Biology subjects

Loog, M.

Publications and source records attributed to Loog, M..

14 recordsLinked to original sources

bioRxiv2023

Bioconversion of Glucose-Rich Lignocellulosic Wood Hydrolysates to 3-Hydroxypropionic Acid and Succinic Acid using Engineered Saccharomyces cerevisiae

Background3-hydroxypropionic acid (3-HP) and succinic acid (SA) were announced as two of the top twelve value-added platform chemicals from biomass out of a group of over 300 potential compounds that could be made from biomass in a government report in 2004 (Werpy and Petersen, 2004) and in an updated report in 2010 (Bozell and Petersen, 2010). The screening criteria used in the report classified 3-HP and SA as direct petroleum replacement building block chemicals. 3-HP is a precursor to several high-value compounds, such as acrylic acid, 1,3-propanediol, acrylamide, and methyl acrylates, that ultimately end up in products such as fibers, contact lenses, diapers, fabric coatings, and other super absorbent polymers (SAPs). SA is a high-value platform chemical used in polyester production and a precursor for nylon and other bioplastics. Additionally, these reports identified pathways to building block compounds from sugars. Yeast fermentations were identified in these reports as a preferred potential pathway to 3-HP and SA production from sugars because of yeasts natural low pH tolerance. ResultsThe laboratory strain Saccharomyces cerevisiae BY4741 was engineered to produce either 3-HP or SA. These yeasts can convert fermentable sugars from glucose-rich lignocellulosic hardwood feedstocks into organic acid products such as 3-HP and SA under low pH conditions using exponential fed-batch cultivation strategies. Glucose-rich wood sugars provided a better growth environment for the engineered yeast strains, increasing production titers by 6.1 and 6.5 times for SA and 3-HP, respectively. ConclusionsThis study shows the potential of locally produced glucose-rich wood sugars to increase the production of platform chemicals necessary in the production of biobased polymers by engineered yeast cell factories.

microbiology↗

bioRxiv2023

Conversion of aromatic compounds from fractionated industrial hydrolysis lignin by Pseudomonas putida and environmental strains

BackgroundThe utilization of Pseudomonas putida was explored in this study as a promising approach for lignin valorization. To this end, dry hydrolysis lignin was used as a feedstock for the first time. Hydrolysis lignin is a product of the enzymatic hydrolysis and separation of cellulose and hemicellulose from the lignin backbone in diverse lignocellulosic sources. Various fractionation techniques were applied to obtain lignin monomers and multimers in solution for use as a growth medium for P. putida, whose tolerance of inhibitory phenolic compounds distinguishes it from most bacteria. ResultsPhysiological evaluations revealed that Pseudomonas putida strains KT2440 and PaW85 exhibited broad pH tolerance ranges, with robust growth observed at elevated pH levels. Batch fermentations using hydrolysis lignin (HL) solutions showed complete consumption of sugars within 24 hours, demonstrating the viability of fractionated HL as a substrate for P. putida cultivation. HPLC analysis of HL monomer concentrations during simulated fed-batch fermentation revealed rapid catabolism of catechol and increased CCMA concentration, followed by stabilization, indicating that CCMA is synthesized more quickly than degraded when the initial catechol concentration is high. Filtered alkaline HL fractionations yielded more than twice as much catechol as unfiltered fractionations. Screening of indigenous bacterial strains isolated from various soil and water samples (CELMS Collection, website http://eemb.ut.ee) identified five new candidate strains for CCMA production, two for PCA production, and three for vanillic acid production. ConclusionsThe novel use of fractionated hydrolysis lignin as a growth medium shows potential for lignin valorization and chemical production. Filtered alkaline fractionation yields more catechol and is superior for cis,cis-muconic acid production; however, unfiltered fractionations may be more suitable for other compounds and upscaling. Further investigation of screened strains could reveal more efficient enzymes, which could be optimized and transformed into P. putida in future research.

microbiology↗

bioRxiv2022

Designing DNA-based predictors of drug response using the signal joint with gene expression

