What should we know about Dr. Abdelhalim Azzi’s Laboratory of Lipids and Chronobiology? Circadian clocks control 24-hour rhythms of several biological and physiological processes such as the sleep-wake cycle, body temperature, hormone release and metabolism. Indeed, metabolic processes in the liver follow a very precise circadian pattern to control and optimize energy use throughout the light/dark cycle. Metabolomic studies in mice and humans revealed that a large portion of metabolites changes in abundance every 24 hours, with daily variations in blood and saliva metabolites independent of sleep or feeding. Interestingly, the most rhythmic metabolites observed were lipids such as phosphatidylinositol (PtdIns). Phosphatidylinositol is a membrane phospholipid that can be phosphorylated at different positions of the inositol ring to generate phosphoinositides (PIs). Phosphoinositides kinases and phosphatases mediate the generation and interconversion of PIs. Despite their role in regulation of glucose and lipid metabolism, PI kinase and phosphatase activity with regards to their temporal circadian regulation of metabolism has not yet been investigated. The laboratory aims to dissect the role of these enzymes in regulation of circadian metabolic processes using in vitro and in vivo systems. The findings will improve knowledge about the role of phosphoinositides in circadian physiology.

 

Professor Agnieszka Chacińska’s Laboratory of Mitochondrial Biogenesis focuses on mitochondria which play a key role in metabolism and regulatory processes within a cell. Thus, the formation of mitochondria is essential for cellular function of every being in the eukaryotic kingdom, from unicellular organisms to mammals. Mitochondria comprise 1000-1500 cellular proteins which are synthesized outside of the mitochondria in the cytosol. The biogenesis of mitochondria relies on the efficient import, sorting, and maturation of proteins, all governed by conserved protein translocases and other complex biological machinery. The research conducted by the Laboratory of Mitochondrial Biogenesis explores novel and exciting links between protein transport mechanisms and mitochondrial protein homeostasis. The research group postulates the presence of unique mechanisms involved in protein biogenesis that involve crosstalk between the cytosol and mitochondrial compartments. The goal is to better understand the complex and dynamic processes involved in the formation of functional organelles, as well as the maintenance of cellular protein homeostasis and its failures, which result in pathology.

 

Homeostasis is the central theme of Dr. Maciej Cieśla’s Laboratory of Stem Cell RNA Metabolism. Homeostasis of all multicellular organisms is maintained by an elite set of cells with an unrestrained capacity to differentiate into all cell types — stem cells. Stem cells rely on a rapid rewiring of molecular pathways to maintain tissue integrity and re-populate pools of undifferentiated cells. Loss of these healthy characteristics is a cornerstone of aging and malignancy. Orchestrated regulation of post-transcriptional layers of gene expression, in particular mRNA translation and splicing, is an emerging determinant of cell fate choices. How these processes are coordinated to ensure fidelity of stem cell activation and allow for homeostatic balance is largely unknown. Activated stem cells embark on a complex programme that is frequently and notably uncoupled from the transcriptional regulation. How these extra-genomic states are acquired in dynamic stem cell transitions remains a key unanswered question. Similarly, the implications of the ‘non-transcriptional’ regulation of stem cell function were not comprehensively addressed so far. That is a fundamental biological conundrum of how post-transcriptional regulatory networks may be co-opted during the initial stages of stem cells activation. What drives these changes? And for which physiological and pathological processes are they important? The research group employs a pallet of stem cell systems to study rapid cellular fate switches during development. By a combination of genetic models, genome-wide sequencing approaches and mechanistic and structural technologies, the lab group studies the function of RNA metabolism during mammalian development. The group aims to understand the molecular basis of the post-transcriptional regulation of stem cell function. The objective of this research is to establish the molecular underpinnings of pathologies related to stem cell dysfunction, including infertility, congenital diseases, and cancer.

