Marek Wieczorek

Age related changes in mitochondrial function and new approaches to study redox regulation in mammalian oocytes in response to age or maturation conditions.

Mammalian oocytes are long-lived cells in the human body. They initiate meiosis already in the embryonic ovary, arrest meiotically for long periods in dictyate stage, and resume meiosis only after extensive growth and a surge of luteinizing hormone which mediates signaling events that overcome meiotic arrest. Few mitochondria are initially present in the primordial germ cells while there are mitogenesis and structural and functional differentiation and stage-specific formation of functionally diverse domains of mitochondria during oogenesis. Mitochondria are most prominent cell organelles in oocytes and their activities appear essential for normal spindle formation and chromosome segregation, and they are one of the most important maternal contributions to early embryogenesis. Dysfunctional mitochondria are discussed as major factor in predisposition to chromosomal nondisjunction during first and second meiotic division and mitotic errors in embryos, and in reduced quality and developmental potential of aged oocytes and embryos. Several lines of evidence suggest that damage by oxidative stress/reactive oxygen species in dependence of age, altered antioxidative defence and/or altered environment and bi-directional signaling between oocyte and the somatic cells in the follicle contribute to reduced quality of oocytes and blocked or aberrant development of embryos after fertilization. The review provides an overview of mitogenesis during oogenesis and some recent data on oxidative defence systems in mammalian oocytes, and on age-related changes as well as novel approaches to study redox regulation in mitochondria and ooplasm. The latter may provide new insights into age-, environment- and cryopreservation-induced stress and mitochondrial dysfunction in oocytes and embryos.

Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation

Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell’s own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors—tapasin for class I and HLA-DM for class II—contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity.

Susceptibility to HLA-DM protein is determined by a dynamic conformation of major histocompatibility complex class II molecule bound with peptide.

Background: HLA-DM-mediated peptide exchange is a key factor in epitope selection, but how HLA-DM selects peptides for editing is not known. Results: Peptide complexes sensitive to HLA-DM editing exhibited conformational alterations. Conclusion: HLA-DM efficiently identifies unstable complexes by sensing MHCII-peptide conformations. Significance: These data emphasize HLA-DM as a conformational editor and provide novel mechanistic insight into its function. HLA-DM mediates the exchange of peptides loaded onto MHCII molecules during antigen presentation by a mechanism that remains unclear and controversial. Here, we investigated the sequence and structural determinants of HLA-DM interaction. Peptides interacting nonoptimally in the P1 pocket exhibited low MHCII binding affinity and kinetic instability and were highly susceptible to HLA-DM-mediated peptide exchange. These changes were accompanied by conformational alterations detected by surface plasmon resonance, SDS resistance assay, antibody binding assay, gel filtration, dynamic light scattering, small angle x-ray scattering, and NMR spectroscopy. Surprisingly, all of those changes could be reversed by substitution of the P9 pocket anchor residue. Moreover, MHCII mutations outside the P1 pocket and the HLA-DM interaction site increased HLA-DM susceptibility. These results indicate that a dynamic MHCII conformational determinant rather than P1 pocket occupancy is the key factor determining susceptibility to HLA-DM-mediated peptide exchange and provide a molecular mechanism for HLA-DM to efficiently target unstable MHCII-peptide complexes for editing and exchange those for more stable ones.

Multivalent Binding of Formin-binding Protein 21 (FBP21)-Tandem-WW Domains Fosters Protein Recognition in the Pre-spliceosome

Background: The role of long proline-rich segments, as they are abundantly present in the spliceosome, is elusive. Results: Cell biological and biophysical data show the significance of multiple motifs for tandem-WW domain recognition. Conclusion: The dynamic assembly of the pre-spliceosome is enabled by transient multivalent interactions. Significance: Our results have general implications for the recognition of proline-rich sequence hubs in modular protein assemblies. The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.

MHC class II complexes sample intermediate states along the peptide exchange pathway

The presentation of peptide-MHCII complexes (pMHCIIs) for surveillance by T cells is a well-known immunological concept in vertebrates, yet the conformational dynamics of antigen exchange remain elusive. By combining NMR-detected H/D exchange with Markov modelling analysis of an aggregate of 275 microseconds molecular dynamics simulations, we reveal that a stable pMHCII spontaneously samples intermediate conformations relevant for peptide exchange. More specifically, we observe two major peptide exchange pathways: the kinetic stability of a pMHCIIs ground state defines its propensity for intrinsic peptide exchange, while the population of a rare, intermediate conformation correlates with the propensity of the HLA-DM-catalysed pathway. Helix-destabilizing mutants designed based on our model shift the exchange behaviour towards the HLA-DM-catalysed pathway and further allow us to conceptualize how allelic variation can shape an individuals MHC restricted immune response.

