Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii.

The growth rate of Borrelia burgdorferi and Borrelia hermsii in BSK II medium prepared with cysteine-free or cysteine-containing (0.185-5.92 mM) CMRL 1066 medium was studied. In media with cysteine-free CMRL 1066, growth of borreliae was detectable, although it was reduced by approximately 80%. Bacterial growth was maximal when the concentration of cysteine in CMRL 1066 reached 1.48 mM, which represents the standard cysteine concentrations of the medium; higher concentrations inhibited the growth of borreliae. Cysteine incorporation, measured by the uptake of radiolabeled cysteine, showed that cysteine enters B. burgdorferi and B. hermsii cells by passive diffusion. Labeling studies of borreliae with [35S]cysteine indicated that B. burgdorferi has several cysteine-containing proteins, including ones at 22, 30 (OspA), and 34 kDa (OspB), whereas B. hermsii showed only two [35S]cysteine-incorporating proteins, at 22 and 24 kDa, which were exposed onto the outer cell surface. In addition, most of the cysteine-incorporating proteins could be biosynthetically radiolabeled when bacterial cells were grown in vitro with [3H]palmitate, and the differences in cysteine incorporation observed between B. burgdorferi and B. hermsii were found to be correlated with differences in lipoproteins.     

Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions

Several fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry, achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum (ER) signal-anchor membrane protein (CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER (OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing enhanced green fluorescent protein (EGFP) or monomeric green fluorescent protein (mGFP) CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric, while a third previously reported as monomeric was found to strongly oligomerize.     

Evaluation of a maleimido derivative of CHX-A' DTPA for site-specific labeling of affibody molecules

Affibody molecules are a new class of small targeting proteins based on a common three-helix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule Z HER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A' DTPA was site-specifically conjugated at the C-terminal cysteine of Z HER2:2395-C, a variant of Z HER2:342, providing a homogeneous conjugate with a dissociation constant of 56 pM. The yield of labeling with (111)In was >99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of (111)In-CHX-A' DTPA-Z 2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all nonspecific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of (111)In-CHX-A' DTPA-Z 2395-C was 17.3 +/- 4.8% IA/g and the tumor-to-blood ratio 86 +/- 46 (4 h postinjection). HER2-expressing xenografts were clearly visualized 1 h postinjection. In conclusion, coupling of maleimido-CHX-A' DTPA to cysteine-containing Affibody molecules provides a well-defined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo.     

Proteomic analysis of mitochondria from the Bos taurus heart. II. Identification of mitochondrial soluble proteins

A fraction of the so-called mitochondrial soluble proteins was obtained after the destruction of purified mitochondria by sonication according to the previously found approach to the identification of protein subsets of the Bos taurus heart proteome. A tryptic destruction of these proteins was achieved. Approximately half of the tryptic hydrolysate was separated into two fractions of cysteine-containing and cysteine-free peptides by covalent chromatography on Thiopropyl Sepharose 4B. The cysteine-containing peptides were modified by iodoacetamide. The peptides were mass-spectrometrically identified in all the three fractions of tryptic hydrolysate, and the proteins were searched for in the amino acid sequence databases. There were 213 unique proteins reliably identified.     

Superfolder GFP is fluorescent in oxidizing environments when targeted via the Sec translocon

The ability to study proteins in live cells using genetically encoded fluorescent proteins (FPs) has revolutionized cell biology (1-3). Researchers have created numerous FP biosensors and optimized FPs for specific organisms and subcellular environments in a rainbow of colors (4,5). However, expressing FPs in oxidizing environments such as the eukaryotic endoplasmic reticulum (ER) or the bacterial periplasm can impair folding, thereby preventing fluorescence (6,7). A substantial fraction of enhanced green fluorescent protein (EGFP) oligomerizes to form non-fluorescent mixed disulfides in the ER (6) and EGFP does not fluoresce in the periplasm when targeted via the SecYEG translocon (7). To overcome these obstacles, we exploited the highly efficient folding capability of superfolder GFP (sfGFP) (8). Here, we report sfGFP does not form disulfide-linked oligomers in the ER and maltose-binding protein (MBP) signal sequence (peri)-sfGFP (9) is brightly fluorescent in the periplasm of Escherichia coli. Thus, sfGFP represents an important research tool for studying resident proteins of oxidizing environments.     

Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans

Tat, the transactivator protein of human immunodeficiency virus-1, has the unusual capacity of being internalized by cells when present in the extracellular milieu. This property can be exploited for the cellular delivery of heterologous proteins fused to Tat both in cell culture and in living animals. Here we provide genetic and biochemical evidence that cell membrane heparan sulfate (HS) proteoglycans act as receptors for extracellular Tat uptake. Cells genetically defective in the biosynthesis of fully sulfated HS are selectively impaired in the internalization of recombinant Tat fused to the green fluorescent protein, as evaluated by both flow cytometry and functional assays. In wild type cells, Tat uptake is competitively inhibited by soluble heparin and by treatment with glycosaminoglycan lyases specifically degrading HS chains. Cell surface HS proteoglycans also mediate physiological internalization of Tat green fluorescent protein released from neighboring producing cells. In contrast to extracellular Tat uptake, both wild type cells and cells genetically impaired in proteoglycan synthesis are equally proficient in the extracellular release of Tat, thus indicating that proteoglycans are not required for this process. The ubiquitous distribution of HS proteoglycans is consistent with the efficient intracellular delivery of heterologous proteins fused with Tat to different mammalian cell types.     

Synthesis of covalent DNA-protein conjugates by expressed protein ligation

Semisynthetic DNA-protein conjugates are versatile tools for many applications in bioanalytics and nanobiotechnology. We here report a method based on expressed protein ligation (EPL) for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins. The latter contain a C-terminal thioester, enabling the mild and highly specific reaction with N-terminal cysteine compounds. To conveniently couple commercially available DNA oligomers with cysteine groups a universal chemical modifier was developed, containing a protected cysteine and an amino-reactive N-hydroxysuccinimide group connected by a hexaethyleneglycol moiety. Using maltose-binding protein (MBP) and green fluorescent protein mutant EYFP as a model systems, we demonstrate the feasibility of this approach, as well as the integrity and functionality of the DNA-protein conjugates synthesized. We anticipate that our concept will enable many applications, such as the generation of large arrays of surface-bound, recombinant proteins assembled by means of DNA-directed immobilization.     

Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors

The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g. EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extracellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes ('activate the fluorogen'). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000-fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (β2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well.     

Thymoquinone suppression of the human hepatocellular carcinoma cell growth involves inhibition of IL-8 expression,elevated levels of TRAIL receptors,oxidative stress and apoptosis

Sod2 is the major salt tolerance plasma membrane protein of Schizosaccharomyces pombe. It functions to remove excess intracellular sodium (or lithium) in exchange for protons. We investigated the role of cysteine residues and created a cysteine-free Sod2 protein. Each cysteine residue of the ten present was individually mutated to serine and the different proteins expressed and characterized in S. pombe. Western blotting revealed that all the individual mutant proteins were expressed. We examined the ability of the mutant proteins to confer salt tolerance to S. pombe with the endogenous Sod2 protein deleted. Only proteins with C26S and C374S mutations were partially reduced in their ability to confer salt tolerance. Additionally, they showed a change in conformation in comparison to the wild-type protein, indicated by differential sensitivity to trypsin. Deletion of all the cysteine residues of Sod2 resulted in a functional protein that was expressed in S. pombe at levels similar to the wild type and also conferred salt tolerance. The conformation of the cysteine-free Sod2 protein was not altered relative to the wild-type protein. We examined the accessibility of amino acids of the cysteineless protein present on putative extracellular loop 2. A cysteine placed at position Ala119 was accessible to externally applied [2-(trimethylammonium)ethyl] methane thiosulfonate bromide. The results demonstrate that cysteines in the Sod2 protein can be changed to serine residues resulting in an expressed, functional protein. The utility of the cysteine-free Sod2 protein for determination of topology and amino acid accessibility is demonstrated.     

Misfolded proteins and human diseases

Though protein folding is a regular phenomenon inside the cellular milieu, slight differences in the folding pattern of proteins may lead to disease-causing forms. Many diseases have been identified that are caused by these misfolded macromolecules and a considerable amount of focus is still directed towards better understanding of the processes that lead to these misfolded structures. Further progress in the field of how soluble proteins begin to misfold and how resultant oligomers begin dysfunction offers exciting prospects for specific molecular intervention.     

