Regulation of Endoplasmic Reticulum Architecture by Protein S-Palmitoylation

The endoplasmic reticulum (ER) is the largest organelle in mammalian cells. More than 50 years ago, its morphology was already described as consisting of two major compartments: the rough ER and the smooth ER, which both fulfil precise and distinct functions to sustain the cellular metabolism. The rough ER localizes in the perinuclear region and is composed of packed membrane sheets with studded ribosomes. The smooth ER extends to the cell periphery as a continuous tubular membrane meshwork. Impairment of the ER structure is related to multiple diseases, in particular psycho-motor degenerations such as hereditary spastic paraplegia. The current model of the ER architecture suggests that specific ER proteins, called ER-shaping proteins, generate and maintain the ER morphology. Different families of ER-shaping proteins support either the curvature or the flatness of the membrane and the exact ER morphology depends on their local concentrations. Although the structural role of these proteins has been extensively characterized, the mechanism of their regulation by the cell is mostly unknown. Moreover, the process has to be very dynamic, as the ER membrane morphology is rapidly interconverted. Our work has discovered that several ER-shaping proteins are modified by a post-translational modification named S-palmitoylation. S-palmitoylation is the covalent but reversible attachment of a 16 carbons chain fatty acid to a cysteine residue of a protein. Interestingly, both ER-sheet and ER-tubule promoting proteins were modified. We observed that these palmitoylated ER-shaping proteins are all controlled by the same palmitoyl-acyltransferase: DHHC6. Thus, our results suggest that DHHC6 is a master regulator of the ER architecture. We also demonstrated that palmitoylation stabilizes the lifetime of one specific substrate: CLIMP-63, which induces ER-sheet proliferation. Transient expression of DHHC6 generated prominent ER-sheets, which were dependent on CLIMP-63's presence and palmitoylation. On the contrary, the absence of DHHC6 diminished the size of the rough ER. Finally, to study protein palmitoylation, we developed a novel assay which was able to quantify the fraction of palmitoylated protein at steady state. Taken together, our data highlight the first general mechanism that mediates the ER architecture. Our future work will focus on understanding how and when the cell uses palmitoylation of ER-shaping proteins and clarifying the effectors of this signalling pathway.

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

Tiziano Dallavilla

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.

EPFL2017

Palmitoylation of membrane proteins (Review)

Françoise Gisou van der Goot Grunberg

S-Palmitoylation is a reversible post-translational modification that results in the addition of a C16-carbon saturated fatty acyl chain to cytoplasmic cysteine residues. This modification is mediated by Palmitoyl-acyl Transferases that are starting to be investigated, and reversed by Protein Palmitoyl Thioesterases, which remain enigmatic. Palmitoylation of cytoplasmic proteins has been well described to regulate the interaction of these soluble proteins with specific membranes or membrane domains. Less is known about the consequences of palmitoylation in transmembrane proteins not only due to the dual difficulty of following a lipid modification and dealing with membrane proteins, but also due to the complexity of the palmitoylation-induced behavior. Moreover, possibly because the available data set is limited, the change in behavior induced by palmitoylation of a transmembrane protein is currently not predictable. We here review the various consequences reported for the palmitoylation of membrane proteins, which include improper folding in the endoplasmic reticulum, retention in the Golgi, inability to assemble into protein platforms, altered signaling capacity, premature endocytosis and missorting in the endocytic pathway. We then discuss the possible underlying mechanisms, in particular the ability of palmitoylation to control the conformation of transmembrane segments, to modify the affinity of a membrane protein for specific membrane domains and to control protein-protein interactions.

Taylor & Francis2008

Roles of Epigenetic Dysregulation in Cancer Progression and Metastasis

Mohammad Sadegh Saghafinia

Nearly all the cells of an organism share the same DNA sequence or genome, and yet they show different phenotypes and carry out different functions. This diversity is made possible by a verity of molecular modifications acting on the DNA sequence that collectively define the cell's epigenome. Failure in the proper regulation of epigenome can lead to abnormal activation or inhibition of vital signaling pathways, which contributes to the initiation and progression of multiple diseases, including cancer. In this thesis, I used a combination of in silico, in vitro, and in vivo techniques to explore the roles of epigenetic dysregulation in tumor progression and metastasis. In the first part, I performed a systematic and unbiased investigation of cancer-associated DNA methylation and gene expression changes across human cancers. I designed a novel algorithmic approach (RESET) to identify aberrant DNA methylation and associated gene expression changes across >6,000 human tumors. We identified a DNA Methylation Instability (DMI) phenotype in tumors acquiring numerous hyper and hypomethylated transcription start sites, which is associated with mutations of chromatin remodeling factors and Wnt-signaling. We showed that silenced genes coalesced in specific pathways including apoptosis, transcriptional regulation, and cell metabolism. The majority of the enhanced genes belonged to cancer-germline antigens (CG), whose expression correlated with response to anti-PD-1 in melanoma patients. Finally, we demonstrated the potential of RESET to explore aberrant DNA methylation in pediatric tumors; pediatric Wilms tumors with diffuse anaplasia exhibit DMI, and specific silencing events predict a worse outcome in cases with favorable histology. These results established a new approach to explore pan-cancer epigenetic modifications and provided a resource of candidate oncogenic events for future functional and therapeutic studies. In the second part, I studied the dynamics of cancer progression and metastasis in Pancreatic Neuroendocrine Tumors (PanNET). This rare tumor is the second most common form of pancreatic cancer. Two principal subtypes of PanNET has been identified: insulinomas (IT) and metastasis-like primaries (MLP), corresponding to the low and high grade of human PanNETs, respectively. From the mouse model of PanNET (RT2), we profiled the single-cell, bulk mRNA and miRNA transcriptomes, and the proteomes of primary and metastasis specimens. We demonstrated that the tumor progression from IT to MLP follows the reverse embryonic and postnatal developmental path. Transcriptomic data from human patients showed that aggressive human PanNETs also follow the same reverse developmental trajectory of dedifferentiation. We established one possible mechanism underlying IT-to-MLP transition. Over-expression of the miR-181cd cluster in IT-like cancer cell-lines resulted in acquisition of the MLP phenotypes. miR-181cd mediated its effect through suppressing the expression of Meis2 and, indirectly, inducing the expression of Hmgb3 and Mycn transcription factors. Inhibiting the expression of Hmgb3 in MLP-like cancer cell-lines resulted in a significant growth decrease both in vitro and in vivo, demonstrating the importance of Hmgb3 in the maintenance of MLP-like cell state. These data presented dedifferentiation as a mechanism by which malignant neuroendocrine cancer cells acquire progenitor-like features, enabling them to become more aggressive and metastatic.