 

The Laboratory of Membrane Machines, led by Dr. Nicola De Franceschi, emphasizes that membranes are one of the fundamental biological polymers and they underpin a myriad of functions in the cell. Yet membranes are also intriguing with respect to their unique biophysical properties. This 5 nm-thick, fluid material is able to bridge scales across biology, holding an entire cell together while integrating the function of individual proteins that work at the nanometer scale. Membranes can expand and re-shape, undergo fusion and fission events, and even self-repair. And they do all of this with grace and elegance, enchanting us with their stunning beauty. The research group uses a bottom-up reconstitution approach to study biological nanomachines that function on membranes, such as membrane pores and membrane-deforming proteins. However, this is not limited to naturally occurring proteins: the group also uses other biopolymers such as nucleic acids to build bio-inspired machines that act in concert with membranes to create new functionalities. Finally, Dr. De Franceschi’s laboratory is interested in studying the still underappreciated role of membranes in the origin of life.

 

As Dr. Piotr Gerlach from the Laboratory of Structural Virology explains, RNA viruses are a diverse group of serious pathogens. Broadening structural insight into their molecular repertoire, and particularly virus-host intracellular interactions, is critical for a complete understanding of infection mechanisms. Host translation is one of the major sites of the virus-host battlefront. Being a center of cellular stress response pathways, it is often abused by viruses to produce viral proteins. Importantly, while the host translation gets switched off during infection, the viral one persists relying on the non-canonical translation strategies. Of particular interest is the fate of cytoplasmic RNA granules, since these compartments serve as a depository of host mRNA, stalled translation initiation complexes, and auxiliary factors that viruses can rely on during infection. Dr. Gerlach’s lab will combine structural biology expertise (cryo-EM, cryo-ET, and X-ray crystallography) with an in vivo platform – mammalian cell cultures transfected with minimal replicon systems, mimicking viral transcription and replication inside the infected cell. This platform will be used as a test tube for various assays, including whole genome CRISPR-Cas9 screens to identify novel host factors involved in viral infection, functional and localization studies, as well as structural analysis. Knowledge acquired on the way will open new research avenues that may ultimately lead to the design of innovative therapies and broad-spectrum antivirals.

 

Professor Magda Konarska’s Laboratory of RNA Biology uses the yeast S. cerevisiae spliceosome as a model of a complex molecular machine. The group aims to understand the details of its architecture and function. The goal of this research is to understand the complex set of substrate - spliceosome interactions during assembly and catalysis, affecting the positioning of reactive groups at the active site. The mechanistic studies in yeast will help the group understand the molecular interactions that influence splicing fidelity and alternative splicing in metazoan systems. The spliceosomal catalytic center undergoes dynamic changes during the catalytic phase of splicing. Changes of relative stabilities of competing conformations at the catalytic center affect splicing catalysis, altering splicing fidelity and thus affecting the selection of splice site sequences for catalysis. These findings have implications for alternative splicing, common to most Eukaryotes. Professor Konarska’s laboratory tests new models of snRNA: snRNA interactions at the catalytic center implicated in the function of the catalytic triplex and positioning of the branch site. The research group also studies spliceosomal factors involved in the substrate positioning for catalysis, in particular, those containing disordered protein domains penetrating the catalytic center. Another project investigates exon sequences that compensate for the defects of the intron 5’SS. Isolated yeast exon motifs are similar to metazoan exon enhancers. This striking sequence similarity suggests common underlying mechanisms of action. The hypothesis is that yeast exon motifs represent substrate binding sites recognized by the spliceosome. Professor Konarska’s group studies the molecular mechanisms underlying their function.

