Kunitz-type protease inhibitors group B from Solanum palustre

Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.     

Synthesis,Antioxidant Activity,and Structure–Activity Relationship of SCAP1 Analogues

Nine analogues of antioxidant peptide SCAP1 were successfully synthesised using a solid-phase method on a 2-chlorotrytil resin. The compounds were obtained in a range of yields of 7.0–57.8%. The occurrence of aggregation during the synthesis is suspected to be responsible for the poor yields. All peptides were characterized by high-resolution time-of-flight mass spectrometry (HR-TOFMS) and nuclear magnetic resonance (NMR). The antioxidant activities of the SCAP1 analogues as well as SCAP1 were analysed utilising the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay. The results revealed that all of the analysed peptides exhibited moderate antioxidant properties. Moreover, the evaluation of the structure–activity relationship showed that the Asn residue is an important requirement for the antioxidant activity of SCAP1. The replacement of Asn with other amino acid residues (Thr, Pro, Tyr, Trp and Phe) resulted in a decrease in the IC₅₀ values of the peptides. Notably, however, the replacement of the Lys residue with Val marginally increased the activity.

     

Comprehensive statistical analysis of residues interaction specificity at protein-protein interfaces

We calculated interchain contacts on the atomic level for nonredundant set of 4602 protein-protein interfaces using an unbiased Voronoi-Delaune tessellation method, and made 20x20 residue contact matrixes both for homodimers and heterocomplexes. The area of contacts and the distance distribution for these contacts were calculated on both the residue and the atomic levels. We analyzed residue area distribution and showed the existence of two types of interresidue contacts: stochastic and specific. We also derived formulas describing the distribution of contact area for stochastic and specific interactions in parametric form. Maximum pairing preference index was found for Cys-Cys contacts and for oppositely charged interactions. A significant difference in residue contacts was observed between homodimers and heterocomplexes. Interfaces in homodimers were enriched with contacts between residues of the same type due to the effects of structure symmetry.     

Spacer structure and hydrophobicity influences transfection activity of novel polycationic gemini amphiphiles

Three novel polycationic gemini amphiphiles with different spacers were developed and evaluated in terms of their physiochemical properties and transfection efficiencies. Cationic liposomes formed by these amphiphiles and the helper lipid DOPE were able to successfully condense DNA, as shown by gel mobility shift and ethidium bromide intercalation assays. Transfection activity of the liposomes was superior to Lipofectamine^® 2000 and was dependent on spacer structure, hydrophobicity, and nucleic acid type (pDNA or siRNA). We demonstrated that the cationic liposomes 2X6/DOPE and 2X7/DOPE are potential non-toxic vehicles for gene delivery.     

Novel Fluorescent Pyrimidine Nucleosides Containing 2,1,3-Benzoxadiazole and Naphtho-[1,2,3-CD] Indole-6 (2H)-One Fragments

A series of novel fluorescent pyrimidine nucleosides containing 2,1,3-benzoxadiazole or naphtho[1,2,3-cd]indole-6 (2h)-one fragments was designed and synthesized. Introduction of fluorescent fragments into the position 5 of the uridine or cytidine heterocycle was carried out in two ways: by Sonogashira Coupling Reaction and CuI-catalyzed cycloaddition (“click” reaction). The obtained nucleoside derivatives became fluorescent due to the inserted fragments. The excitation wavelength (440–450 nm) was outside the absorption band of many biomolecules and significantly differed from the emission wavelength (560–600 nm). In addition, the intended nucleoside analogs were shown to kill cultured human tumor cells at submicromolar concentrations.     

Fructose-1,6-bisphosphatase degradation in the methylotrophic yeast Komagataella phaffii occurs in autophagy pathway

Many enzymes of methanol metabolism of methylotrophic yeasts are located in peroxisomes whereas some of them have the cytosolic localization. After shift of methanol-grown cells of methylotrophic yeasts to glucose medium, a decrease in the activity of cytosolic (formaldehyde dehydrogenase, formate dehydrogenase, and fructose-1,6-bisphosphatase [FBP]) along with peroxisomal enzymes of methanol metabolism is observed. Mechanisms of inactivation of cytosolic enzymes remain unknown. To study the mechanism of FBP inactivation, the changes in its specific activity of the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of Komagataella phaffii with or without the addition of the MG132 (proteasome degradation inhibitor) were investigated after shift of methanol-grown cells in glucose medium. Western blot analysis showed that inactivation of FBP in GS200 occurred due to protein degradation whereas inactivation in the strains SMD1163 and gss1Δ was negligible in such conditions. The effect of the proteasome inhibitor MG132 on FBP inactivation was insignificant. To confirm FBP degradation pathway, the recombinant strains with GFP-labeled Fbp1 of K. phaffii and red fluorescent protein-labeled peroxisomes were constructed on the background of GS200 and SMD1163. The fluorescent microscopy analysis of the constructed strains was performed using the vacuolar membrane dye FM4-64. Microscopic data confirmed that Fbp1 degrades by autophagy pathway in K. phaffii. K. phaffii transformants, which express heterologous β-galactosidase under FLD promoter, have been constructed.