Microinjected ribonuclease A as a probe for lysosomal pathways of intracellular protein degradation

There are multiple pathways of intracellular protein degradation, and molecular determinants within proteins appear to target them for particular pathways of breakdown. We use red cell-mediated microinjection to introduce radiolabeled proteins into cultured human fibroblasts in order to follow their catabolism. A well-characterized protein, bovine pancreatic ribonuclease A (RNase A), is localized initially in the cytosol of cells after microinjection, but it is subsequently taken up and degraded by lysosomes. This lysosomal pathway of proteolysis is subject to regulation in that RNase A is taken up and degraded by lysosomes at twice the rate when serum is omitted from the culture medium. Subtilisin cleaves RNase A between residues 20 and 21, and the separated fragments are termed RNase S-peptide (residues 1–20) and RNase S-protein (residues 21–124). Microinjected RNase S-protein is degraded in a serum-independent manner, while RNase S-peptide microinjected alone shows a twofold increase in degradation in response to serum withdrawal. Furthermore, covalent linkage of S-peptide to other proteins prior to microinjection causes degradation of the conjugate to become serum responsive. These results show that recognition of RNase A and certain other proteins for enhanced lysosomal degradation during serum withdrawal is based on some feature of the amino-terminal 20 amino acids. The entire S-peptide is not required for enhanced lysosomal degradation during serum withdrawal because degradation of certain fragments is also responsive to serum. We have identified the essential region to be within residues 7–11 of RNase S-peptide (Lys-Phe-Glu-Arg-Gln; KFERQ). To determine whether related peptides exist in cellular proteins, we raised antibodies to the pentapeptide. Affinity-purified antibodies to KFERQ specifically precipitate 25–35% of cellular proteins, and these proteins are preferentially degraded in response to serum withdrawal. Computer analyses of known protein sequences indicate that proteins degraded by lysosomes at an enhanced rate in response to serum withdrawal contain peptide regions related, but not identical, to KFERQ. We suggest two possible peptide motifs related to KFERQ and speculate about possible mechanisms of selective delivery of proteins to lysosomes based on such peptide regions.     

The design and production of semisynthetic ribonucleases with increased thermostability by incorporation of S-peptide analogues with enhanced helical stability

Recent work has shown that, with synthetic analogues of C-peptide (residues 1-13 of ribonuclease A), the stability of the peptide helix in H2O depends strongly on the charge on the N-terminal residue. We have asked whether, in semisynthetic ribonuclease S reconstituted from S-protein plus an analogue of S-peptide (1-15), the stability of the peptide helix is correlated with the Tm of the reconstituted ribonuclease S. Six peptides have been made, which contain Glu9----Leu, a blocked alpha-COO- group (-CONH2), and either Gln11 or Glu11. The N-terminal residue has been varied; its charge varies from +2 (Lys) to -1 (succinyl-Ala). We have measured the stability of the peptide helix, the affinity of the peptide for S-protein (by C.D. titration), and the thermal stability of the reconstituted ribonuclease S. All six peptide analogues show strongly enhanced helix formation compared to either S-peptide (1-15) or (1-19), and the helix content increases as the charge on the N-terminal residue changes from +2 to -1. All six peptides show increased affinity for S-protein compared to S-peptide (1-19), and all six reconstituted ribonucleases S show an increase in Tm compared to the protein with S-peptide (1-19). The Tm increases as the charge on residue 1 changes from +2 to -1. The largest increment in Tm is 6 degrees. The results suggest that the stability of a protein can be increased by enhancing the stability of its secondary structure.     

A 'cassette' RNase: site-selective cleavage of RNA by RNase S equipped with RNA-recognition segment

RNase S is a unique protein comprising the non-covalent association of two components, the S-peptide and the S-protein. An RNA-recognition segment derived from the human immunodeficiency virus (HIV)-1 Rev protein was conjugated with the S-peptide to form a complex with the S-protein. The resulting RNase S bearing the RNA-recognition segment preferentially hydrolyzed a single position of the RNA stem-loop derived from the specific binding site for the Rev protein.     

Ribonuclease S-peptide as a carrier in fusion proteins.

S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).     