Endoplasmic reticulum–to–Golgi trafficking of procollagen III via conventional vesicular and tubular carriers

Collagen is the major protein component of the extracellular matrix. Synthesis of procollagens starts in the endoplasmic reticulum (ER), and three α chains form a rigid triple helix 300–400 nm in length. It remains unclear how such a large cargo is transported from the ER to the Golgi apparatus. In this study, to elucidate the intracellular transport of fibril-forming collagens, we fused cysteine-free GFP to the N-telopeptide region of procollagen III (GFP-COL3A1) and analyzed transport by live-cell imaging. We found that the maturation dynamics of procollagen III was largely different from that of network-forming procollagen IV. Proline hydroxylation of procollagen III uniquely triggered the formation of intralumenal droplet-like structures, similarly to events caused by liquid–liquid phase separation, and ER exit sites surrounded large droplets containing chaperones. Procollagen III was transported to the Golgi apparatus via vesicular and tubular carriers containing ERGIC53 and RAB1B; this process required TANGO1 and CUL3, which we previously reported to be dispensable for procollagen IV. GFP-COL3A1 and mCherry-α1AT were cotransported in the same vesicle. Based on these findings, we propose that shortly after ER exit, enlarged carriers containing procollagen III fuse to ERGIC for transport to the Golgi apparatus by conventional cargo carriers.     

Disulphide bonds in casein micelle from milk

Mammary epithelial cells synthesised and secreted caseins, the major milk proteins in most mammals, as large aggregates called micelles into the alveolar lumen they surround. We investigated the implication of the highly conserved cysteine(s) of kappa-casein in disulphide bond formation in casein micelles from several species. Dimers were found in all milks studied, confirming previous observation in ruminants. More importantly, the study of interchain disulphide bridges in mouse and rat casein micelles revealed that any casein possessing a cysteine is engaged in disulphide bond interchange; these species express four or five cysteine-containing caseins, respectively. We found that the main rodent caseins form both homo- and heterodimers. Additionally, disulphide bond formation among milk proteins was specific since the interaction of the caseins with cysteine-containing whey proteins was not observed in native casein micelles.     

Rapid directed molecular evolution of fluorescent proteins in mammalian cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 10⁷ independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near‐infrared fluorescent proteins with enhanced intracellular brightness. The developed near‐infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near‐infrared fluorescent proteins enabled crosstalk‐free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual‐color near‐infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.     

Subcellular localization of VDAC in mitochondria and ER in the cerebellum

The voltage-dependent anion channel (VDAC) provides passage for adenine nucleotides, Ca2+ and other metabolites into and from mitochondria. Here, the intracellular localization and oligomeric organization of VDAC in brain mitochondria and ER are demonstrated. Immunohistochemical staining of VDAC in rat cerebellum showed high labeling of the Purkinje neurons. Immunogold labeling and EM analysis of the cerebellar molecular layer showed specific VDAC immunostaining of the mitochondrial outer membrane, highly enhanced in contact sites between mitochondria or between mitochondria and associated ER. Purified ER membranes contain VDAC, but not other mitochondrial proteins. Chemical cross-linking of isolated mitochondria, ER or purified VDAC demonstrated the existence of VDAC in oligomeric form. Based on the enrichment of VDAC in the junctional face of closely associated mitochondrial and ER membranes and the existence of VDAC oligomers, we propose an involvement of VDAC in specialized intermembrane communication between mitochondria or between ER and mitochondria, serving to complement the tight structural and functional coupling observed between these organelles.     

Development of an ON/OFF switchable fluorescent probe targeting His tag fused proteins in living cells

The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occasionally affects their functions and/or localization in living cells. Therefore, an imaging method that uses short peptide tags such as hexa-histidine (the His tag) has been attracting increasing attention. Few studies have investigated ON/OFF switchable fluorescent probes for intracellular His-tagged proteins. We herein developed a novel ON/OFF switchable probe for imaging targeted intracellular proteins fused with a CH6 tag, which is composed of one cysteine residue and six histidine residues.     

Redox signalling via the cellular thiolstat

Research conducted during the last two decades has provided evidence for the existence of an extensive intracellular redox signalling, control and feedback network based on different cysteine-containing proteins and enzymes. Together, these proteins enable the living cell to sense and respond towards external and internal redox changes in a measured, gradual, appropriate and mostly reversible manner. The (bio)chemical basis of this regulatory 'thiolstat' is provided by the complex redox chemistry of the amino acid cysteine, which occurs in vivo in various sulfur chemotypes and is able to participate in different redox processes. Although our knowledge of the biological redox behaviour of sulfur (i.e. cysteine or methionine) is expanding, numerous questions still remain. Future research will need to focus on the individual proteins involved in this redox system, their particular properties and specific roles in cellular defence and survival. Once it is more fully understood, the cellular thiolstat and its individual components are likely to form prominent targets for drug design.     