MotivationAnti-cancer drugs may elicit resistance or sensitivity through mechanisms which involve several genomic layers. Nevertheless, we have demonstrated that gene expression contains most of the predictive capacity compared to the remaining omic data types. Unfortunately, this comes at a price: gene expression biomarkers are often hard to interpret and show poor robustness. ResultsTo capture the best of both worlds, i.e. the accuracy of gene expression and the robustness of other genomic levels, such as mutations, copy-number or methylation, we developed Percolate, a computational approach which extracts the joint signal between gene expression and the other omic data types. We developed an out-of-sample extension of Percolate which allows predictions on unseen samples without the necessity to recompute the joint signal on all data. We employed Percolate to extract the joint signal between gene expression and either mutations, copy-number or methylation, and used the out-of sample extension to perform response prediction on unseen samples. We showed that the joint signal recapitulates, and sometimes exceeds, the predictive performance achieved with each data type individually. Importantly, molecular signatures created by Percolate do not require gene expression to be evaluated, rendering them suitable to clinical applications where only one data type is available. AvailabilityPercolate is available as a Python 3.7 package and the scripts to reproduce the results are available here.

bioinformatics↗

bioRxiv2022

Identifying commonalities between cell lines and tumors at the single cell level using Sobolev Alignment of deep generative models

Preclinical models are essential to cancer research, however, key biological differences with patient tumors result in reduced translatability to the clinic and high attrition rates in drug development. Variability among and between patients, preclinical models, and individual cells obscures commonalities which could otherwise be exploited therapeutically. To discover the shared biological processes between cell line models and clinical tumors we developed Sobolev Alignment, a computational framework which uses deep generative models to capture non-linear processes in single-cell RNA sequencing data and kernel methods to align and interpret these processes. We show that our approach faithfully captures shared processes on a set of three synthetic datasets. Exploiting two large panels of untreated non-small cell lung cancer cell lines and patients, we identify the similarities between cell lines and tumors and show the conservation of key mitotic and immune-related pathways. Employing our approach on a large in-vitro perturbation screen, we show that processes captured by our method faithfully recapitulate the known modes of action of clinically approved drugs and allow investigation into the mode of action of an uncharacterized drug.

bioinformatics↗

bioRxiv2021

Cdc6 is sequentially regulated by PP2A-Cdc55, Cdc14 and Sic1 for origin licensing in S. cerevisiae

Cdc6, a subunit of the pre-replicative complex, contains multiple regulatory Cdk1 consensus sites, SP or TP motifs. In S. cerevisiae, Cdk1 phosphorylates Cdc6-T7 to recruit Cks1, the Cdk1 phospho-adaptor in S-phase, for subsequent multisite phosphorylation and protein degradation. Cdc6 accumulates in mitosis and is tightly bound by Clb2 through N-terminal phosphorylation in order to prevent premature origin licensing and degradation. It has been extensively studied how Cdc6 phosphorylation is regulated by the Cyclin-Cdk1 complex. However, a detailed mechanism on how Cdc6 phosphorylation is reversed by phosphatases has not been elucidated. Here, we show that PP2ACdc55 dephosphorylates Cdc6 N-terminal sites to release Clb2. Cdc14 dephosphorylates the C-terminal phospho-degron, leading to Cdc6 stabilization in mitosis. In addition, the Cdk1 inhibitor, Sic1, releases Clb2{middle dot}Cdk1{middle dot}Cks1 from Cdc6 to load Mcm2-7 on the chromatin upon mitotic exit. Thus, pre-RC assembly and origin licensing is promoted by the attenuation of distinct CDK-dependent Cdc6 inhibitory mechanisms.

cell biology↗

bioRxiv2021

Differentially accessible Cdc4 phospho-degrons regulate Ctf19CCAN kinetochore subunit stability in mitosis

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. How effective, yet strictly centromere-dependent kinetochore assembly is coupled to cell cycle progression is incompletely understood. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-Phase and targets the protein for degradation. Binding of the Mtw1 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature-sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic to cells, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits protects against ectopic kinetochore assembly and contributes to mitotic checkpoint silencing. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.

cell biology↗

bioRxiv2021

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates1-3. The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle4-7, together with variations in substrate docking to sites on associated cyclin and Cks subunits3, 6, 8-10. Many substrates are modified at multiple sites to provide more complex regulation9, 11-14. Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of genes required for mitotic progression15, 16. As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback17-20. Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that CLB2 expression is inhibited by high levels of Cdk1 activity in a mitotic arrest. Inhibition is accompanied by Ndd1 degradation, and we present evidence that high mitotic Cdk1-Clb2 activity generates phosphodegrons on Ndd1, leading to its degradation. Complete Ndd1 phosphorylation by the Clb2-Cdk1-Cks1 complex requires the phosphothreonine-binding site of Cks1, as well as a novel phosphate-binding pocket on the cyclin Clb221. We therefore propose that initial phosphorylation by Cdk1 primes the protein for secondary phosphorylation at phosphodegrons, resulting in degradation only at high Cdk1 activity. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.

cell biology↗

bioRxiv2020

Regulation of trehalase activity by multi-site phosphorylation and 14-3-3 interaction

Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown. Here, we study multi-site phosphorylation using the yeast trehalase Nth1 and its activator, the 14-3-3 protein Bmh1, as a model. Nth1 is known to be phosphorylated by the metabolic kinase PKA on four serine residues and by the cell cycle kinase CDK on one residue. However, how these five phospho-sites adjust Nth1 activity remains unclear. Using a novel reporter construct, we investigated the contribution of the individual sites for the regulation of the trehalase and its 14-3-3 interactor. In contrast to the constitutively phosphorylated S20 and S83, the weaker sites S21 and S60 are only phosphorylated by increased PKA activity. For binding Bmh1, S83 functions as the high-affinity "gatekeeper" site, but successful binding of the Bmh1 dimer and thus Nth1 activation requires S60 as a secondary site. Under nutrient-poor conditions with low PKA activity, S60 is not efficiently phosphorylated and the cell cycle dependent phosphorylation of S66 by Cdk1 contributes to Nth1 activity, likely by providing an alternative Bmh1 binding site. Additionally, the PKA sites S20 and S21 modulate the dephosphorylation of Nth1 on downstream Bmh1 sites. In summary, our results expand our molecular understanding of Nth1 regulation and provide a new aspect of the interaction of 14-3-3 proteins with their targets.

biochemistry↗

bioRxiv2020

PRECISE+ predicts drug response in patients by non-linear subspace-based transfer from cell lines and PDX models

Pre-clinical models have been the workhorse of cancer research for decades. While powerful, these models do not fully recapitulate the complexity of human tumors. Consequently, translating biomarkers of drug response from pre-clinical models to human tumors has been particularly challenging. To explicitly take these differences into account and enable an efficient exploitation of the vast pre-clinical drug response resources, we developed TRANSACT, a novel computational framework for clinical drug response prediction. First, TRANSACT employs non-linear manifold learning to capture biological processes active in pre-clinical models and human tumors. Then, TRANSACT builds predictors on cell line response only and transfers these to Patient-Derived Xenografts (PDXs) and human tumors. TRANSACT outperforms four competing approaches, including Deep Learning approaches, for a set of 15 drugs on PDXs, TCGA cohorts and 226 metastatic tumors from the Hartwig Medical Foundation data. For only four drugs Deep Learning outperforms TRANSACT. We further derived an algorithmic approach to interpret TRANSACT and used it to validate the approach by identifying known biomarkers to targeted therapies and we propose novel putative biomarkers of resistance to Paclitaxel and Gemcitabine.

bioinformatics↗

bioRxiv2020

Biochemical evidence of furin specificity and potential for phospho-regulation at Spike protein S1/S2 cleavage site in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV

The Spike protein of the novel coronavirus SARS-CoV2 contains an insertion 680SPRRAR{downarrow}SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous insertion was present in the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert SPRRAR{downarrow}SV can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furins ability to cleave the site. Although phosphoregulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.

molecular biology↗

bioRxiv2020

Comprehensive analysis of fungal G1 cyclin docking motif sequences that control CDK regulatory potency in vivo

