Biology subjects

Ruskamo, S.

Publications and source records attributed to Ruskamo, S..

10 recordsLinked to original sources

bioRxiv2025

A novel DNA repair protein, N-Myc downstream regulated gene 1 (NDRG1), links stromal tumour microenvironment to chemoresistance

In pancreatic ductal adenocarcinoma cancer (PDAC) drug resistance is a severe clinical problem and patients relapse within a few months after receiving the standard-of-care chemotherapy. One contributing factor to treatment resistance is the desmoplastic nature of PDAC; the tumours are surrounded by thick layers of stroma composing up to 90% of the tumour mass. This stroma, which is mostly comprised of extracellular matrix (ECM) proteins, is secreted by cancer-associated fibroblasts (CAFs) residing in the tumour microenvironment. However, the mechanistic basis by which the tumour stroma directly contributes to chemoresistance remains unclear. Here, we show that CAF-secreted ECM proteins induce chemoresistance by blunting chemotherapy-induced DNA damage. Mechanistically, we identify N-myc downstream regulated gene 1 (NDRG1) as a key protein required for stroma-induced chemoresistance that responds to signals from the ECM and adhesion receptors. We further show that NDRG1 is a novel DNA repair protein that physically interacts with replication forks, maintains DNA replication and functions to resolve stalled forks caused by chemotherapy. More specifically, NDRG1 reduces R-loops, RNA-DNA hybrids that are known to cause genomic instability. R-loops occur during replication-transcription conflicts in S-phase and after chemotherapy treatments, thus posing a major threat to normal replication fork homeostasis. We identify NDRG1 as highly expressed in PDAC tumours, and its high expression correlates with chemoresistance and poor disease-specific survival. Importantly, knock-out of NDRG1 or inhibition of its phosphorylation restores chemotherapy-induced DNA damage and resensitizes tumour cells to treatment. In conclusion, our data reveal an unexpected role for CAF-secreted ECM proteins in enhancing DNA repair via NDRG1, a novel DNA repair protein, directly linking tumour stroma to replication fork homeostasis and R-loop biology, with important therapeutic implications for restoring DNA damage response pathways in pancreatic cancer. Summary paragraphDrug resistance is a severe clinical problem in stroma-rich tumours, such as pancreatic ductal adenocarcinoma (PDAC), and patients often relapse within a few months on chemotherapy1-9. The stroma, comprised of extracellular matrix (ECM) proteins, is secreted by cancer-associated fibroblasts (CAFs) residing in the tumour microenvironment10-13. Prior work show that ECM proteins provide survival benefits to cancer cells14,15. However, the precise role of CAF-secreted ECM in resistance to DNA damaging chemotherapies remains poorly understood. Here, we link ECM proteins to chemoresistance by enhanced DNA damage repair (DDR). Mechanistically, we identify N-myc downstream-regulated gene 1 (NDRG1) as a key effector downstream of ECM and the integrin-Src-SGK1-signalling axis that mediates enhanced DDR. We show that NDRG1 loss, mutation of conserved His194, or inhibition of NDRG1 phosphorylation by SGK1 lead to replication fork stalling, increased R-loops, and higher transcription-replication conflicts, resulting in genomic instability and sensitivity to chemotherapies. Our analysis of PDAC patient cohorts16 found that high NDRG1 expression correlates with chemoresistance and poor patient survival. In conclusion, we uncover an unexpected role for CAF-secreted ECM proteins in promoting therapeutic resistance by enhancing DDR and establish NDRG1 as a novel DNA repair protein directly linking tumour stroma to DDR.

cancer biology↗

bioRxiv2024

On the synergy between myelin proteins P0, MBP, and P2 in peripheral nerve major dense line formation

Myelin is a proteolipid membrane multilayer held together by a set of proteins. The proper formation and function of the myelin sheath relies on the coordinated action of several key myelin proteins. Research exploring how proteins from the peripheral myelin cytoplasmic apposition - myelin basic protein (MBP), the cytoplasmic tail of myelin protein zero (P0ct), and peripheral myelin protein 2 (P2) - interact with each other and with myelin-like membranes was conducted using various techniques, such as small-angle X-ray diffraction (SAXD), differential scanning calorimetry (DSC), surface plasmon resonance (SPR), as well as electron and live epifluorescence microscopy. DSC revealed changes in lipid interactions depending on the protein combination, with MBP and P0ct binding more tightly to lipid membranes than P2, resulting in altered membrane fluidity and stability. These results were supported by SPR, which indicated that the myelin proteins may compete for membrane surface binding. Analysis of the Bragg peaks induced by the myelin proteins in lipidic environments showed both lamellar and non-lamellar phases in protein-lipid complexes. The results indicate both synergy and competition between the three main proteins residing in the PNS myelin major dense line. Furthermore, the observed direct effects of myelin proteins on lipid membrane properties may be relevant to their function in myelinating cells.

