Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing.

Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, NaV1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods.

Optical control of the antigen translocation by synthetic photo-conditional viral inhibitors.

The immune system makes use of major histocompatibility complex class I (MHC I) molecules to present peptides to other immune cells, which can evoke an immune response. Within this process of antigen presentation, the MHC I peptide loading complex, consisting of a transporter associated with antigen processing TAP, MHC I, and chaperones, is key to the initiation of immune response by shuttling peptides from the cytosol into the ER lumen. However, it is still enigmatic how the flux of antigens is precisely coordinated in time and space, limiting our understanding of antigen presentation pathways. Here, we report on the development of a synthetic viral TAP inhibitor that can be cleaved by light. This photo-conditional inhibitor shows temporal blockade of TAP-mediated antigen translocation, which is unleashed upon illumination. The recovery of TAP activity was monitored at single-cell resolution both in human immune cell lines and primary cells. The development of a photo-conditional TAP inhibitor thus expands the repertoire of chemical intervention tools for immunological processes.

Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies.

Accurate labeling of endogenous proteins for advanced light microscopy in living cells remains challenging. Nanobodies have been widely used for antigen labeling, visualization of subcellular protein localization and interactions. To facilitate an expanded application, we present a scalable and high-throughput strategy to simultaneously target multiple endogenous proteins in living cells with micro- to nanometer resolution. For intracellular protein labeling, we advanced nanobodies by site-specific and stoichiometric attachment of bright organic fluorophores. Their fast and fine-tuned intracellular transfer by microfluidic cell squeezing enabled high-throughput delivery with less than 10% dead cells. This strategy allowed for the dual-color imaging of distinct endogenous cellular structures, and culminated in super-resolution imaging of native protein networks in genetically non-modified living cells. The simultaneous delivery of multiple engineered nanobodies does not only offer exciting prospects for multiplexed imaging of endogenous protein, but also holds potential for visualizing native cellular structures with unprecedented accuracy.

Dynamic blue light-switchable protein patterns on giant unilamellar vesicles.

The blue light-dependent interaction between the proteins iLID and Nano allows recruiting and patterning proteins on GUV membranes, which thereby capture key features of patterns observed in nature. This photoswitchable protein interaction provides non-invasive, reversible and dynamic control over protein patterns of different sizes with high specificity and spatiotemporal resolution.

Vapor Phase Exchange of Self-Assembled Monolayers for Engineering of Biofunctional Surfaces.

We show that 4'-nitro-1,1'-biphenyl-4-thiol self-assembled monolayers (NBPT SAMs) on gold can be exchanged with 11-(mercaptoundecyl)triethylene glycol (C11EG3OH) SAMs via vapor deposition (VD). The pristine and the exchanged SAMs obtained by VD as well as solution method (SM) were characterized by X-ray photoelectron spectroscopy (XPS) and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). Using surface plasmon resonance (SPR), it is shown that C11EG3OH SAMs on gold obtained by vapor deposition exchange (VDEx) have the same protein resistivity as SAMs obtained by the direct self-assembly process. As expected, the cross-linked NBPT SAM are found to be resistive to both exchange processes, VDEx and solution method exchange (SMEx). In this way, VDEx opens up an elegant and new approach of patterning SAM surfaces in situ at vacuum conditions without using any solvents. By combining electron irradiation-induced chemical lithography of NBPT SAMs with VDEx, biofunctional patterned substrates were engineered and used for immobilization of protein arrays.

Nanomolar affinity protein trans-splicing monitored in real-time by fluorophore-quencher pairs.

High background originating from non-reacted, 'always-on' fluorescent probes remains a key unsolved problem in life science since washing procedures are not easily applicable. Covalent labeling approaches with simultaneous activation of fluorescence are advantageous to increase sensitivity and to reduce background signal. Here, we combined high-affinity protein trans-splicing with fluorophore/quencher pairs for online detection of covalent N-terminal 'traceless' protein labeling under physiological conditions in cellular environments. Substantial fluorescence enhancement at nanomolar probe concentrations was achieved.

'Traceless' tracing of proteins - high-affinity trans-splicing directed by a minimal interaction pair.

Protein trans-splicing mediated by split inteins is a powerful technique for site-specific protein modification. Despite recent developments there is still an urgent need for ultra-small high-affinity intein tags for in vitro and in vivo approaches. To date, only very few in-cell applications of protein trans-splicing have been reported, all limited to C-terminal protein modifications. Here, we developed a strategy for covalent N-terminal intein-mediated protein labeling at (sub) nanomolar probe concentrations. Combined with a minimal synthetic lock-and-key element, the affinity between the intein fragments was increased more than 50-fold to 10 nM. Site-specific and efficient 'traceless' protein modification by high-affinity trans-splicing is demonstrated at nanomolar concentrations in living mammalian cells.

Silicon-on-insulator based nanopore cavity arrays for lipid membrane investigation.

We present the fabrication and characterization of nanopore microcavities for the investigation of transport processes in suspended lipid membranes. The cavities are situated below the surface of silicon-on-insulator (SOI) substrates. Single cavities and large area arrays were prepared using high resolution electron-beam lithography in combination with reactive ion etching (RIE) and wet chemical sacrificial underetching. The locally separated compartments have a circular shape and allow the enclosure of picoliter volume aqueous solutions. They are sealed at their top by a 250 nm thin Si membrane featuring pores with diameters from 2 µm down to 220 nm. The Si surface exhibits excellent smoothness and homogeneity as verified by AFM analysis. As biophysical test system we deposited lipid membranes by vesicle fusion, and demonstrated their fluid-like properties by fluorescence recovery after photobleaching. As clearly indicated by AFM measurements in aqueous buffer solution, intact lipid membranes successfully spanned the pores. The nanopore cavity arrays have potential applications in diagnostics and pharmaceutical research on transmembrane proteins.

The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation.

A challenging task for the adaptive immune system of vertebrates is to identify and eliminate intracellular antigens. Therefore a highly specialized antigen presentation machinery has evolved to display fragments of newly synthesized proteins to effector cells of the immune system at the cell surface. After proteasomal degradation of unwanted proteins or defective ribosome products, resulting peptides are translocated into the endoplasmic reticulum by the transporter associated with antigen processing and loaded onto major histocompatibility complex (MHC) class I molecules. Peptide-MHC I complexes are transported via the secretory pathway to the cell surface where they are then inspected by cytotoxic T lymphocytes, which can trigger an immune response. This review summarizes the current view of the intracellular machinery of antigen processing and of viral immune escape mechanisms to circumvent destruction by the host.

Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus.

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.

Herpes viral proteins blocking the transporter associated with antigen processing TAP--from genes to function and structure.

In adaptation to the immune system, viruses have developed manifold mechanisms to evade the immune response, causing lifelong persistence in the host. Several members of the herpesvirus family are known to interfere with antigen presentation via MHC class I molecules. Here we compare the mechanistic and structural aspects of two unrelated herpesviral proteins, both of which have selected the transporter associated with antigen processing (TAP) as target for immune evasion. However, ICP47 (IE12) encoded by the herpes simplex virus and US6 from human cytomegalovirus utilize entirely different strategies to block TAP function. Detailed knowledge of the function and structure of these viral factors will help to understand TAP function and to design novel immune suppressors or vectors for gene transfer.

Immune escape of melanoma: first evidence of structural alterations in two distinct components of the MHC class I antigen processing pathway.

Sequence analyses of the transporter associated with antigen processing (TAP) in tumor cell lines with deficient MHC class I surface expression identified a bp deletion at position 1489 near the ATP-binding domain of Tap1, causing a frameshift in one melanoma cell line. The impaired TAP1 protein expression was associated with deficient TAP2 protein expression, peptide binding, translocation, and MHC class I surface expression. Stable TAP1 gene transfer reconstitutes the described defects, whereas lysis by HLA-A2-restricted CTLs was still abrogated. This was attributable to a 2-bp insertion at position 890 in the HLA-A2 gene and was corrected after HLA-A2 cotransfection. This study describes for the first time mutations in two distinct components of the MHC class I antigen processing pathway, suggesting an immune selection against CTLs recognizing both TAP-dependent and -independent T-cell epitopes.

Molecular mechanism and structural aspects of transporter associated with antigen processing inhibition by the cytomegalovirus protein US6.

The human cytomegalovirus (HCMV) has evolved a set of elegant strategies to evade host immunity. The HCMV-encoded type I glycoprotein US6 inhibits peptide trafficking from the cytosol into the endoplasmic reticulum and subsequent peptide loading of major histocompatibility complex I molecules by blocking the transporter associated with antigen processing (TAP). We studied the molecular mechanism of TAP inhibition by US6 in vitro. By using purified US6 and human TAP co-reconstituted in proteoliposomes, we demonstrate that the isolated endoplasmic reticulum (ER)-luminal domain of US6 is essential and sufficient to block TAP-dependent peptide transport. Neither the overall amount of bound peptides nor the peptide affinity of TAP is affected by US6. Interestingly, US6 causes a specific arrest of the peptide-stimulated ATPase activity of TAP by preventing binding of ATP but not ADP. The affinity of the US6-TAP interaction was determined to 1 microm. The ER-luminal domain of US6 is monomeric in solution and consists of 19% alpha-helices, 25% beta-sheets, and 27% beta-turns. All eight cysteine residues are involved in forming a stabilizing network of four intramolecular disulfide bridges. Glycosylation of US6 is not required for function. These findings point to fascinating mechanistic and structural properties, by which specific binding of US6 at the ER-luminal loops of TAP signals across the membrane to the nucleotide-binding domains to prevent ATP hydrolysis of TAP.

Enhanced expression of human ABC-transporter tap is associated with cellular resistance to mitoxantrone.

Multidrug resistance (MDR) phenotypes have been associated with the overexpression of various members of the superfamily of ATP binding cassette (ABC) transporters. Here we demonstrate that a member of the ABC-transporter family, the heterodimer 'transporter associated with antigen processing' (TAP), physiologically involved in major histocompatibility complex class I-restricted antigen presentation, is significantly overexpressed in the human gastric carcinoma cell line EPG85-257RNOV exhibiting a mitoxantrone-resistant phenotype. This tumor cell line shows an atypical MDR phenotype in the absence of 'P-glycoprotein' or 'MDR-associated protein' overexpression but with an enforced 'breast cancer resistance protein' expression level. Transfection of both TAP subunits encoding cDNA molecules, TAP1 and TAP2, into the drug-sensitive parental gastric carcinoma cell line EPG85-257P conferred a 3.3-fold resistance to mitoxantrone but not to alternative anti-neoplastic agents. Furthermore, cell clones transfected with both, but not singularly expressed TAP1 or TAP2, reduced cellular mitoxantrone accumulation. Taken together, the data suggest that the heterodimeric TAP complex possesses characteristics of a xenobiotic transporter and that the TAP dimer contributes to the atypical MDR phenotype of human cancer cells.

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP.

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide-MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAP-specific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis-Menten kinetics with a maximal velocity V(max) of 2 micromol/min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.

Downregulation of the constitutive tapasin expression in human tumor cells of distinct origin and its transcriptional upregulation by cytokines.

Human tumor cells frequently exhibit abnormalities in the major histocompatibility complex (MHC) class I surface expression which can be due to structural alterations and/or dysregulation of various components of the MHC class I antigen processing machinery, such as HLA class I heavy and light chains, the peptide transporter and the proteasome subunits. Although several cofactors critical for proper MHC class I assembly have been identified, their contribution to the immune escape phenotype of tumor cells has not been analyzed. In order to determine whether tapasin deficits are an integral part of immune escape mechanisms of human tumors, we studied the constitutive and cytokine-regulated expression pattern of tapasin in malignant cells of distinct histology. Heterogeneous and reduced expression levels of tapasin were found in small-cell lung carcinoma, pancreatic carcinoma, colon carcinoma, head an neck squamous cell carcinoma and renal cell carcinoma cell lines. Tapasin downregulation was also prominent in surgically removed tumor lesions when compared to normal controls. The impaired tapasin expression is often associated with low MHC class I cell surface expression. In addition, various cytokines, including interferon (IFN)-alpha, IFN-gamma, tumor necrosis factor (TNF)-alpha and interleukin (IL)-4, but not granulocyte-macrophage colony stimulating factor (GM-CSF), transcriptionally upregulate to a distinct extent and in a time-dependent manner tapasin expression in tumor cells. Thus, deficient tapasin expression appears to be a frequent event in human tumor cells. Its restoration by cytokines further suggests that impaired tapasin expression in tumors is rather due to dysregulation than to structural alterations.

Single molecule research on surfaces: from analytics to construction and back.