Cyclin-dependent kinases (CDKs) control the ordered series of events during eukaryotic cell division. The stage at which individual CDK substrates are phosphorylated can be dictated by cyclin-specific docking motifs. In budding yeast, substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs and the conservation of this mechanism were unknown. Here we comprehensively analyzed LP motif requirements in vivo by combining a competitive growth assay with mutational scanning and deep sequencing. We quantified the impact of all single-residue replacements in five different LP motifs, using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results reveal the basis for variations in potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the potency of other candidate motifs. In one protein, Whi5, we found overlapping LP and phosphorylation motifs with partly redundant effects. In another protein, the CDK inhibitor Sic1, we found that its LP motif is inherently weak due to unfavorable residues at key positions, and this imposes a beneficial delay in its phosphorylation and degradation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in LP motif potency can tune the strength and timing of CDK regulation.

molecular biology↗

bioRxiv2019

Cell cycle-balanced expression of pluripotency regulators via cyclin-dependent kinase 1 activity

Embryonic stem cells (ESCs) have a unique ability to remain pluripotent while undergoing rapid rounds of cell division required for self-renewal. However, it is not known how cell cycle and pluripotency regulatory networks co-operate in ESCs. Here, we used stable isotope labeling with amino acids in cell culture (SILAC) combined with mass spectrometry to determine pluripotency proteome dynamics during cell cycle in mouse ESCs. We found the S/G2M-fluctuating pluripotency transcription factors (ESRRB, REST), chromatin regulators (JARID2, TRIM24) and proteins with E3 ligase activity (NEDD4L, PIAS2) to peak in S phase. This expression balance was disrupted upon inhibition of cyclin-dependent kinase 1 (CDK1) activity resulting in the shift of the expression peak from S to G2M. Our results demonstrate that mouse ESCs require CDK1 activity to maintain high S to G2M ratio of pluripotency regulators revealing critical role of cell cycle dynamics in balancing ESC identity.

cell biology↗

bioRxiv2019

PRECISE: A domain adaptation approach to transfer predictors of drug response from pre-clinical models to tumors

MotivationCell lines and patient-derived xenografts (PDX) have been used extensively to understand the molecular underpinnings of cancer. While core biological processes are typically conserved, these models also show important differences compared to human tumors, hampering the translation of findings from pre-clinical models to the human setting. In particular, employing drug response predictors generated on data derived from pre-clinical models to predict patient response, remains a challenging task. As very large drug response datasets have been collected for pre-clinical models, and patient drug response data is often lacking, there is an urgent need for methods that efficiently transfer drug response predictors from pre-clinical models to the human setting. ResultsWe show that cell lines and PDXs share common characteristics and processes with human tumors. We quantify this similarity and show that a regression model cannot simply be trained on cell lines or PDXs and then applied on tumors. We developed PRECISE, a novel methodology based on domain adaptation that captures the common information shared amongst pre-clinical models and human tumors in a consensus representation. Employing this representation, we train predictors of drug response on pre-clinical data and apply these predictors to stratify human tumors. We show that the resulting domain-invariant predictors show a small reduction in predictive performance in the pre-clinical domain but, importantly, reliably recover known associations between independent biomarkers and their companion drugs on human tumors. AvailabilityPRECISE and the scripts for running our experiments are available on our GitHub page (https://github.com/NKI-CCB/PRECISE). Contactl.wessels@nki.nl Supplementary informationSupplementary data are available. online.

bioinformatics↗

bioRxiv2018

Synthetic-evolution reveals that phosphoregulation of the mitotic kinesin-5 Cin8 is constrained

Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions. These phosphorylation sites do not appear to require precise positioning for their function. The mitotic kinesin-5 Cin8 was shown to be phosphoregulated at three Cdk1 sites in disordered loops within its catalytic motor domain. Here, we examined the flexibility of Cin8 phosphoregulation by analyzing the phenotypes of synthetic Cdk1-sites that were systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites that we created, eight were non-functional; 19 were neutral, similar to the phosphodeficient variant; and two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one site resulted in novel phosphoregulation, and only one site, immediately adjacent to a native Cdk1 site, produced phosphoregulation similar to wild-type. This study shows that, while the gain of a single phosphorylation site can confer regulation and modulate the dynamics of the spindle, to achieve optimal regulation of a mitotic kinesin-5 motor protein, phosphoregulation has to be site-specific and precise.

cell biology↗