 

Dr. Anna Marusiak’s Laboratory of Molecular OncoSignalling is interested in cancer biology, cellular signalling during oncogenesis and the identification of novel targets for cancer therapies. Signal transduction plays an important role in cancer development, and protein kinases, being the master regulators of signalling pathways, represent key targets in cancer treatment. The research group wants to understand how aberrant signalling in cancer cells contributes to cancer development, metastasis or therapy resistance, and how that knowledge can be used to design novel anticancer treatments.  In particular, the group focuses on investigating oncogenic signalling activated by MLK4 in breast cancer. MLK4 is a member of the Mixed-Lineage Kinase family of serine/threonine kinases that are activated by environmental stress, cytokines and growth factors, and play a role in a variety of cellular processes. The members of the MLK family have been involved in the regulation of a wide range of disorders including cancer, inflammation, metabolic and neurobiological disorders. The lab investigates MLK4 signalling in breast cancer where MLK4 is highly upregulated and contributes to malignant progression. One of the group’s projects is focused on understanding the role of MLK4 in the response to chemotherapy in breast cancer. Furthermore, Dr Marusiak’s laboratory aims to develop and validate the potency and efficacy of first-in-class MLK4-targeting compounds. The group is also interested in uncovering the importance of MLK4-dependent cross-talk signalling between components of the tumor microenvironment and breast cancer cells. The studies use human and mouse cancer cell lines, as well as syngeneic and xenograft tumor models in mice. Methods such as 3D cell cultures, flow cytometry, mass spectrometry and various phenotypic assays including invasion and migration assays are also used.

 

Dr. Karolina Szczepanowska's Laboratory of Metabolic Quality Control points out that metabolism is firmly defined by the mitochondria. They dictate the bioenergetic capacity of our cells and provide us with critical metabolites. The bulk of cellular energy is generated by the elaborative molecular machines embedded inside the mitochondrial membranes, jointly known as the OXPHOS system. The everyday stress impacts the composition and functionality of OXPHOS, leading to its dysfunction. Consequently, compromised OXPHOS fitness has been implicated in a broad spectrum of disorders, including cancer, diabetes, obesity, ischemia-reperfusion injury, neurodegeneration, or aging. What are the mechanisms that sculpture OXPHOS in response to challenges? Is there a way to repair it when injured by stress? The research group recently started to recognize that mitochondrial bioenergetics stay under the constant surveillance of dedicated proteostatic machinery. Yet the exact mechanisms and responsible players remain mostly elusive. The group’s research aims to dissect the mechanisms that mediate the quality control of OXPHOS exposed to disease-associated conditions and trace the molecular signals that steer its degradation. With the help of modern proteomics and advanced biochemical and molecular biology approaches, the research group can discover damage hot-spots in mammalian mitochondria and identify the factors responsible for their recognition and repair. The findings will help to understand how metabolic quality control contributes to the known cellular stress-responses. Furthermore, OXPHOS salvage can constitute an attractive target for novel therapies against a broad range of human diseases.

 

As Dr. Carlo Vascotto’s from the Laboratory of Mitochondrial Nucleic Acids explains, mitochondria are the primary endogenous source of reactive oxygen species (ROS): highly reactive molecules that can modify proteins, lipids, and nucleic acids. To ensure the stability of mitochondrial DNA and the functionality of transcription and translation processes, cells have evolved repair mechanisms and signaling pathways to detect and respond to ROS production. The lab studies the molecular mechanisms responsible for the repair of mtDNA lesions, the degradation processes of damaged mitochondrial mRNAs, and the characterization of mitochondrial-nucleus crosstalk in the regulation of mitochondrial functions.

 

Proteomics Core Facility led by Dr. Remigiusz Serwa: a closer look at proteins

IMol has its own proteomics laboratory where researchers conduct analyses that determine the level of protein expression in human cells or model organisms. The rate of protein synthesis or degradation is quantified, and its intracellular location is determined. Using proteomic methods to identify protein profiles, researchers analyse protein-protein or protein-small molecule interactions (e.g. a metabolite or a drug). All of the measurements are carried out using a Thermo Fisher Scientific mass spectrometer and an Orbitrap flow nanoliquid chromatography-tandem mass spectrometry set-up. A mass spectrometer analyses the substance’s elemental composition, and the Orbitrap determines the mass of ions. Measurements carried out with these instruments have a very high sensitivity, depth and repeatability of results. Both researchers at IMol and from outside the institute use the proteomics laboratory to support their research.