Engineering S-protein fragments of bovine ribonuclease A for targeted drug delivery

High affinity interaction between S-protein and S-peptide fragments of bovine pancreatic RNase A has been recently used for construction of molecular vehicles for targeted drug delivery. The vehicle is assembled as a complex of drug carrier conjugated S-protein with S-peptide-tagged targeting protein. To avoid random chemical crosslinking of drug carriers to S-protein, we constructed a mutant 16-124aa fragment of RNase A in which 122ala is replaced with a cysteine residue. The mutant and the corresponding wild type fragments expressed in Escherichia coli are refolded into functional conformations only in the presence of S-peptide. After the removal of S-peptide, both fragments retain the ability to bind S-peptide and S-peptide-tagged proteins. The 122cys residue in the mutant fragment is available for site-specific conjugation.     

Thermodynamics of the helix-coil transition: Binding of S15 and a hybrid sequence, disulfide stabilized peptide to the S-protein

Pancreatic ribonuclease A may be cleaved to produce two fragments: the S-peptide (residues 1-20) and the S-protein (residues 21-124). The S-peptide, or a truncated version designated as the S15 peptide (residues 1-15), combines with the S-protein to produce catalytically active complexes. The conformation of these peptides and many of their analogues is predominantly random coil at room temperature; however, they populate a significant fraction of helical form at low temperature under certain solution conditions. Moreover, they adopt a helical conformation when bound to the S-protein. A hybrid sequence, disulfide-stabilized peptide (ApaS-25), designed to stabilize the helical structure of the S-peptide in solution, also combines with the S-protein to yield a catalytically active complex. We have performed high-precision titration microcalorimetric measurements to determine the free energy, enthalpy, entropy, and heat capacity changes for the binding of ApaS-25 to S-protein within the temperature range 5-25 degrees C. The thermodynamic parameters for both the complex formation reactions and the helix-to-coil transition also were calculated, using a structure-based approach, by calculating changes in accessible surface area and using published empirical parameters. A simple thermodynamic model is presented in an attempt to account for the differences between the binding of ApaS-25 and the S-peptide. From this model, the thermodynamic parameters of the helix-to-coil transition of S15 can be calculated.     

Protein and peptide binding and stimulation of in vitro lysosomal proteolysis by the 73-kDa heat shock cognate protein.

Lysosomal degradation of intracellular proteins during serum withdrawal is stimulated by a member of the 70-kDa heat shock protein (hsp70) family (Chiang, H.-L., Terlecky, S. R., Plant, C. P., and Dice, J. F. (1989) Science 246, 382-385). This hsp70, isolated by affinity chromatography with RNase S-peptide-Sepharose, is referred to as the 73-kDa peptide recognition protein (prp73). We now report that prp73 binds to several proteins and peptides whose degradative rates are increased during serum withdrawal. prp73 also binds to the pentapeptide, KFERQ, and more weakly to most modified RNase S-peptide derivatives with a single amino acid substitution within the KFERQ sequence. Taken together, these results suggest that prp73 binds to a variety of proteins at peptide regions biochemically related to KFERQ. Three lines of evidence indicate that prp73 is the heat shock cognate protein of 73 kDa (hsc73): (a) among five hsp70s tested, hsc73 binds to RNase S-peptide most avidly, (b) both prp73 and hsc73 also bind to RNase A and aspartate aminotransferase but not to ovalbumin, lysozyme, or ubiquitin, and (c) both prp73 and hsc73 promote uptake and degradation of [3H] RNase S-peptide by lysosomes in vitro, while three other hsp70s are without activity in this assay.     

Preparation of ribonuclease S domain-swapped dimers conjugated with DNA and PNA: modulating the activity of ribonucleases

Obtaining highly specific and active ribonuclease activities is an important goal with numerous medical and biochemical applications. As a step toward more active and specific ribonucleases, we describe the preparation and the enzymatic and structural properties of RNase S monomers and dimers conjugated to DNA and PNA molecules. Poly(dT)n (2'-oligodeoxyribonucleotides, n = 8, 15) and t8 peptide nucleic acid (PNA) chains have been conjugated to the S-peptide of ribonuclease S. Monomers and dimers of the conjugated enzyme have been obtained and characterized by 1H NMR spectroscopy, showing that DNA or PNA conjugation does not alter the native structure of ribonuclease S. The oligonucleotide-conjugated RNase S monomer and dimer show significant activity against single-stranded RNA and very low/negligible hydrolysis of double-stranded poly(A).poly(U). In contrast, the t8-conjugated RNase S monomer and dimer show substantial activity against both ssRNA and dsRNA. These results highlight the importance of positive charges near but not in the active site in enhancing activity against dsRNA and reveal the promise of PNA-RNase conjugates for modulating RNase activity.     

Separation of Albumin–Ribonuclease Conjugates

A method for separation of albumin–ribonuclease (RNase) conjugates has been proposed based on the use of macroporous silicates. It was established that about 76% of ligand-free human serum albumin (LFHSA) formed complexes with enzymes. It was shown that most of the conjugates of albumin and pancreatic RNase contained up to 2 mol enzyme per 1 mol LFHSA. The conjugates of albumin and bacterial RNase isolated from cells of the strain Bacillus intermedius 7P displayed higher specific activities, containing, on average, 2.3 mol RNase per 1 mol LFHSA (for the conjugates with molecular weights below 92 kDa) or 3.3 mol RNase per 1 mol protein carrier (for the conjugates with higher molecular weight).     

Isolation of conjugates of albumin and ribonuclease

A method for separation of albumin-ribonuclease (RNase) conjugates has been proposed, based on the use of macroporous silicates. It was established that about 76% of ligand-free human serum albumin (LFHSA) formed complexes with enzymes. It was shown that most of the conjugates of albumin and pancreatic RNase contained up to 2 mol enzyme per 1 mol LFHSA. The conjugates of albumin and bacterial RNase, isolated from the cells of the strain Bacillus intermedius 7P, displayed higher specific activities, containing, on average, 2.3 mol RNase per 1 mol LFHSA (for the conjugates with molecular weights below 92 kDa) or 3.3 mol RNase per 1 mol protein carrier (for the conjugates with higher molecular weight).     

Functionalization of osmium arene anticancer complexes with (poly)arginine: effect on cellular uptake, internalization, and cytotoxicity

Attaching peptides to metallodrugs may result in improved biological properties of the complexes. The potential use of cell penetrating peptides (CPPs) as cell delivery vectors is attractive, since directed cell uptake of (metallo)drugs remains a major challenge in anticancer drug design. In this work, we report the synthesis of peptide conjugates of the organometallic Os(II) anticancer complex [(η(6)-biphenyl)Os(picolinate)Cl] with different arginine (Arg) chain lengths. Complexes conjugated to Arg(5) or Arg(8) at the 5-position of the picoline ring increase Os uptake into A2780 human ovarian cancer cells by ca. 2× and 10×, respectively, whereas a single Arg had no effect. Furthermore, a 15-fold increase in binding of Os to DNA, a potential target for these complexes, was observed for Arg(8) compared to the Arg(1) conjugate. The Arg(5) and Arg(8) conjugates exhibited fast kinetics of binding to calf thymus DNA and an ability to precipitate DNA at very low concentrations. In serum-free medium, the Arg(8) complex was cytotoxic (IC(50) 33 μM) and appears to be a rare example of a bioactive organometallic peptide conjugate. Experiments on CHO cells deficient in DNA repair suggested that unrepaired DNA damage contributes to the cytotoxicity of the Arg(5) and Arg(8) conjugates. These studies demonstrate the potential for use of cell- and nucleus-penetrating peptides in targeting organometallic arene anticancer complexes.     

Targeting DNA mismatches with rhodium intercalators functionalized with a cell-penetrating peptide

Cell-penetrating peptides are widely used to deliver cargo molecules into cells. Here we describe the synthesis, characterization, DNA binding, and cellular uptake studies of a series of metal-peptide conjugates containing oligoarginine as a cell-penetrating peptide. d-Octaarginine units are appended onto a rhodium intercalator containing the sterically expansive chrysenequinone diimine (chrysi) ligand to form Rh(chrysi)(phen)(bpy)(3+)-tethered oligoarginine conjugates, where the peptide is attached to the ancillary bpy ligand; some conjugates also include a fluorescein or thiazole orange tag. These complexes bind and with photoactivation selectively cleave DNA neighboring single-base mismatches. The presence of the oligoarginines is found to increase the nonspecific binding affinity of the complexes for both matched and mismatched DNA, but for these conjugates, photocleavage remains selective for the mismatched site, as assayed using both gel electrophoresis and mass spectrometry experiments. Significantly, the rhodium complex does not interfere with the delivery properties of the cell-penetrating peptide. Confocal microscopy experiments show rapid nuclear localization of the metal-peptide conjugates containing the tethered fluorescein. Mass spectrometry experiments confirm the association of the rhodium with the HeLa cells. These results provide a strategy for targeting mismatch-selective metal complexes inside cell nuclei.     

