Effects of Lipidation on a Proline-Rich Antibacterial Peptide

The emergence of multidrug-resistant bacteria is a worldwide health problem. Antimicrobial peptides have been recognized as potential alternatives to conventional antibiotics, but still require optimization. The proline-rich antimicrobial peptide Bac7(1-16) is active against only a limited number of Gram-negative bacteria. It kills bacteria by inhibiting protein synthesis after its internalization, which is mainly supported by the bacterial transporter SbmA. In this study, we tested two different lipidated forms of Bac7(1-16) with the aim of extending its activity against those bacterial species that lack SbmA. We linked a C12-alkyl chain or an ultrashort cationic lipopeptide Lp-I to the C-terminus of Bac7(1-16). Both the lipidated Bac-C12 and Bac-Lp-I forms acquired activity at low micromolar MIC values against several Gram-positive and Gram-negative bacteria. Moreover, unlike Bac7(1-16), Bac-C12, and Bac-Lp-I did not select resistant mutants in E. coli after 14 times of exposure to sub-MIC concentrations of the respective peptide. We demonstrated that the extended spectrum of activity and absence of de novo resistance are likely related to the acquired capability of the peptides to permeabilize cell membranes. These results indicate that C-terminal lipidation of a short proline-rich peptide profoundly alters its function and mode of action and provides useful insights into the design of novel broad-spectrum antibacterial agents.

НЕЙРОДЕГЕНЕРАТИВНЫЕ ИЗМЕНЕНИЯ В ВЕСТИБУЛЯРНЫХ НЕЙРОНАХ ПОСЛЕ ОДНОСТОРОННЕЙ ЛАБИРИНТЭКТОМИИ (UL) И ИХ ЗАЩИТА ГИПОТАЛАМИЧЕСКИМ ПРОЛИН БОГАТЫМ ПЕПТИДОМ (PRP) И ЗМЕИНЫМ ЯДОМ NAJA NAJA OXIANA (NOX) / NEURODEGENERATIVE CHANGES IN VESTIBULAR NEURONS AFTER UNILATERAL LABYRINTHECTOMY (UL) AND THEIR PROTECTION WITH HYPOTHALAMIC PROLINE RICH PEPTIDE (PRP) AND SNAKE VENOM NAJA NAJA OXIANA (NOX)

Мы протестировали реакции нейронов ядра Дейтерса на двустороннюю высокочастотную стимуляцию паравентрикулярных и супраоптических ядер гипоталамуса (PVN & SON) в норме и после односторонней лабиринтэктомии (UL). Анализ спайковой активности проводился с помощью on-line выборки и специальной программы. Комплексные усредненные гистограммы времени и частоты перисобытий показывают усиление тормозных и возбуждающих реакций нейронов Дейтерса на ранней стадии вестибулярной компенсации после инъекции богатого пролином пептида (PRP-1) и яда кобры Naja Naja Oxiana (NOX), достигая норме по окончании испытаний. При гистохимическом исследовании обнаружено изменение активности Са2+-зависимой кислой фосфатазы (КФ) в нейронах. Было показано, что у UL животных полное исчезновение или задержка обесцвечивания нейронов Дейтерса приводит к нейродегенеративному паттерну в виде клеточного «оттенка». Активность КФ после UL и инъекции PRP-1 вызывает более эффективное восстановление нейронов по сравнению с событиями, наблюдаемыми после введения NOX. Наблюдения за поведением в «открытом поле» показывают, что PRP-1 и NOX являются протекторами, которые могут успешно восстанавливать нарушенные вестибулярные функции. / We tested the reactions of Deiters’ nucleus neurons to bilateral high frequency stimulation of hypothalamic paraventricualar and supraoptic nuclei (PVN & SON) in norm and following unilateral labyrinthectomy (UL). The analysis of spike activity was carried out by mean of on-line selection and special program. The complex averaged perievent time and frequency histograms shows the increase of inhibitory and excitatory reactions of Deiters’ neurons at early stage of vestibular compensation following proline-rich peptide (PRP-1) and cobra venom Naja Naja Oxiana (NOX) injection, reaching the norm at the end of tests. In histochemical study the changes in Ca2+-dependent acidic phosphatase (AP) activity in neurons was discovered. It was shown that in UL animals the total disappearance or delay of decolorizing of Deiters’ neurons lead to neurodegenerative pattern as cellular “shade”. AP activity after UL and PRP-1 injection exerts more effective recovery of neurons in comparison with events, observed after the administration of NOX. The behavioral observations in “open fieald” indicate that PRP-1 and NOX are protectors, which may successfully recover the disturbed vestibular functions.

Polyproline-Rich Peptides Organize Four Cholinesterase Subunits into a Tetramer; BChE and AChE Scavenge Polyproline Peptides Released during Metabolic Turnover

The genes for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) encode the proteins responsible for enzyme activity. Additional gene products, PRiMA and ColQ, anchor AChE and BChE proteins into membranes. Soluble AChE and BChE tetramers are composed of four identical subunits plus one polyproline-rich peptide. Dilution does not release the polyproline-rich peptide from tetramers. However, protein denaturation, for example, heating in a boiling water bath, dissociates the polyproline-rich peptide. Using mass spectrometry to sequence peptides released from soluble AChE and BChE tetramers, we find sequences that correspond to proline-rich regions from a variety of proteins. A typical peptide sequence contains 20 consecutive prolines in a 23-residue peptide, LPPPPPPPPPPPPPPPPPPPPLP. There is no single, common consensus sequence, i.e., no specific gene appears to be responsible for the polyproline-rich peptides found in soluble AChE and BChE tetramers. We propose that during metabolic turnover, protein fragments containing polyproline-rich sequences are scavenged by AChE and BChE dimers, to make stable AChE and BChE tetramers. The 40-residue, alpha-helical C-terminus of AChE or BChE is the tetramerization domain that binds the polyproline-rich peptide. Four parallel alpha helices wrap around a single antiparallel polyproline peptide to lock the tetramer in place. This organization was established by classical X-ray crystallography for isolated C-termini in complex with a proline-rich peptide. The organization was confirmed for intact, tetrameric human BChE using cryoelectron microscopy. When 40 amino acids are deleted from the carboxy terminus, monomeric enzymes are created that retain full enzymatic activity.

Salivary proline rich peptide decreases cell growth in HCC38 triple negative breast cancer cell line

Objective: The objective of this study is to determine the effects of p1978 on the growth rate of a triple receptor negative breast cancer cell line.Methods: Three cell lines, 185B5 normal tissue, HCC38, and AU585, were seeded with peptide p1978. Corresponding plates were seeded with PBS to serve as controls. Baseline line cell counts were taken at 30% confluence prior to seeding. Counts were again made 48 hours later.Results: The HCC38 cell line showed decreased growth when exposed to the low and high dose of p1978 peptide.Conclusion: The results suggest that p1978 may have potential in treating triple receptor negative breast cancer.

Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein–protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB–CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.