The study of single molecules opens a new dimension in understanding nature down to its finest ramifications. While much progress was achieved in the last decade concerning the detection techniques, suitable techniques for manipulating and handling the biomolecules still bear a challenge. Primarily, the task is keeping an individual, active molecule of a certain lifespan in the spot. Here, we will focus on techniques for the functional immobilization of (single) molecules on surfaces to enable their observation at one position over a time period. Presenting the main methods of reversible immobilization we will accentuate the chelator lipid concept as combining all features prerequisite for functional, reversible and well-defined immobilization. This will also show that single molecule research in principle is the synthesis of an insight into the function of nature and nano-biotechnology (manipulation): thus of analytics, construction, and back.

Design of supported membranes tethered via metal-affinity ligand-receptor pairs.

Model lipid layers are very promising in investigating the complex network of recognition, transport and signaling processes at membranes. We have developed a novel and generic approach to create supported lipid membranes tethered by metal-affinity binding. By self-assembly we have generated various interfaces that display histidine sequences (6xHis) via polymer spacers. These histidine-functionalized interfaces are designed to allow specific docking and fusion of vesicles containing metal-chelating lipids. By means of surface plasmon resonance and atomic force microscopy we analyzed the formation and subsequently the structure of these solid-supported membranes. Although the affinity constant of single ligand-receptor pairs is only in the micromolar range, very stable immobilization of these membranes was observed. This behavior can be explained by multivalent interactions resembling many features of cell adhesion. The process is highly specific, because vesicle docking and bilayer formation are strictly dependent on the presence of metal-affinity ligand-receptor pairs. The surface accessibility and geometry of these tethered membranes were probed by binding of histidine-tagged polypeptides. The supported membranes show adsorption kinetics and values similar to planar supported monolayers. Using various combinations of metal-chelating and histidine-tagged lipids or thiols these metal-affinity-tethered membranes should make a great impact on probing and eventually understanding the dynamic dialog of reconstituted membrane proteins.

Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of Lactococcus lactis.

The oligopeptide transport system (Opp) of Lactococcus lactis has the unique capacity to mediate the transport of peptides from 4 up to at least 18 residues. The substrate specificity of this binding protein-dependent ATP-binding cassette transporter is determined mainly by the receptor protein OppA. To study the specificity and ligand-binding mechanism of OppA, the following strategy was used: (i) OppA was purified and anchored via the lipid moiety to the surface of liposomes; (ii) the proteoliposomes were used in a rapid filtration-based binding assay with radiolabeled nonameric bradykinin as a reporter peptide; and (iii) combinatorial peptide libraries were used to determine the specificity and selectivity of OppA. The studies show that (i) OppA is able to bind peptides up to at least 35 residues, but there is a clear optimum in affinity for nonameric peptides; (ii) the specificity for nonameric peptides is not equally distributed over the whole peptide, because positions 4, 5, and 6 in the binding site are more selective; and (iii) the differences in affinity for given side chains is relatively small, but overall hydrophobic residues are favored-whereas glycine, proline, and negatively charged residues lower the binding affinity. The data indicate that not only the first six residues (enclosed by the protein) but also the C-terminal three residues interact in a nonopportunistic manner with (the surface of) OppA. This binding mechanism is different from the one generally accepted for receptors of ATP-binding cassette-transporter systems.

Gene transfer of human interferon gamma complementary DNA into a renal cell carcinoma line enhances MHC-restricted cytotoxic T lymphocyte recognition but suppresses non-MHC-restricted effector cell activity.

Even though renal cell carcinomas (RCC) are thought to be immunogenic, many tumors express variations in surface molecules and intracellular proteins that hinder induction of optimal antitumor responses. Interferon gamma (IFNgamma) stimulation can correct some of these deficiencies. Therefore, we introduced the complementary DNA (cDNA) encoding human IFNgamma into a well-characterized RCC line that has been selected for development of an allogeneic tumor cell vaccine for treatment of patients with metastatic disease. Studies were performed to determine how endogenous IFNgamma expression influences tumor cell immunogenicity. IFNgamma transductants showed minimal increases in surface expression of MHC class I and adhesion molecules but expression of class II molecules was induced. Proteins of the transporter associated with antigen processing (TAP) and low molecular weight polypeptide (LMP) were constitutively expressed at high levels. The transductants stimulated allospecific cytotoxic T lymphocytes (CTL); however, they were not better than unmodified tumor cells in this capacity. Endogenous IFNgamma expression enhanced tumor cell recognition by MHC-restricted, tumor antigen-specific CTL but suppressed recognition by non-MHC-restricted cytotoxic cells. Thus, the functional consequences of IFNgamma expression varied with respect to the type of effector cell and were not always beneficial for tumor cell recognition.