Novel systems biology approaches for design and analysis of palmitoylation networks reveal its regulatory effects on protein stability, activity and localization

Communication and environment sensing are fundamental for the regulation of the majority of the cellular functions. In cells there are dedicated networks that are responsible for signalling, which gives the ability to the cell of sensing the external and internal environment allowing rapid response and adaptation to changes and stress events. A major component of these networks are post translational modifications. Their attachment to proteins in response to a change in the surrounding conditions can have very drastic consequences on modified proteins. Since post translational modification can heavily influence the cellular fate it is essential to identify these modifications and to understand their functioning, to better comprehend how cells respond to environments changes and different types of stress events to which they are continuously subjected. In this work we analyse a type of modification called S-palmitoylation, which is recently emerging as signalling/regulatory modification. S-Palmitoylation, or more generally âacylationâ, is the addition of an acyl chain to the SH-group of a cysteine via a thioester bond. This type of modification has a big impact at the cellular level. In different studies about palmitoylated proteins, functional consequences are observed at the cellular level, like altered signalling capacity, activity modulation, regulation of protein stability and localization. Palmitoylation was studied using a mathematical modelling approach. Based on experimental data we developed models of the palmitoylation process of different proteins. Using a bottom up approach we first investigated the consequences of protein palmitoylation on the endoplasmic reticulum chaperone calnexin, to better understand the consequences of palmitoylation at the protein level. Following the same approach we investigated the effects of palmitoylation on DHHC6, a palmitoyltransferase that is responsible for palmitoylation of calnexin, which itself undergoes palmitoylation from the palmitoyltransferase DHHC16. In the last part of the project we focused on the study of palmitoylation at the network level. For this purpose we reconstructed the palmitoylation cascade of calnexin, including in the model all the proteins involved in the palmitoylation process of this protein. This network of palmitoylation allowed us to investigate the dynamics of palmitoylation at the network level, focusing in the characterization of those mechanisms that are responsible for regulation of palmitoylation on different substrates. This work provides an unprecedented level of understanding of palmitoylation dynamics, both at the protein and network level. Moreover all the models and the tools for model analysis used in this work have been developed keeping in mind the final goal of reconstructing the entire palmitoylation network. Thanks to the approach we chose for modelling, the calnexin network model developed during this project can be easily expanded to include other portions of the palmitoylation networks. Similarly the tools developed for model analysis can be easily adapted to work on post-translational modification assays and analysis of biological functions from the smaller to the larger scales

Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin

Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non- palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC

<i>In silico</i> analysis of the calnexin species distribution.

<p><b>A:</b> We replicated <i>in silico</i> the ³⁵S pulse-chase experiment on WT calnexin. During the experiment we monitored the relative distribution of calnexin in the different palmitoylation states. Solid line is the mean of the simulations of 382 models; error bars are defined by the first and the third quartile of the simulations of 382 models (details of the <i>in silico</i> labelling experiment can be found in the Expanded View). <b>B:</b> The model was used to predict the steady state distribution of WT calnexin and of the mutants in the cell. rCAL correspond to calnexin during the synthesis, fCAL represent folded calnexin while c1CAL and c2CAL denote the two single palmitoylated species. c12CAL represents the double palmitoylated calnexin. Error bars correspond to first and third quartile of the simulations of 382 models (details of the <i>in silico</i> labelling experiment can be found in the Supplementary Information).</p

Experiments and modelling of Palmitoylation/depalmitoylation of calnexin.

<p><b>AB</b>: HeLa cells were transfected for 24h with calnexin-WT-HA, calnexin-CA-HA, or calnexin-AC-HA. Cells were incubated with ³H-palmitic acid for 2h, washed and further incubated for different times at 37°C in complete medium prior to immunoprecipitation using anti-calnexin or anti-HA antibodies. Immunoprecipitates were split into two, run on SDS-PAGE and analyzed either by autoradiography (³H-palmitate) or Western blotting (anti-HA). Autoradiograms were quantified using the Typhoon Imager (Image QuantTool, GE healthcare). Errors correspond to standard deviations (n = 3). <b>CD:</b> HeLa cells were incubated with ³H-palmitic acid for different times at 37°C, washed prior to immunoprecipitation using anti-calnexin antibodies. Immunoprecipitates were split into two, run on SDS-PAGE and analyzed either by autoradiography (³H-palmitate) or Western blotting (anti-calnexin). Errors correspond to standard deviations (n = 4). <b>E:</b> Validation of the model output though comparison of the <i>in silico</i> experiments with experimental data that was not used in the calibration of the model. Solid line is the mean of the simulations of 382 models; the shaded area is defined by the first and the third quartile of the simulations of 382 models (details of the <i>in silico</i> labelling experiment can be found in the Supplemental Information).</p

Calnexin palmitoylation model and kinetics of decay.

<p><b>A:</b> Calnexin is first synthesized (rCAL). During the folding process the protein assumes the proper conformation (fCAL), which then can be palmitoylated twice by DHHC6. The first palmitoylation can occur on either of the two sites leading to c1CAL or c2CAL. The second palmitoylation events leads to c12CAL. We assume that both palmitoylation steps are reversible. Degradation can occur from all states. <b>B-E:</b> HeLa cells were transfected or not for 24h with calnexin-WT-HA, HA-calnexin-KDEL, calnexin-CA-HA, calnexin-AC-HA, calnexin-AA-HA and with or without DHHC6-myc, or additional transfected for 72h with DHHC6 siRNA. Cells were incubated 20 min pulse at 37°C with ³⁵S-methionine/cysteine, washed and further incubated for different times at 37°C. Calnexin was immunoprecipitated and analyzed by SDS-PAGE. Autoradiography <b>(B)</b> and western blotting were quantified using the Typhoon Imager (Image QuantTool, GE healthcare). <b>C:</b> Decay profile of WT calnexin (Calx-WT) and of a calnexin mutant in which the transmembrane and cytosolic domain were removed and replaced by the KDEL sequence for ER retention (Calx-KDEL). Errors correspond to standard deviations (n = 7 for Calx-WT, n = 3 for Calx-KDEL). <b>D:</b> Decay profile of WT calnexin was observed under normal condition (Ctrl) and after overexpression (DHHC6 Overexpression) or silencing (DHHC6 Silencing) of DHHC6. Errors correspond to standard deviations (n = 7, 3, 3 for Crtl, DHHC6 overexpression and silencing respectively). <b>E:</b> Decay profile of WT calnexin (Calx-WT) and different mutants, in which site c1 (Calx-AC), site c2 (Calx-CA), or both palmitoylation sites (Calx-AA) were mutated. Errors correspond to standard deviations (n = 8, 3, 3, 3 for Calx-WT, AC, CA and AA respectively).</p

CRB1-Related Cystic Maculopathy in Twins Conceived Through Heterologous Fertilization With Variant-Carrying Oocytes

Cystic maculopathy has been associated with genetic disorders such as retinitis pigmentosa, X-linked retinoschisis, cone dystrophy, and foveal retinoschisis. Familial foveal retinoschisis was recently described as a rare disease caused by CRB1 variants. The authors report the phenotype-genotype pattern of a pair of dizygotic twins with early-onset cystic maculopathy due to CRB1 pathogenic variants. The twins were conceived by heterologous fertilization with variant-carrying oocytes. The probands were monitored for a period of 4 years. Next generation sequencing of a panel of genes responsible for retinal dystrophies was performed. Both children carried three pathogenic variants in CRB1: a novel heterozygous truncating variant p.(Val855*) inherited from the father and two known heterozygous missense variants, p.[(Phe144Val; Thr745Met)], inherited from the oocyte donor. The findings confirm that CRB1 variants can be responsible for foveal retinoschisis with variable clinical expressivity ranging from schitic macular alteration to early-onset forms of cystic maculopathy. The authors highlight the importance of exome analysis of gamete donors to assess the likelihood of recessively inherited disorders by means of a prediction algorithm able to combine parent and donor exome data. [J Pediatr Ophthalmol Strabismus. 2020;57:e19-e24.]

Kinetics of calnexin palmitoylation and stability.

<p><b>A:</b> The decay curves were predicted for the different palmitoylation species: fCAL represent folded calnexin, c1CAL and c2CAL the single palmitoylated species, c12CAL double palmitoylated calnexin. Solid line is the mean of the simulations of 382 models; shaded area is defined by the first and the third quartile of the simulations of 382 models (details of the <i>in silico</i> labelling experiment can be found in the Expanded View). <b>B:</b> Decay profiles were determined for WT calnexin either by ³⁵S Cys/Meth labelling (³⁵S calx WT) and by SNAP labeling (Calx WT SNAP). <b>C:</b> The model was simulated until it reached steady state and the values of the palmitoylation rates under steady state conditions were plotted. Error bars correspond to first and third quartile of the simulations of 382 models (details of the <i>in silico</i> labelling experiment can be found in the Expanded View). <b>D:</b> The model was simulated until it reached steady state and the values of the degradation rates under steady state conditions were plotted. Error bars were determined as in <b>(C).</b></p

Prediction of depalmitoylation kinetics.

<p><b>A:</b> WT calnexin was labelled with 3H-palmitate in silico either for two hrs or to reach the steady state distribution of palmitoylation species. <b>B:</b> The kinetics of palmitate loss starting from the two distributions in A were determined. The solid lines represent the means of the simulations of 382 models and the shaded areas are defined by the first and the third quartile of the simulations of 382 models (details of the <i>in silico</i> labelling experiment can be found in the Expanded View). <b>C:</b> The rate constants for depalmitoylation from c1CAL, c2CAL and c1c2CAL where determined. Error bars correspond to first and third quartile of the simulations of 382 models.</p

Stochastic simulations reveal the average palmitoylation time for calnexin.

<p>Single proteins were tracked in 5000 stochastic simulations of the labelling method described in Expanded View. From the simulations we estimated the average and median time requested for a single molecule of calnexin to undergo double palmitoylation.</p

Location and condition based reconstruction of colon cancer microbiome from human RNA sequencing data

Abstract Background The association between microbes and cancer has been reported repeatedly; however, it is not clear if molecular tumour properties are connected to specific microbial colonisation patterns. This is due mainly to the current technical and analytical strategy limitations to characterise tumour-associated bacteria. Methods Here, we propose an approach to detect bacterial signals in human RNA sequencing data and associate them with the clinical and molecular properties of the tumours. The method was tested on public datasets from The Cancer Genome Atlas, and its accuracy was assessed on a new cohort of colorectal cancer patients. Results Our analysis shows that intratumoural microbiome composition is correlated with survival, anatomic location, microsatellite instability, consensus molecular subtype and immune cell infiltration in colon tumours. In particular, we find Faecalibacterium prausnitzii, Coprococcus comes, Bacteroides spp., Fusobacterium spp. and Clostridium spp. to be strongly associated with tumour properties. Conclusions We implemented an approach to concurrently analyse clinical and molecular properties of the tumour as well as the composition of the associated microbiome. Our results may improve patient stratification and pave the path for mechanistic studies on microbiota-tumour crosstalk