Piotr Mucha

Separation of siderophores by capillary electrophoresis

Abstract Capillary electrophoresis (CE) was applied as a fast method of siderophore separation. Siderophores are iron binding and regulating cell products, which facilitate iron transport into cells. A fast and efficient method of siderophore analysis is important for better understanding of the iron pathways in a sea environment or marine organisms. The best results of CE analysis were obtained using free zone CE in 25 m M phosphate buffer at basic pH using a constant voltage of 20 kV. Under these conditions it was possible to detect the presence of siderophores in seawater.

Functional Recognition of the Modified Human tRNALys3UUU Anticodon Domain by HIV’s Nucleocapsid Protein and a Peptide Mimic

The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNA(Lys3)(UUU) as the primer for reverse transcription. NCp7 also remodels the htRNAs amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNAs unique composition of post-transcriptional modifications on NCp7 recognition of htRNA(Lys3)(UUU), the proteins binding and functional remodeling of the human anticodon stem and loop domain (hASL(Lys3)) were studied. NCp7 bound the hASL(Lys3)(UUU) modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm(5)s(2)U(34)) and 2-methylthio-N(6)-threonylcarbamoyladenosine at position-37 (ms(2)t(6)A(37)) with a considerably higher affinity than the unmodified hASL(Lys3)(UUU) (K(d)=0.28±0.03 and 2.30±0.62 μM, respectively). NCp7 denatured the structure of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) more effectively than that of the unmodified hASL(Lys3)(UUU). Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K(d)=0.55±0.10 μM) and specificity for the ASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37) comparable to that of NCp7. The peptides recognized a t(6)A(37)-modified ASL with an affinity (K(d)=0.60±0.09 μM) comparable to that for hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37), indicating a preference for the t(6)A(37) modification. Significantly, one of the peptides was capable of relaxing the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNA(Lys3)(UUU) have been found to be important determinants of NCp7s recognition prior to the tRNA(Lys3)(UUU) being annealed to the viral genome as the primer of reverse transcription.

Experimental Models of Protein-RNA Interaction: Isolation and Analyses of tRNAPhe and U1 snRNA-Binding Peptides from Bacteriophage Display Libraries

Peptides that bind either U1 small nuclear RNA (U1 snRNA) or the anticodon stem and loop of yeast tRNAPhe (tRNAACPhe) were selected from a random-sequence, 15-amino acid bacteriophage display library. An experimental system, including an affinity selection method, was designed to identify primary RNA-binding peptide sequences without bias to known amino acid sequences and without incorporating nonspecific binding of the anionic RNA backbone. Nitrocellulose binding assays were used to evaluate the binding of RNA by peptide-displaying bacteriophage. Amino acid sequences of RNA-binding bacteriophage were determined from the foreign insert DNA sequences, and peptides corresponding to the RNA-binding bacteriophage inserts were chemically synthesized. Peptide affinities for the RNAs (Kd ≍ 0.1–5.0 μM) were analyzed successfully using fluorescence and circular dichroism spectroscopies. These methodologies demonstrate the feasibility of rapidly identifying, isolating, and initiating the analyses of small peptides that bind to RNAs in an effort to define better the chemistry, structure, and function of protein–RNA complexes.

Structural requirements for conserved Arg52 residue for interaction of the human immunodeficiency virus type 1 trans-activation responsive element with trans-activator of transcription protein (49–57): Capillary electrophoresis mobility shift assay

A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the interaction between the trans-activation response element (TAR) and the trans-activator of transcription protein (Tat) has been presented. The human immunodeficiency virus type 1 (HIV-1) Tat promotes elongation of viral mRNAs binding to the TAR. It has been suggested that a single, conserved arginine residue (presumably Arg52) within the arginine-rich region (ARR) of Tat plays the major role for the Tat-TAR recognition. To study structural requirements of the Arg52 position, Tat(49-57)-NH2 analogues substituted with nonencoded amino acids at the Arg52 position have been synthesized and their interaction with TAR has been studied by CEMSA. Using a linear polyacrylamide-coated capillary and a sieving polymer containing separation buffer, well separated and shaped peaks of free and bound TAR RNA were obtained. In the presence of Tat1 peptide bearing the native sequence of Tat(49-57) a significant shift of migration time of TAR from 18.66 min (RSD=1.4%) to 20.12 min (RSD=2.4%) was observed. We have found that almost every substitution within the guanidino group of the Arg52 [L-Arg52-->Cit, -->Orn, -->Arg(NO2), -->Arg(Me2)] strongly disrupted or abolished the TAR-Tat peptide interaction. Enantiomeric substitution, L-Arg52-->D-Arg was the only one which notably promoted TAR-Tat peptide interaction. The results demonstrate that the specific net of hydrogen bonds created by the guanidinio group of conserved Arg52 plays a crucial role for TAR-Tat HIV-1 recognition. The newly developed procedure describes for the first time use of CE to monitor RNA-peptide complex formation. The methodology presented should be generally applicable to study RNA-peptide (protein) interaction.

Cell-penetrating peptides as a promising tool for delivery of various molecules into the cells

Many biologically active compounds, including macromolecules that are used as various kinds of drugs, must be delivered to the interior of cell or organelles such as mitochondria or nuclei to achieve a therapeutic effect. However, very often, lipophilic cell membrane is impermeable for these molecules. A new method in the transport of macromolecules through the cell membrane is the one based on utilizing cell-penetrating peptides (CPPs). Invented 25 years ago, CPPs are currently the subject of intensive research in many laboratories all over the world. CPPs are short compounds comprising up to 30 amino acid residues, which penetrate the cell membrane but do not cause cell damage. Additionally, CPPs can transfer hydrophilic molecules (peptides, proteins, nucleic acids) which exceed their mass, and for which the cell membrane is generally impermeable. In this review, we concentrate on the cellular uptake mechanism of CPPs and a method of conjunction of CPPs to the transported molecules. We also highlight the potential of CPPs in delivering various kinds of macromolecules into cells, including compounds of therapeutic interest.

Monitoring of the purification of systemin by capillary electrophoresis

Capillary electrophoresis (CE) was applied as a method for monitoring of the separation process of systemin, an endogenous peptide from tomato plants. Systemin, an 18-amino-acid polypeptide, was isolated from tomato leaves and purified. A systemin peak was identified using synthetic systemin as a marker, and traced by CE and reversed-phase high-performance liquid chromatography (RP-HPLC) during all chromatographic steps. The best results of CE analysis were obtained in 25 mM phosphate buffer at acidic pH using a constant operating voltage of 30 kV.

A peptide nucleic acid (PNA)-mediated polymerase chain reaction clamping allows the selective inhibition of the ERVWE1 gene amplification.

A new peptide nucleic acid (PNA) mediated QPCR technique for the detection and quantification of the Multiple Sclerosis-Associated Retrovirus (MSRV) belonging to the human endogenous retrovirus-W (HERV-W) family has been developed. The assay utilizes a PNA probe which is fully complementary to the ERVWE1 sequence, another member of the HERV-W family which is closely related to MSRV. Due to its excellent affinity to a completely matched template, PNA probe selectively blocks the amplification of ERVWE1 thus allowing the specific and exclusive detection and quantification of the MSRV as PNA does not interfere with the amplification of MSRV. Using this newly developed method we found that MSRV is predominantly expressed over ERWVE1 in astrocyte-derived U-87 MG cell line but not in human umbilical vein endothelial cells or human placental cells.

‘Click’ chemistry synthesis and capillary electrophoresis study of 1,4-linked 1,2,3-triazole AZT-systemin conjugate

The Cu(I) catalyzed Huisgen 1,3‐dipolar azide‐alkyne cycloaddition (CuAAC) was applied for a nucleoside‐peptide bioconjugation. Systemin (Sys), an 18‐aa plant signaling peptide naturally produced in response to wounding or pathogen attack, was chemically synthesized as its N‐propynoic acid functionalized analog (Prp‐Sys) using the SPPS. Next, CuAAC was applied to conjugate Prp‐Sys with 3′‐azido‐2′,3′‐dideoxythymidine (AZT), a model cargo molecule. 1,4‐Linked 1,2,3‐triazole AZT‐Sys conjugate was designed to characterize the spreading properties and ability to translocate of cargo molecules of systemin. CuAAC allowed the synthesis of the conjugate in a chemoselective and regioselective manner, with high purity and yield. The presence of Cu(I) ions generated in situ drove the CuAAC reaction to completion within a few minutes without any by‐products. Under typical separation conditions of phosphate ‘buffer’ at low pH and uncoated fused bare‐silica capillary, an increasing peak intensity assigned to triazole‐linked AZT‐Sys conjugate was observed using capillary electrophoresis (CE) during CuAAC. CE analysis showed that systemin peptides are stable in tomato leaf extract for up to a few hours. CE‐ESI‐MS revealed that the native Sys and its conjugate with AZT are translocated through the tomato stem and can be directly detected in stem exudates. The results show potential application of systemin as a transporter of low molecular weight cargo molecules in tomato plant and of CE method to characterize a behavior of plant peptides and its analogs. Copyright

Interaction of Native RNAs with Tat Peptides

We present experimental data on the interaction of arginine-rich Tat protein of human immunodeficiency virus (HIV-Tat) and its short fragments with three different RNAs: HIV-TAR RNA, yeast tRNAPhe and lupin ribosomal 5S rRNA. The aim of the studies was to learn about the basis of the specificity of RNA-protein interaction. All of these RNA molecules contain the same structural motif, the single stranded nucleotide sequence UGGG and form complexes with the Tat peptide of the amino acid sequence GRKKRRQRRRA and its derivatives, where R is substituted by D-arginine, citrulline or ornithine. The structure of the RNA-Tat peptide complexes were probed with specific RNases and Pb2+-induced specific cleavage of the RNA phosphodiester bond. The nucleotide sequence UGGG located in the dihydrouridine loop of tRNAPhe and in the loop D of plant 5S rRNA is involved in binding of Tat-peptide and in the complex it is resistant to RNases and a hydrolysis by Pb2+ ion. It seems that the specificity of the peptide - RNA complex formation depends on direct recognition of guanine moieties of tRNA with arginine residues of Tat peptide. The nature of the interactions are similar to those observed in many DNA - protein complexes, but are different from those previously observed for TAR RNA-Tat complexes. We also described a method for preparation of TAR RNA which uses hammerhead ribozymes, to cut off the required RNA from the in vitro synthesised transcript.

Systemin — An inducer of proteinase inhibitor synthesis can be responsible for biological activity of a lupin extract against insects

Summary Lupin seed extract has been shown to possess a variety of biological properties involving pesticide activity. Using high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC) analyses, we found that systemin, a polypeptide defence signal in plants, is one of the components of the lupin extract. This peptide may be responsible for some of its activities.