumulates up to 60% of seed oil and is a non-toxic alternative to castor (Ricinus communis) seeds as a source for HFA for the chemical industry. Domestication and improvement of P. fendleri seed oil requires characterization of genes involved in developing seed metabolism. Tissue culture-based transformation of P. fendleri is laborious, low-efficiency, and time-consuming (T1 [~]18 months). Additionally, P. fendleri is self-incompatible requiring laborious hand pollination for propagation and seed collection from transgenic lines. We developed a rapid virus-induced gene silencing (VIGS) method to characterize genes within developing seeds. Identification of the self-incompatibility mechanisms in P. fendleri allowed the use of self-compatibility as a novel visual selectable marker by co-targeting the gene of interest (GOI) with the self-incompatibility gene S-locus receptor kinase (SRK). Seeds develop without cross-pollination from silenced SRK and each of those seeds contain the GOI silenced, allowing rapid phenotypic characterization of the seeds in the first generation. Through this methodology we confirmed the in vivo function of two key genes (FAH12, FAE1) involved in lesquerolic acid production. Thus, this self-compatibility based functional genomics approach is a rapid methodology for in vivo reverse-genetic gene characterization in self-incompatible plants.

plant biology↗

bioRxiv2023

The α/β hydrolase domain-containing protein 1 (ABHD1) acts as a lysolipid lipase and is involved in lipid droplet formation

Lipid droplets (LDs) are the major sites of lipid and energy homeostasis. However, few LD biogenesis proteins have been identified. Here, using Chlamydomonas as a model, we show that ABHD1, a member of the /{beta} hydrolase domain-containing protein family, is a novel type of LD-associated protein which stimulates LD formation through two distinct actions on the LD surface, one enzymatic and the other structural. ABHD1 was localized to LD surface in Chlamydomonas cells. The knockout mutants contained similar amounts of triacylglycerols (TAG) but their LDs showed an increased content in lyso- derivatives of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). Over-expression of ABHD1 in Chlamydomonas induced LD formation and boosted TAG content, suggesting a key role in LD biogenesis. The purified recombinant ABHD1 protein hydrolyzed lyso-DGTS, producing a free fatty acid and a glyceryltrimethylhomoserine moiety. In vitro experiments using droplet- embedded vesicles showed that ABHD1 promoted LD emergence. Taken together, these results identify ABHD1 as a new player in LD formation by its lipase activity on lyso-DGTS and by its distinct biophysical property. This study further suggests that lipases targeted to LDs and able to act on their polar lipid coat may be interesting tools to promote LD assembly in eukaryotic cells. Significant statementLipid droplets are subcellular organelles specialized for triacylglycerol storage. Their dynamic turnover is key to managing energy homeostasis in response to cell cycle states and environmental cues. To gain insights into LD biogenesis, we characterized a putative /{beta}- hydrolase (ABHD1) in the model algae Chlamydomonas reinhardtii and show it is located at the LD surface. We found that ABHD1 overexpression promotes LD formation and acts as a lipase mainly on lyso derivatives of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), the major lipid constituent of the LD hemi-membrane. We also show that ABHD1 has a remarkable biophysical property favoring LD budding. This work thus identifies a novel type of lipase acting on betaine lipid and provides a first example of a protein with a dual function nvolved in LD formation.

biochemistry↗

bioRxiv2023

Triacylglycerol stability limits futile cycles and inhibition of carbon capture in oil-accumulating tobacco leaves

Engineering plant vegetative tissue to accumulate triacylglycerols (TAG, e.g., oil) can increase the amount of oil harvested per acre to levels that exceed current oilseed crops. Engineered Nicotiana tabacum (tobacco) lines that accumulate 15% to 30% oil of leaf dry weight resulted in starkly different metabolic phenotypes. In-depth analysis of the leaf lipid accumulation and 14CO2 metabolic tracing mechanistically described metabolic adaptations to the leaf oil engineering. An oil-for-membrane lipid tradeoff in the 15% oil line (referred to as HO) was surprisingly not further exacerbated when lipid production was enhanced to 30% (LEC2 line). The HO line exhibited a futile cycle that limited TAG yield through exchange with starch, altered carbon flux into various metabolite pools and end products, and suggested overlapping pathways of the glyoxylate cycle and photorespiration that limited CO2 assimilation by 50%. In contrast, inclusion of the LEC2 transcription factor in tobacco improved TAG stability, alleviated the TAG-to-starch futile cycle, and recovered CO2 assimilation and plant growth comparable to wild type but with much higher lipid levels in the leaves. Thus, the unstable production of storage reserves and futile cycling limit vegetative oil engineering approaches. The capacity to overcome futile cycles and maintain enhanced stable TAG levels in LEC2 demonstrated the importance of considering unanticipated metabolic adaptations while engineering vegetative oil crops.

plant biology↗

bioRxiv2023

Comparative omics reveals unanticipated metabolic rearrangements in a high-oil mutant of plastid acetyl-CoA carboxylase

Heteromeric acetyl-CoA carboxylase (ACCase) catalyzes the ATP-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, the committed step for de novo fatty acid synthesis. In plants, ACCase activity is controlled at multiple levels, including negative regulation by biotin attachment domain-containing (BADC) proteins, of which the badc1/3 double mutant leads to increased seed triacylglycerol accumulation. Unexpectedly, the Arabidopsis badc1/3 mutant also accumulates more protein. The metabolic consequences from both higher oil and protein was investigated in developing badc1/3 seed using global transcriptomics, translatomics, proteomics, and metabolomics. Changes include: reduced plastid pyruvate dehydrogenase; increased acetyl-CoA synthetase; increased storage and lipid-droplet packaging proteins; increased lipases; and increased {beta}-oxidation fatty acid catabolism. We present a model of how Arabidopsis adapted to deregulated ACCase, limiting total oil accumulation, and altering flux through pathways of carbon accumulation that presents possible targets for future bioengineering of valuable seed storage reserves.

plant biology↗

bioRxiv2020

Shifting carbon flux from non-transient starch to lipid allows oil accumulation in transgenic tobacco leaves

Plant leaf biomass is composed predominantly of carbohydrate and protein with less than 5% dry weight allocated to lipid and less than 1% of total lipid in the form of triacylglycerols (TAGs). The combined overexpression of multiple genes involved in different aspects of TAG synthesis and stabilization can result in TAG accumulation to over 30% dry weight in tobacco leaves, presumably requiring many metabolic adjustments within plant cells. The metabolic consequences to the combined source and sink capacities of high oil accumulating transgenic tobacco leaves compared to wild-type were inspected across development and photoperiod by utilizing foliar biomass components and 13CO2 flux through central carbon intermediates. Lipid biosynthesis was investigated through assessment of acyl-acyl carrier protein (ACP) pools using a recently derived quantification method that was extended to accommodate isotopic labeling. Lipids accumulated stepwise over plant development in the high-oil leaves, with 13CO2-labeling studies confirming increased carbon flux to lipids. The large increase in lipid content was concurrent with a decrease in foliar starch, with limited contribution from non-sucrose soluble sugars, indicating a redirection of carbon from starch to lipids. Starch accumulated non-transiently with plant age in wild-type leaves, suggesting an inherent capacity for a developmentally-regulated carbon sink in tobacco leaves that may have enabled the programmed altered carbon partitioning to lipids in transgenics. These studies provide insight into the metabolic plasticity of dual source-sink leaves over development and may in part explain recent successful leaf lipid engineering efforts in tobacco. One sentence summaryEngineering high oil accumulation in tobacco leaves is enabled by inherent source-sink plasticity associated with non-transient foliar starch accumulation over development.

plant biology↗