ATP‐independent molecular chaperone activity generated under reducing conditions

Molecular chaperones are essential to maintain proteostasis. While the functions of intracellular molecular chaperones that oversee protein synthesis, folding and aggregation, are established, those specialized to work in the extracellular environment are less understood. Extracellular proteins reside in a considerably more oxidizing milieu than cytoplasmic proteins and are stabilized by abundant disulfide bonds. Hence, extracellular proteins are potentially destabilized and sensitive to aggregation under reducing conditions. We combine biochemical and mass spectrometry experiments and elucidate that the molecular chaperone functions of the extracellular protein domain Bri2 BRICHOS only appear under reducing conditions, through the assembly of monomers into large polydisperse oligomers by an intra‐ to intermolecular disulfide bond relay mechanism. Chaperone‐active assemblies of the Bri2 BRICHOS domain are efficiently generated by physiological thiol‐containing compounds and proteins, and appear in parallel with reduction‐induced aggregation of extracellular proteins. Our results give insights into how potent chaperone activity can be generated from inactive precursors under conditions that are destabilizing to most extracellular proteins and thereby support protein stability/folding in the extracellular space.SignificanceChaperones are essential to cells as they counteract toxic consequences of protein misfolding particularly under stress conditions. Our work describes a novel activation mechanism of an extracellular molecular chaperone domain, called Bri2 BRICHOS. This mechanism is based on reducing conditions that initiate small subunits to assemble into large oligomers via a disulfide relay mechanism. Activated Bri2 BRICHOS inhibits reduction‐induced aggregation of extracellular proteins and could be a means to boost proteostasis in the extracellular environment upon reductive stress.     

Role of cysteine residues in the NCKX2 Na+/Ca(2+)-K+ Exchanger: generation of a functional cysteine-free exchanger

Cysteine residues play an important role in many proteins, either in enzymatic activity or by mediating inter- or intramolecular interactions. The Na(+)/Ca(2+)-K(+) exchanger plays a critical role in Ca(2+) homeostasis in retinal rod (NCKX1) and cone (NCKX2) photoreceptors by extruding Ca(2+) that enters rod and cone cells via the cGMP-gated channels. NCKX1 and NCKX2 contain five highly conserved cysteine residues. The objectives of this study were threefold: (1) to examine the importance of cysteine residues in NCKX2 protein function; (2) to examine their role in the interaction between NCKX2 and the CNGA subunit of the cGMP-gated channel; and (3) to generate a functional cysteine-free NCKX2 protein. The latter will facilitate structural studies taking advantage of the unique chemistry of the thiol group following insertion of cysteine residues at specific positions in the cysteine-free background. We generated a cysteine-free NCKX2 mutant protein that showed normal protein synthesis and processing and approximately 50% wild-type cation transport function. Cysteine residues were also not critical for the formation of NCKX2 homo-oligmers or NCKX2 hetero-oligomers with the CNGA subunit of the cGMP-gated channel. Our results appear to rule out a critical importance of an intramolecular disulfide linkage in NCKX2 protein synthesis and folding as had been reported before.     

Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real‐time bioluminescence imaging

It is known that endoplasmic reticulum (ER) stress in cells and extracellular vesicles (EVs) plays a significant role in cancer cells, therefore the evaluation of compounds that can regulate ER stress and EV secretion would be a suitable system for further screening and development of new drugs. In this study, we evaluated chemical chaperones derived from natural products based on monitoring Bip/GRP78 promoter activity during cancer cell growth, at the level of the single cell, by a bioluminescence microscopy system that had several advantages compared with fluorescence imaging. It was found that several chemical chaperones, such as ferulic acid (FA), silybin, and rutin, affected the activity. We visualized EVs from cancer cells using bioluminescence imaging and showed that several EVs could be observed when using CD63 fused with NanoLuc luciferase, which has a much smaller molecular weight and higher intensity than conventional firefly luciferase. We then examined the effects of the chemical chaperones on EVs from cancer cells by bioluminescence imaging and quantified the expression of CD63 in these EVs. It was found that the chemical chaperones examined in this study affected CD63 levels in EVs. These results showed that imaging at the level of the single cell using bioluminescence is a powerful tool and could be used to evaluate chemical chaperones and EVs from cancer cells. This approach may produce new information in this field when taken together with conventional and classical methods.     

Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening

The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5+/-1.5% (mean+/-SEM) and 8.0+/-0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8+/-2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1+/-1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals.