Dynamic imaging of protease activity with fluorescently quenched activity-based probes

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.     

Near-infrared fluorescent deoxyglucose analogue for tumor optical imaging in cell culture and living mice

2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has extensively been used for clinical diagnosis, staging, and therapy monitoring of cancer and other diseases. Nonradioactive glucose analogues enabling the screening of the glucose metabolic rate of tumors are of particular interest for anticancer drug development. A nonradioactive fluorescent deoxyglucose analogue may have many applications for both imaging of tumors and monitoring therapeutic efficacy of drugs in living animals and may eventually translate to clinical applications. We found that a fluorescent 2-deoxyglucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), can be delivered in several tumor cells via the glucose transporters (GLUTs). We therefore conjugated D-glucosamine with a near-infrared (NIR) fluorphor Cy5.5 and tested the feasibility of the Cy5.5-D-glucosamine (Cy5.5-2DG) conjugate for NIR fluorescence imaging of tumors in a preclinical xenograft animal model. Cy5.5-2DG was prepared by conjugating Cy5.5 monofunctional N-hydroxysuccinimide ester (Cy5.5-NHS) and D-glucosamine followed by high-performance liquid chromatography purification. The accumulation of Cy5.5-2DG and Cy5.5-NHS in different tumor cell lines at 37 and 4 degrees C were imaged using a fluorescence microscope. Tumor targeting and retention of Cy5.5-2DG and Cy5.5-NHS in a subcutaneous U87MG glioma and A375M melanoma tumor model were evaluated and quantified by a Xenogen IVIS 200 optical cooled charged-coupled device system. Fluorescence microscopy imaging shows that Cy5.5-2DG and Cy5.5-NHS are taken up and trapped by a variety of tumor cell lines at 37 degrees C incubation, while they exhibit marginal uptake at 4 degrees C. The tumor cell uptake of Cy5.5-2DG cannot be blocked by the 50 mM D-glucose, suggesting that Cy5.5-2DG may not be delivered in tumor cells by GLUTs. U87MG and A375M tumor localization was clearly visualized in living mice with both NIR fluorescent probes. Tumor/muscle contrast was clearly visible as early as 30 min postinjection (pi), and the highest U87MG tumor/muscle ratios of 2.81 +/- 0.10 and 3.34 +/- 0.23 were achieved 24 h pi for Cy5.5-2DG and Cy5.5-NHS, respectively. While as a comparison, the micropositron emission tomography imaging study shows that [18F]FDG preferentially localizes to the U87MG tumor, with resulting tumor/muscle ratios ranging from 3.89 to 4.08 after 30 min to 2 h postadministration of the probe. In conclusion, the NIR fluorescent glucose analogues, Cy5.5-2DG and Cy5.5-NHS, both demonstrate tumor-targeting abilities in cell culture and living mice. More studies are warranted to further explore their application for optical tumor imaging. To develop NIR glucose analogues with the ability to target GLUTs/hexokinase, it is highly important to select NIR dyes with a reasonable molecular size.     

Changes in the Nuclear Envelope Environment Affect Spindle Pole Body Duplication in Saccharomyces cerevisiae

The Saccharomyces cerevisiae nuclear membrane is part of a complex nuclear envelope environment also containing chromatin, integral and peripheral membrane proteins, and large structures such as nuclear pore complexes (NPCs) and the spindle pole body. To study how properties of the nuclear membrane affect nuclear envelope processes, we altered the nuclear membrane by deleting the SPO7 gene. We found that spo7Δ cells were sickened by the mutation of genes coding for spindle pole body components and that spo7Δ was synthetically lethal with mutations in the SUN domain gene MPS3. Mps3p is required for spindle pole body duplication and for a variety of other nuclear envelope processes. In spo7Δ cells, the spindle pole body defect of mps3 mutants was exacerbated, suggesting that nuclear membrane composition affects spindle pole body function. The synthetic lethality between spo7Δ and mps3 mutants was suppressed by deletion of specific nucleoporin genes. In fact, these gene deletions bypassed the requirement for Mps3p entirely, suggesting that under certain conditions spindle pole body duplication can occur via an Mps3p-independent pathway. These data point to an antagonistic relationship between nuclear pore complexes and the spindle pole body. We propose a model whereby nuclear pore complexes either compete with the spindle pole body for insertion into the nuclear membrane or affect spindle pole body duplication by altering the nuclear envelope environment.THE nuclear envelope is composed of distinct outer and inner nuclear membranes. The outer nuclear membrane is continuous with the endoplasmic reticulum. The inner nuclear membrane is associated with a unique set of proteins, some of which mediate interactions between the nuclear envelope and chromatin (reviewed in Zhao et al. 2009). Nuclear pore complexes traverse both membranes and allow transport of proteins and solutes between the cytoplasm and the nucleus. The inner and outer nuclear membranes fuse in the region surrounding each nuclear pore complex.In animal cells, the nuclear envelope disassembles as cells enter mitosis and reassembles upon mitotic exit. Nuclear envelope breakdown allows the association of chromosomes with spindle microtubules, which are nucleated from centrosomes that reside in the cytoplasm. In contrast, certain types of fungi, such as the budding yeast Saccharomyces cerevisiae, undergo closed mitosis, where the nuclear envelope remains intact throughout the entire cell cycle. Closed mitosis is possible because the yeast centrosome-equivalent, the spindle pole body (SPB), is embedded in the nuclear envelope, allowing the SPB to nucleate both cytoplasmic and nuclear microtubules.SPB duplication requires a mechanism for inserting the new SPB into the nuclear envelope (reviewed in Jaspersen and Winey 2004). The new SPB begins to form in late G₁/early S phase as satellite material deposited on the cytoplasmic face of an electron-dense region of the nuclear envelope, called the half-bridge. The satellite material matures into a duplication plaque, which is then inserted into the nuclear membrane and becomes the daughter SPB. Many genes are known to be required for SPB duplication, and this process has been carefully examined cytologically (Rose and Fink 1987; Winey et al. 1991, 1993; Spang et al. 1995; Bullitt et al. 1997; Adams and Kilmartin 1999; Elliott et al. 1999; Schramm et al. 2000; Jaspersen et al. 2002; Nishikawa et al. 2003; Araki et al. 2006). However, the exact mechanisms by which SPB duplication and insertion occur remain a mystery.Equally unclear is how nuclear pore complexes are inserted into an intact nuclear envelope (reviewed in Hetzer and Wente 2009). For both the SPB and nuclear pore complexes, the inner and outer nuclear membranes must fuse to allow insertion into the nuclear envelope. Yeast and vertebrate nuclear pore complexes each have four pore membrane (POM) nucleoporins containing transmembrane domains. Other nucleoporins have motifs with potential for bending membranes or sensing membrane curvature. Thus, certain nuclear pore complex components may have the ability to alter the nuclear membrane or stabilize particular membrane conformations (Devos et al. 2004, 2006; Alber et al. 2007; Drin et al. 2007). It is interesting to note that, in S. cerevisiae, nuclear pore complexes are enriched in the vicinity of the SPB (Heath et al. 1995; Winey et al. 1997; Adams and Kilmartin 1999), but the significance of this phenomenon is not known. The SPB and nuclear pore complexes share at least two common components, the integral membrane protein Ndc1p and the small calcium-binding protein Cdc31p (Chial et al. 1998; Fischer et al. 2004). Ndc1p is thought to play a role in insertion of both SPBs and nuclear pore complexes into the nuclear membrane.SUN domain proteins are a conserved family of inner nuclear membrane proteins that interact with specific outer nuclear membrane proteins to form a physical bridge across the nuclear envelope (reviewed in Hiraoka and Dernburg 2009; Razafsky and Hodzic 2009). One of the components of the S. cerevisiae SPB is the SUN domain protein Mps3p. The N terminus of Mps3p is in the nucleoplasm, while the C terminus, containing the SUN domain, is found in the space between the inner and outer nuclear membranes. In addition to the SPB, Mps3p localizes to multiple foci at the nuclear periphery, and these two pools of Mps3p have distinct functions (Jaspersen et al. 2002, 2006; Nishikawa et al. 2003). At the SPB, Mps3p is required for half-bridge formation and early steps of SPB duplication, and cells compromised for Mps3p function accumulate in mitosis with a single SPB and a monopolar spindle (Jaspersen et al. 2002; Nishikawa et al. 2003). At the nuclear periphery, Mps3p is involved in tethering telomeres to the nuclear envelope in mitosis and meiosis, sequestering DNA double-strand breaks away from recombination factors, and associating with soluble chromatin proteins (Antoniacci et al. 2004, 2007; Bupp et al. 2007; Conrad et al. 2007, 2008; Oza et al. 2009; Schober et al. 2009).While many structural features of the yeast nucleus have been identified, little is known about how the physical properties of the nuclear membrane contribute to processes that occur at the nuclear envelope. As noted above, resident proteins of the nuclear envelope may affect nuclear membrane properties. In addition, the nuclear membrane is affected by altering lipid biosynthesis, for example, by inactivating the phosphatidic acid (PA) phosphohydrolase Pah1p or by inactivating the phosphates complex, made of Spo7p and Nem1p, which activates Pah1p. In the absence of Spo7p, Nem1p, or Pah1p, cells exhibit nuclear envelope extensions and extensive ER membrane sheets, and they also have altered membrane lipid composition, including a decrease in phosphatidylcholine and an increase in PA, phosphatidylethanolamine, and phosphatidylinositol (Siniossoglou et al. 1998; Santos-Rosa et al. 2005; Campbell et al. 2006; Han et al. 2006). These three proteins are unique among phospholipid biosynthesis proteins in their ability to affect nuclear morphology upon gene disruption (Han et al. 2008). A similar phenotype was seen upon overexpression of DGK1, which counteracts the activity of Pah1p by converting diacylglycerol to PA, leading to an increase in PA levels at the nuclear envelope (Han et al. 2008). Consistent with a conserved role for Pah1p in regulating nuclear envelope processes, deletion of either NEM1 or SPO7 is synthetically lethal with deletions of certain nucleoporin genes (Siniossoglou et al. 1998), and inactivation of the PAH1 homolog in Caenorhabditis elegans, LPIN-1, results in defects in nuclear envelope disassembly and reassembly (Golden et al. 2009; Gorjanacz and Mattaj 2009).To identify processes that are affected by altered nuclear membrane properties, we screened for pathways that are compromised in spo7Δ cells. We found that SPO7 inactivation strongly influences the SPB. By screening for proteins that could alleviate spo7Δ-induced SPB defects, we uncovered an unexpected inhibitory role for nucleoporins in SPB function, revealing that nuclear pore complexes, or components thereof, act antagonistically to the SPB in the nuclear envelope. Taken together, our findings indicate that the nuclear envelope environment is important for the function of protein complexes and biological processes occurring at the nuclear periphery.     

Specific interactions of divalent metal ions with a DNA duplex containing the d(CA)n/(GT)n tandem repeat

Divalent metal ions are essential for maintaining functional states of the DNA molecule. Their participation in DNA structure is modulated by the base sequence and varies depending on the nature of the ion. The present investigation addresses the interaction of Ca2+ ions with a tandem repeat of two CA dinucleotides, (CA)2/(TG)2. The binding of Ca2+ to the repeat is monitored by nuclear magnetic resonance (NMR) spectroscopy using chemical shift mapping. Parallel experiments monitor binding of Mg2+ ions to the repeat as well as binding of each ion to a DNA duplex in which the (CA)2/(TG)2 repeat is eliminated. The results reveal that the direction and the magnitude of chemical shift changes induced by Ca2+ ions in the NMR spectra of the repeat are different from those induced by Mg2+ ions. The differences between the two cations are significantly diminished by the elimination of the (CA)2/(TG)2 repeat. These findings suggest a specific interaction of Ca2+ ions with the (CA)2/(TG)2 motif. The specificity of the interaction resides in the two A-T base pairs of the repeat, and it involves the major groove of the first A-T base pair and both grooves of the second A-T base pair.     

Conjugation to Nedd8 instigates ubiquitylation and down-regulation of activated receptor tyrosine kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.     

Yeast nuclear envelope subdomains with distinct abilities to resist membrane expansion

Little is known about what dictates the round shape of the yeast Saccharomyces cerevisiae nucleus. In spo7Delta mutants, the nucleus is misshapen, exhibiting a single protrusion. The Spo7 protein is part of a phosphatase complex that represses phospholipid biosynthesis. Here, we report that the nuclear protrusion of spo7Delta mutants colocalizes with the nucleolus, whereas the nuclear compartment containing the bulk of the DNA is unaffected. Using strains in which the nucleolus is not intimately associated with the nuclear envelope, we show that the single nuclear protrusion of spo7Delta mutants is not a result of nucleolar expansion, but rather a property of the nuclear membrane. We found that in spo7Delta mutants the peripheral endoplasmic reticulum (ER) membrane was also expanded. Because the nuclear membrane and the ER are contiguous, this finding indicates that in spo7Delta mutants all ER membranes, with the exception of the membrane surrounding the bulk of the DNA, undergo expansion. Our results suggest that the nuclear envelope has distinct domains that differ in their ability to resist membrane expansion in response to increased phospholipid biosynthesis. We further propose that in budding yeast there is a mechanism, or structure, that restricts nuclear membrane expansion around the bulk of the DNA.     

Peptide Derived from HIV-1 TAT Protein Destabilizes a Monolayer of Endothelial Cells in an in Vitro Model of the Blood-Brain Barrier and Allows Permeation of High Molecular Weight Proteins

Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3–0.6 μmol/ml of this C-TAT peptide, for a period of 1–2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.