Formation, structure, and dissociation of the ribonuclease S three-dimensional domain-swapped dimer

Post-translational events, such as proteolysis, are believed to play essential roles in amyloid formation in vivo. Ribonuclease A forms oligomers by the three-dimensional domain-swapping mechanism. Here, we demonstrate the ability of ribonuclease S, a proteolytically cleaved form of ribonuclease A, to oligomerize efficiently. This unexpected capacity has been investigated to study the effect of proteolysis on oligomerization and amyloid formation. The yield of the RNase S dimer was found to be significantly higher than that of RNase A dimers, which suggests that proteolysis can activate oligomerization via the three-dimensional domain-swapping mechanism. Characterization by chromatography, enzymatic assays, and NMR spectroscopy indicate that the structure of the RNase S dimer is similar to that of the RNase A C-dimer. The RNase S dimer dissociates much more readily than the RNase A C-dimer does. By measuring the dissociation rate as a function of temperature, the activation enthalpy and entropy for RNase S dimer dissociation were found to resemble those for the release of the small fragment (S-peptide) from monomeric RNase S. Excess S-peptide strongly slows RNase S dimer dissociation. These results strongly suggest that S-peptide release is the rate-limiting step of RNase S dimer dissociation.     

Polyarginine as a multifunctional fusion tag

Fusion to cationic peptides, such as nonaarginine (R(9)), provides a means to deliver molecular cargo into mammalian cells. Here, we provide a thorough analysis of the effect of an R(9) tag on the attributes of a model protein: bovine pancreatic ribonuclease (RNase A). The R(9) tag diminishes the conformational stability of RNase A (DeltaT(m)=-8 degrees C in phosphate-buffered saline). This effect is nearly mitigated by the addition of salt. The tag does not compromise the enzymatic activity of RNase A. An R(9) tag facilitates the purification of RNase A by cation-exchange chromatography and enables the adsorption of RNase A on glass slides and silica resin with the retention of enzymatic activity. The tag can be removed precisely and completely by treatment with carboxypeptidase B. Finally, the R(9) tag increases both the cellular uptake of RNase A and the cytotoxicity of G88R RNase A, a variant that evades the cytosolic ribonuclease inhibitor protein. Thus, we conclude that polyarginine is a versatile protein fusion tag.     

Conformationally constrained peptides for drug delivery

Peptides have shown great potential in acting as template for developing versatile carrier platforms in nanomedicine, aimed at selective delivery of drugs to only pathological tissues saving its normal neighbors. Cell‐penetrating peptides (CPPs) are short oligomeric peptides capable of translocating across the cell membrane while simultaneously employing multiple mechanisms of entry. Most CPPs exist as disordered structures in solution and may adopt a helical conformation on interaction with cell membrane, vital to their penetrative capability. Herein, we report a series of cationic helical amphipathic peptides (CHAPs), which are topologically constrained to be helical. The peptides were tested against cervical and breast cancer cells for their cell penetration and drug delivery potential. The cellular uptake of CHAP peptides is independent of temperature and energy availability. The activity of the peptides is biocompatible in bovine serum. CHAPs delivered functional methotrexate (MTX) inside the cell as CHAP‐MTX conjugates. CHAP‐MTX conjugates were more toxic to cancer cells than MTX alone. However, the CHAP‐MTX conjugates were less toxic to HEK‐293 cells compared with the cancer cells suggesting higher affinity towards cancer cells.     

A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides

Most bioactive macromolecules, such as protein, DNA and RNA, basically cannot permeate into cells freely from outside the plasma membrane. Cell-penetrating peptides (CPPs) are a group of short peptides that possess the ability to traverse the cell membrane and have been considered as candidates for mediating gene and drug delivery into living cells. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) are able to form stable complexes with plasmid DNA and deliver DNA into insect Sf9 cells in a noncovalent manner. The transferred plasmid DNA containing enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) coding regions could be expressed in cells functionally assayed at both the protein and RNA levels. Furthermore, treatment of cells with CPPs and CPP/DNA complexes resulted in a viability of 84-93% indicating these CPPs are not cytotoxic. These results suggest that arginine-rich CPPs appear to be a promising tool for insect transgenesis.     

Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains

Gold nanoparticles modified with nuclear localization peptides were synthesized and evaluated for their subcellular distribution in HeLa human cervical epithelium cells, 3T3/NIH murine fibroblastoma cells, and HepG2 human hepatocarcinoma cells. Video-enhanced color differential interference contrast microscopy and transmission electron microscopy indicated that transport of nanoparticles into the cytoplasm and nucleus depends on peptide sequence and cell line. Recently, the ability of certain peptides, called protein transduction domains (PTDs), to transclocate cell and nuclear membranes in a receptor- and temperature-independent manner has been questioned (see for example, Lundberg, M.; Wikstrom, S.; Johansson, M. (2003) Mol. Ther. 8, 143-150). We have evaluated the cellular trajectory of gold nanoparticles carrying the PTD from HIV Tat protein. Our observations were that (1) the conjugates did not enter the nucleus of 3T3/NIH or HepG2 cells, and (2) cellular uptake of Tat PTD peptide-gold nanoparticle conjugates was temperature dependent, suggesting an endosomal pathway of uptake. Gold nanoparticles modified with the adenovirus nuclear localization signal and the integrin binding domain also entered cells via an energy-dependent mechanism, but in contrast to the Tat PTD, these signals triggered nuclear uptake of nanoparticles in HeLa and HepG2 cell lines.     

Dendritic oligoguanidines as intracellular translocators

A series of polyguanidylated dendritic structures that can be used as molecular translocators have been designed and synthesized based on nonpeptide units. The dendritic oligoguanidines conjugated with fluorescein or with a green fluorescent protein (GFP) mutant as cargos were isolated and characterized. Quantification and time-course analyses of the cellular uptake of the conjugates using HeLa S3 and human cervical carcinoma cells reveal that the polyguanidylated dendrimers have comparable translocation efficiency to the Tat(49-57) peptide. Furthermore, the deconvolution microscopy image analysis shows that they are located inside the cells. These results clearly show that nonlinear, branched dendritic oligoguanidines are capable of translocation through the cell membrane. This work also demonstrates the potential of these nonpeptidic dendritic oligoguanidines as carriers for intracellular delivery of small molecule drugs, bioactive peptides, and proteins.     

Mechanism of ribonuclease A endocytosis: analogies to cell-penetrating peptides

Pancreatic-type ribonucleases can exert toxic activity by catalyzing the degradation of cellular RNA. Their ability to enter cells is essential for their cytotoxicity. Here, we determine the mechanism by which bovine pancreatic ribonuclease (RNase A) enters human cells. Inhibiting clathrin-dependent endocytosis with dynasore or chlorpromazine decreases RNase A-uptake by ~70%. Limited colocalization between RNase A and transferrin indicates that RNase A is not routed through recycling endosomes. Instead, vesicular staining of RNase A overlaps substantially with that of nona-arginine and the cationic peptide corresponding to residues 47-57 of the HIV-1 TAT protein. At low concentrations (<5 μM), internalization of RNase A and these cell-penetrating peptides (CPPs) is inhibited by chlorpromazine as well as the macropinocytosis inhibitors cytochalasin D and 5-(N-ethyl-N-isopropyl)amiloride to a similar extent, indicative of common endocytic mechanism. At high concentrations, CPPs adopt a nonendocytic mechanism of cellular entry that is not shared by RNase A. Collectively, these data suggest that RNase A is internalized via a multipathway mechanism that involves both clathrin-coated vesicles and macropinosomes. The parallel between the uptake of RNase A and CPPs validates reference to RNase A as a "cell-penetrating protein".     

Zinc(II)cyclen-peptide conjugates interacting with the weak effector binding state of Ras

Zinc(II)cyclen-peptide hybrid compounds and bis-zinc(II)cyclen complexes are prepared as potential binders of the guanine nucleotide binding protein Ras, an important molecular switch in cellular signal transduction. The design of the compounds is based on the previous observation that zinc(II)cyclen complexes could serve as lead compounds for inhibitors of Ras-effector interaction and thus be able to interrupt Ras induced signal transduction. Zinc(II)cyclen selectively stabilizes conformational state 1 of active Ras, a conformational state with drastically decreased affinity to effector proteins like Raf-kinase. To achieve higher binding affinities of such Ras-Raf interaction inhibitors, zinc(II)cyclen conjugates with short peptides, derived from the sequence of the Ras-activator SOS, were prepared by solid phase synthesis protocols. Dinuclear bis-zinc(II)cyclen complexes were obtained from alkyne-azide cycloaddition reactions. NMR investigations of the prepared compounds revealed that the peptide conjugates do not lead to an increase in Ras binding affinity of the metal complex-peptide conjugates. The dinuclear zinc complexes lead to an immediate precipitation of the protein prohibiting spectroscopic investigations of their binding.