Peptide Linkage to the α-Subunit of MHCII Creates a Stably Inverted Antigen Presentation Complex

Class II proteins of the major histocompatibility complex (MHCII) typically present exogenous antigenic peptides to cognate T cell receptors of CD4-T lymphocytes. The exact conformation of peptide-MHCII complexes (pMHCII) can vary depending on the length, register and orientation of the bound peptide. We have recently found the self-peptide CLIP (class-II-associated invariant chain-derived peptide) to adopt a dynamic bidirectional binding mode with regard to the human MHCII HLA-DR1 (HLA, human leukocyte antigen). We suggested that inversely bound peptides could activate specific T cell clones in the context of autoimmunity. As a first step to prove this hypothesis, pMHC complexes restricted to either the canonical or the inverted peptide orientation have to be constructed. Here, we show that genetically encoded linkage of CLIP and two other antigenic peptides to the HLA-DR1 α-chain results in stable complexes with inversely bound ligands. Two-dimensional NMR and biophysical analyses indicate that the CLIP-bound pMHC(inv) complex (pMHC(inv), inverted MHCII-peptide complex) displays high thermodynamic stability but still allows for the exchange against higher-affinity viral antigen. Complemented by comparable data on a corresponding β-chain-fused canonical HLA-DR1/CLIP complex, we further show that linkage of CLIP leads to a binding mode exactly the same as that of the corresponding unlinked constructs. We suggest that our approach constitutes a general strategy to create pMHC(inv) complexes. Such engineering is needed to create orientation-specific antibodies and raise T cells to study phenomena of autoimmunity caused by isomeric pMHCs.

HLA-DMA Polymorphisms Differentially Affect MHC Class II Peptide Loading

During the adaptive immune response, MHCII proteins display antigenic peptides on the cell surface of APCs for CD4+ T cell surveillance. HLA-DM, a nonclassical MHCII protein, acts as a peptide exchange catalyst for MHCII, editing the peptide repertoire. Although they map to the same gene locus, MHCII proteins exhibit a high degree of polymorphism, whereas only low variability has been observed for HLA-DM. As HLA-DM activity directly favors immunodominant peptide presentation, polymorphisms in HLA-DM (DMA or DMB chain) might well be a contributing risk factor for autoimmunity and immune disorders. Our systematic comparison of DMA*0103/DMB*0101 (DMA-G155A and DMA-R184H) with DMA*0101/DMB*0101 in terms of catalyzed peptide exchange and dissociation, as well as direct interaction with several HLA-DR/peptide complexes, reveals an attenuated catalytic activity of DMA*0103/DMB*0101. The G155A substitution dominates the catalytic behavior of DMA*0103/DMB*0101 by decreasing peptide release velocity. Preloaded peptide–MHCII complexes exhibit ∼2-fold increase in half-life in the presence of DMA*0103/DMB*0101 when compared with DMA*0101/DMB*0101. We show that this effect leads to a greater persistence of autoimmunity-related Ags in the presence of high-affinity competitor peptide. Our study therefore reveals that HLA-DM polymorphic residues have a considerable impact on HLA-DM catalytic activity.

Human leukocyte Antigen-DM polymorphisms in autoimmune diseases.

Classical MHC class II (MHCII) proteins present peptides for CD4+ T-cell surveillance and are by far the most prominent risk factor for a number of autoimmune disorders. To date, many studies have shown that this link between particular MHCII alleles and disease depends on the MHCIIs particular ability to bind and present certain peptides in specific physiological contexts. However, less attention has been paid to the non-classical MHCII molecule human leucocyte antigen-DM, which catalyses peptide exchange on classical MHCII proteins acting as a peptide editor. DM function impacts the presentation of both antigenic peptides in the periphery and key self-peptides during T-cell development in the thymus. In this way, DM activity directly influences the response to pathogens, as well as mechanisms of self-tolerance acquisition. While decreased DM editing of particular MHCII proteins has been proposed to be related to autoimmune disorders, no experimental evidence for different DM catalytic properties had been reported until recently. Biochemical and structural investigations, together with new animal models of loss of DM activity, have provided an attractive foundation for identifying different catalytic efficiencies for DM allotypes. Here, we revisit the current knowledge of DM function and discuss how DM function may impart autoimmunity at the organism level.

Peptide–polymer ligands for a tandem WW-domain, an adaptive multivalent protein–protein interaction: lessons on the thermodynamic fitness of flexible ligands

Summary Three polymers, poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), hyperbranched polyglycerol (hPG), and dextran were investigated as carriers for multivalent ligands targeting the adaptive tandem WW-domain of formin-binding protein (FBP21). Polymer carriers were conjugated with 3–9 copies of the proline-rich decapeptide GPPPRGPPPR-NH2 (P1). Binding of the obtained peptide–polymer conjugates to the tandem WW-domain was investigated employing isothermal titration calorimetry (ITC) to determine the binding affinity, the enthalpic and entropic contributions to free binding energy, and the stoichiometry of binding for all peptide–polymer conjugates. Binding affinities of all multivalent ligands were in the µM range, strongly amplified compared to the monovalent ligand P1 with a K D > 1 mM. In addition, concise differences were observed, pHPMA and hPG carriers showed moderate affinity and bound 2.3–2.8 peptides per protein binding site resulting in the formation of aggregates. Dextran-based conjugates displayed affinities down to 1.2 µM, forming complexes with low stoichiometry, and no precipitation. Experimental results were compared with parameters obtained from molecular dynamics simulations in order to understand the observed differences between the three carrier materials. In summary, the more rigid and condensed peptide–polymer conjugates based on the dextran scaffold seem to be superior to induce multivalent binding and to increase affinity, while the more flexible and dendritic polymers, pHPMA and hPG are suitable to induce crosslinking upon binding.