Prothymosin-alpha: Features of the protein sequence that contribute to the anti-HIV activity

Prothymosin-alpha (ProTalpha) is a protein mainly located in the nucleus of the cells. Though its exact function is not known, it is believed to be involved in cell proliferation. The ability of this protein to inhibit HIV replication in macrophages cells was discovered recently. Apart from the full length protein, a peptide derived from central region of it (from residues 50-89) is shown to inhibit HIV in a dose dependent manner.;The central region of the protein (residues 50-89) has a combined total of 26 glutamic acid (Glu) and aspartic acid (Asp) residues which imparts high negative charge and acidity to the peptide. ProTalpha 50-89 peptide was synthesized to reconfirm its anti-HIV activity. Along with it three other peptide sequences (Polyglutamic acid, ProTalpha(50-105) and AlexFluor488-ProTalpha(51-89)) were synthesized in order to understand the features that give the protein its anti-HIV activity. They were synthesized to understand if high negative charge and entry of the peptide into the nucleus are required for protein's anti-HIV activity. All the sequences were synthesized using Solid Phase Peptide Synthesis coupled with a 9-Fluorenylmethoxycarbonyl (Fmoc) protecting strategy. Synthesis of ProTa(50-89) was carried out on Microwave synthesizer whereas ProTalpha(50-105) and the Polyglutamic acid sequence were synthesized using an automated synthesizer. During the synthesis of ProTalpha(50-105) with a new protecting group strategy, we encountered unexpected formation of a glutarimide between residues 68 and 69 which was further resolved. Purification of these sequences was performed using Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) or Strong Anion Exchange Chromatography (SAX) and characterized with Electrospray Ionization-Mass Spectrometry (ESI-MS). Samples obtained from SAX contained salt and were desalted by dialysis for 6 hours.;Activity testing was done on two of the sequences. Results from these experiments reconfirmed the anti-HIV activity of ProTalpha(50-89)N50W and also suggest that there is some sequence specificity to the peptide's ability to inhibit HIV replication in the post-integration step. Results demonstrating the importance of the NLS will also be discussed.