biochemistry↗

bioRxiv2023

Conformational analysis of membrane-proximal segments of GDAP1 in a lipidic environment using synchrotron radiation suggests a mode of assembly at the mitochondrial outer membrane

The mitochondrial outer membrane creates a diffusion barrier between the cytosol and mitochondrial intermembrane space, allowing the exchange of metabolic products, important for efficient mitochondrial function in neurons. The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial outer membrane protein with a critical role in mitochondrial dynamics and metabolic balance in neurons. Missense mutations in the GDAP1 gene are linked to the most common human peripheral neuropathy, Charcot-Marie-Tooth disease (CMT). GDAP1 is a distant member of the glutathione-S-transferase (GST) superfamily, with unknown enzymatic properties or functions at the molecular level. The structure of the cytosol-facing GST-like domain has been described, but there is no consensus on how the protein interacts with the mitochondrial outer membrane. Here, we describe a model for GDAP1 assembly on the membrane using peptides vicinal to the GDAP1 transmembrane domain. We used oriented circular dichroism spectroscopy (OCD) with synchrotron radiation to study the secondary structure and orientation of GDAP1 segments at the outer and inner surfaces of the outer mitochondrial membrane. These experiments were complemented by small-angle X-ray scattering, providing the first experimental structural models for full-length human GDAP1. The results indicate that GDAP1 is bound into the membrane via a single transmembrane helix, flanked by two peripheral helices interacting with the outer and inner leaflet of the mitochondrial outer membrane in different orientations. Impairment of such interactions could be a mechanism for CMT in the case of missense mutations affecting these segments instead of the GST-like domain.

biochemistry↗

bioRxiv2022

Conserved intramolecular networks in GDAP1 are closely connected to CMT-linked mutations and protein stability

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its subtypes are linked to mutations in dozens of different genes, including the gene coding for ganglioside-induced differentiation-associated protein 1 (GDAP1). The main GDAP1-linked CMT subtypes are the demyelinating CMT4A and the axonal CMT2K. Over a hundred different missense CMT mutations in the GDAP1 gene have been reported. However, despite implications for mitochondrial fission and fusion, cytoskeletal interactions, and response to reactive oxygen species, the etiology of GDAP1-linked CMT is poorly understood at the protein level. Based on earlier structural data, CMT-linked mutations could affect intramolecular interaction networks within the GDAP1 protein. We carried out structural and biophysical analyses on several CMT-linked GDAP1 protein variants and describe new crystal structures of the autosomal recessive R120Q and the autosomal dominant A247V and R282H GDAP1 variants. These mutations reside in the structurally central helices 3, 7, and 8. In addition, solution properties of the CMT mutants R161H, H256R, R310Q, and R310W were analysed. All disease variant proteins retain close to normal structure and solution behaviour. All mutations, apart from those affecting Arg310 outside the folded GDAP1 core domain, decreased thermal stability. In addition, a bioinformatics analysis was carried out to shed light on the conservation and evolution of GDAP1, which is an outlier member of the GST superfamily. Many CMT mutation sites are highlighted in the bioinformatics analyses focusing on sequence conservation and entropy, and the analyses support the outlier nature of GDAP1 in the GST superfamily. A central role for the 6-7 loop, within a conserved interaction network, is identified for GDAP1 protein stability. To conclude, we have expanded the structural analysis on GDAP1, strengthening the hypothesis that alterations in conserved intramolecular interactions may alter GDAP1 stability and function, eventually leading to mitochondrial dysfunction, impaired protein-protein interactions, and neuronal degeneration.

biochemistry↗

bioRxiv2022

Human myelin proteolipid protein structure and lipid bilayer stacking

The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, -helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure-function differences, as well as fundamental protein-lipid interplay in CNS compact myelin.

biochemistry↗

bioRxiv2022

Structural insights into Charcot-Marie-Tooth disease-linked mutations in human GDAP1

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its different subtypes are linked to mutations in dozens of different genes. Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) cause two types of CMT, demyelinating CMT4A and axonal CMT2K. The GDAP1-linked CMT genotypes are mainly missense point mutations. Despite clinical profiling and in vivo studies on the mutations, the etiology of GDAP1-linked CMT is poorly understood. Here, we describe the biochemical and structural properties of the Finnish founding CMT2K mutation H123R as well as CMT2K-linked R120W, both of which are autosomal dominant mutations. The disease variant proteins retain close to normal structure and solution behaviour, but both present a large decrease in thermal stability. Using GDAP1 variant crystal structures, we identify a side chain interaction network between helices 3, 6, and 7, which is affected by CMT mutations, as well as a hinge in the long helix 6, which is linked to structural flexibility. Structural analysis of GDAP1 indicates that CMT may arise from disruption of specific intra- and intermolecular interaction networks, leading to alterations in GDAP1 structure and stability, and eventually, insufficient motor and sensory neuron function.

biochemistry↗

bioRxiv2021

Human myelin protein P2: From crystallography to time-lapse membrane imaging and neuropathy-associated variants

Peripheral myelin protein 2 (P2) is a fatty acid-binding protein expressed in vertebrate peripheral nervous system myelin, as well as in human astrocytes. Suggested functions of P2 include membrane stacking and lipid transport. Mutations in the PMP2 gene, encoding P2, are associated with Charcot-Marie-Tooth disease (CMT). Recent studies have revealed three novel PMP2 mutations in CMT patient families. To shed light on the structure and function of the corresponding P2 variants, we used X-ray and neutron crystallography, small-angle X-ray scattering, circular dichroism spectroscopy, computer simulations, and lipid binding assays. The crystal and solution structures of the I50del, M114T, and V115A variants of P2 showed only minor differences to the wild-type protein, whereas the thermal stability of the disease variants was reduced. Lipid vesicle aggregation assays revealed no change in membrane stacking characteristics, while the variants showed slightly altered fatty acid binding. Time-lapse imaging of lipid bilayers indicated membrane blebbing induced by P2, which could be related to its function in stacking of two curved membrane surfaces in myelin in vivo. All variants caused blebbing of membranes on similar timescales. In order to better understand the links between structure, dynamics, and function, the crystal structure of perdeuterated P2 was refined from room temperature data collected using both neutrons and X-rays, and the results were compared to molecular dynamics simulations and cryocooled crystal structures. Taken together, our data indicate similar properties of all known CMT variants of human P2; while crystal structures are nearly identical, stability and function of the disease variants are impaired compared to the wild-type protein. Our data provide new insights into the structure-function relationships and dynamics of P2 in health and disease.

biochemistry↗

bioRxiv2020

Determinants for forming a supramolecular myelin-like proteolipid lattice

Myelin protein P2 is a peripheral membrane protein of the fatty acid binding protein family. It functions in the formation and maintenance of the peripheral nerve myelin sheath, and several P2 mutations causing human Charot-Marie-Tooth neuropathy have been reported. Here, electron cryomicroscopy of myelin-like proteolipid multilayers revealed a three-dimensionally ordered lattice of P2 molecules between stacked lipid bilayers, visualizing its possible assembly at the myelin major dense line. A single layer of P2 is inserted between two bilayers in a tight intermembrane space of [~]3 nm, implying direct interactions between P2 and two membrane surfaces. Further details on lateral protein organization were revealed through X-ray diffraction from bicelles stacked by P2. Surface mutagenesis of P2 coupled to structural and functional experiments revealed a role for both the portal region and the opposite face of P2 in membrane interactions. Atomistic molecular dynamics simulations of P2 on myelin-like and model membrane surfaces suggested that Arg88 is an important residue for P2-membrane interactions, in addition to the helical lid domain on the opposite face of the molecule. Negatively charged myelin lipid headgroups anchor P2 stably on the bilayer surface. Membrane binding may be accompanied by opening of the P2 {beta} barrel structure and ligand exchange with the apposing lipid bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step towards deciphering the 3-dimensional assembly of a mature myelin sheath at the molecular level.

biochemistry↗

bioRxiv2019

Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail

Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment.

biophysics↗

bioRxiv2018

Molecular structure and function of myelin protein P0 in membrane stacking

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in fluidity changes of the bilayers themselves, but also potentially involves the rearrangement of the Ig-like domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease.

biochemistry↗