The Lateral Membrane Organization and Dynamics of Myelin Proteins PLP and MBP Are Dictated by Distinct Galactolipids and the Extracellular Matrix

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC),and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS),while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP's diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies

Virus Evolution Reveals an Exclusive Role for LEDGF/p75 in Chromosomal Tethering of HIV

Retroviruses by definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular cofactors has been proposed. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 in integration has been proposed. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for the interaction with integrase. In an attempt to select HIV strains that can overcome the inhibition, we now have used T-cell lines that stably express a C-terminal fragment of LEDGF/p75. Despite resistance development, the affinity of integrase for LEDGF/p75 is reduced and replication kinetics in human primary T cells is impaired. Detection of the integrase mutations A128T and E170G at key positions in the LEDGF/p75–integrase interface provides in vivo evidence for previously reported crystallographic data. Moreover, the complementary inhibition by LEDGF/p75 knockdown and mutagenesis at the integrase–LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 function during proviral integration. Altogether, the data provide a striking example of the power of viral molecular evolution. The results underline the importance of the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites

The polymeric glyco-linker controls the signal outputs for plasmonic gold nanorod biosensors due to biocorona formation

Gold nanorods (GNRs) are a promising platform for nanoplasmonic biosensing. The localised surface plasmon resonance (LSPR) peak of GNRs is located in the near-infrared optical window and is sensitive to local binding events, enabling label-free detection of biomarkers in complex biological fluids. A key challenge in the development of such sensors is achieving target affinity and selectivity, while both minimizing non-specific binding and maintaining colloidal stability. Herein, we reveal how GNRs decorated with galactosamine-terminated polymer ligands display significantly different binding responses in buffer compared to serum, due to biocorona formation, and how biocorona displacement due to lectin binding plays a key role in their optical responses. GNRs were coated with either poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) or poly(N-hydroxyethyl acrylamide) (PHEA) prepared via reversible addition–fragmentation chain-transfer (RAFT) polymerisation and end-functionalised with galactosamine (Gal) as the lectin-targeting unit. In buffer Gal-PHEA-coated GNRs aggregated upon soybean agglutinin (SBA) addition, whereas Gal-PHPMA-coated GNRs exhibited a red-shift of the LSPR spectrum without aggregation. In contrast, when incubated in serum Gal-PHPMA-coated nanorods showed no binding response, while Gal-PHEA GNRs exhibited a dose-dependent blue-shift of the LSPR peak, which is the opposite direction (red-shift) to what was observed in buffer. This differential behaviour was attributed to biocorona formation onto both polymer-coated GNRs, shown by differential centrifugal sedimentation and nanoparticle tracking analysis. Upon addition of SBA to the Gal-PHEA coated nanorods, signal was generated due to displacement of weakly-bound biocorona components by lectin binding. However, in the case of Gal-PHPMA which had a thicker corona, attributed to lower polymer grafting densities, addition of SBA did not lead to biocorona displacement and there was no signal output. These results show that plasmonic optical responses in complex biological media can be significantly affected by biocorona formation, and that biocorona formation itself does not prevent sensing so long as its exact nature (e.g. ‘hard versus soft’) is tuned

Hidden States within Disordered Regions of the CcdA Antitoxin Protein

The bacterial toxin–antitoxin system CcdB–CcdA provides a mechanism for the control of cell death and quiescence. The antitoxin protein CcdA is a homodimer composed of two monomers that each contain a folded N-terminal region and an intrinsically disordered C-terminal arm. Binding of the intrinsically disordered C-terminal arm of CcdA to the toxin CcdB prevents CcdB from inhibiting DNA gyrase and thereby averts cell death. Accurate models of the unfolded state of the partially disordered CcdA antitoxin can therefore provide insight into general mechanisms whereby protein disorder regulates events that are crucial to cell survival. Previous structural studies were able to model only two of three distinct structural states, a closed state and an open state, that are adopted by the C-terminal arm of CcdA. Using a combination of free energy simulations, single-pair Förster resonance energy transfer experiments, and existing NMR data, we developed structural models for all three states of the protein. Contrary to prior studies, we find that CcdA samples a previously unknown state where only one of the disordered C-terminal arms makes extensive contacts with the folded N-terminal domain. Moreover, our data suggest that previously unobserved conformational states play a role in regulating antitoxin concentrations and the activity of CcdA’s cognate toxin. These data demonstrate that intrinsic disorder in CcdA provides a mechanism for regulating cell fate

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices

The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering

Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein–protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting

Aryl-hydrocarbon receptor-interacting protein regulates tumorigenic and metastatic properties of colorectal cancer cells driving liver metastasis

12 p.-5 fig.-1 tab.Background: Liver metastasis is the primary cause of colorectal cancer (CRC)-associated death. Aryl-hydrocarbon receptor-interacting protein (AIP), a putative positive intermediary in aryl-hydrocarbon receptor-mediated signalling, is overexpressed in highly metastatic human KM12SM CRC cells and other highly metastatic CRC cells.Methods: Meta-analysis and immunohistochemistry were used to assess the relevance of AIP. Cellular functions and signalling mechanisms mediated by AIP were assessed by gain-of-function experiments and in vitro and in vivo experiments.Results: A significant association of high AIP expression with poor CRC patients’ survival was observed. Gain-of-function and quantitative proteomics experiments demonstrated that AIP increased tumorigenic and metastatic properties of isogenic KM12C (poorly metastatic) and KM12SM (highly metastatic to the liver) CRC cells. AIP overexpression dysregulated epithelial-to-mesenchymal (EMT) markers and induced several transcription factors and Cadherin-17 activation. The former induced the signalling activation of AKT, SRC and JNK kinases to increase adhesion, migration and invasion of CRC cells. In vivo, AIP expressing KM12 cells induced tumour growth and liver metastasis. Furthermore, KM12C (poorly metastatic) cells ectopically expressing AIP became metastatic to the liver.Conclusions: Our data reveal new roles for AIP in regulating proteins associated with cancer and metastasis to induce tumorigenic and metastatic properties in colon cancer cells driving liver metastasis.This work was supported by the financial support of the PI17CIII/00045 and PI20CIII/00019 grants from the AES-ISCIII program to RB. J Hendrix acknowledges funding by UH-BOF (BOF20TT06). J Hofkens acknowledges financial support from the Research Foundation-Flanders (FWO, Grant No. ZW15_09-G0H6316N), the Flemish government through long-term structural funding Methusalem (CASAS2, Meth/15/04) and the MPI as MPI fellow. S.R. acknowledges the financial support of the KU Leuven through the internal C1 funding (KU Leuven (C14/16/053)). GSF is the recipient of a predoctoral contract (grant number 1193818 N) supported by The Flanders Research Foundation (FWO). The FPU predoctoral contract to AMC is supported by the Spanish Ministerio de Educación, Cultura y Deporte.Peer reviewe

Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers

Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly

Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods