Pro Residues Research Articles

Analysis of the ability of pramlintide to inhibit amyloid formation by human islet amyloid polypeptide reveals a balance between optimal recognition and reduced amyloidogenicity.

The hormone human islet amyloid polypeptide (hIAPP or amylin) plays a role in glucose metabolism, but forms amyloid in the pancreas in type 2 diabetes (T2D) and is associated with β-cell death and dysfunction in the disease. Inhibitors of islet amyloid have therapeutic potential; however, there are no clinically approved inhibitors, and the mode of action of existing inhibitors is not well understood. Rat IAPP (rIAPP) differs from hIAPP at six positions, does not form amyloid, and is an inhibitor of amyloid formation by hIAPP. Five of the six differences are located within the segment of residues 20-29, and three of them are Pro residues, which are well-known disruptors of β-sheet structure. rIAPP is thus a natural example of a "β-breaker inhibitor", a molecule that combines a recognition element with an entity that inhibits β-sheet formation. Pramlintide (PM) is a peptide drug approved for use as an adjunct to insulin therapy for treatment of diabetes. PM was developed by introducing the three Pro substitutions found in rIAPP into hIAPP. Thus, it more closely resembles the human peptide than does rIAPP. Here we examine and compare the ability of rIAPP, PM, and a set of designed analogues of hIAPP to inhibit amyloid formation by hIAPP, to elucidate the factors that lead to effective peptide-based inhibitors. Our results reveal, for this class of molecules, a balance between the reduced amyloidogenicity of the inhibitory sequence on one hand and its ability to recognize hIAPP on the other.

A Double Heterozygous Mutation of TNNI3 Causes Hypertrophic Cardiomyopathy in a Han Chinese Family

Objectives: To investigate the variations in the TNNI3 gene in a Chinese Han family affected by hypertrophic cardiomyopathy (HCM) and the potential molecular mechanism linking these mutations with disease. Methods: Peripheral venous blood was acquired from family members, and TNNI3 mutations were identified by DNA sequencing. The pathophysiology of TNNI3 mutations was investigated using bioinformatics, subcellular localization determination and Western blotting. Results: Sanger sequencing revealed that the proband possessed 2 heterozygous mutations, c.235C>T and c.470C>T, located at exons 4 and 6 of the TNNI3 gene. The proband (II-2) and her brother (II-1), who had been previously diagnosed with HCM, harbored both mutations whereas their healthy parents harbored only 1. Alignment of the TNNI3 amino acid sequence indicated that the two Pro residues were highly conserved across species. Subcellular localization showed that both wild-type (WT) and mutant TNNI3 proteins were localized at the cell nucleus. Western blot analysis of expression in human embryonic kidney 293T cells showed that the intracellular levels of the mutant proteins were significantly decreased compared to WT TNNI3 (p < 0.01). Conclusions: Our findings showed that a double heterozygous mutation in the TNNI3 gene is involved in the pathogenesis of HCM via haploinsufficiency. These results will inspire further studies to investigating the link between the TNNI3 gene and HCM.

Low energy CID and action IRMPD provide insights into a minor subpopulation of the gas-phase conformers of triply charged bradykinin

Unique fragmentation of the IMS-resolved conformers of triply charged bradykinin is shown to occur at very low collision energies by quadrupole CID. For the major C conformation of bradykinin, the unique fragmentation appears to result from a subpopulation that has a different average CCS than the dominant C population that has been previously identified as TTC (trans, trans, cis at the three Pro residues). The three major gas-phase isomers exhibit minimal differences in fragmentation at collision energies greater than 12eV, presumably because the amide bonds of the proline residues isomerize prior to dissociation at these higher energies. The differences in fragmentation observed in the low collision energy regime (<12eV) are attributed to the conformational differences of the fragmenting subpopulations of the mobility resolved populations associated with the cis or trans isomerization state of the three proline residues. Action IRMPD of the b2 ion supports a cis-Pro2 isomerization state for the subpopulation of conformation C fragmenting at low energies, assuming no isomerization was induced by the low energy CID. Substitution of N-methyl alanine, an acyclic proline mimic, independently, for each of the three prolines of bradykinin and low energy fragmentation of the minor subpopulation of conformation C allowed for tentative assignments of the isomerization state of two of the three proline resides of this subpopulation. Density functional theory calculations support the conclusion made from NMA-substitutions and together suggest that the subpopulation of conformation C is cis at Pro2 and trans at Pro7. It is not clear whether the minor subpopulation is a typical feature of triply charged bradykinin or whether unintended activation in the instrument used here produces this population. The results illustrate the utility of ion mobility coupled with low-energy CID for the determination of a minor subpopulation that was not visible by MS or IM alone.

A newly developed molecularly imprinted polymer on the surface of TiO2 for selective extraction of triazine herbicides residues in maize, water, and soil.

A new surface molecularly imprinted polymer (MIP) based on nano-TiO2 was developed using propazine (Pro) as a template molecule, ethyleneglycol dimethacrylate (EGDMA) as a crosslinker, methacrylic acid (MAA) as a functional monomer, and 2,2'-azobis (isobutyronitrile) (AIBN) as an initiator. Structures of the newly synthesized surface MIPs were characterized by Fourier transmission infrared spectrometry (FT-IR), scanning electron microscope (SEM), transmission electron microscope (TEM), and X-ray diffraction (XRD). The MIP had a good adsorption capacity and high recognition selectivity to propazine. Meanwhile, it exhibited a cross-selectivity for simazine (Sim) and atrazine (Atr). The MIPs were used as a solid phase extraction (SPE) material. Concomitant extraction, purification, and determination of three pesticides (Pro, Sim, and Atr) residues in water, soil, and maize plant and grain samples were performed by MIP-SPE coupled with high performance liquid chromatography (HPLC). The highly selective separation and enrichment of Pro, Atr, and Sim from the complex environmental media can be achieved. Thus, the newly developed technique provides an analytical platform to quantify the trace amount of Pro, Sim, and Atr residues in multi environmental media and food source.

Transport of Antihypertensive Peptide RVPSL, Ovotransferrin 328-332, in Human Intestinal Caco-2 Cell Monolayers.

The objective of this study was to investigate the transepithelial transport of RVPSL (Arg-Val-Pro-Ser-Leu), an egg-white-derived peptide with angiotensin I-converting enzyme (ACE) inhibitory and antihypertensive activity, in human intestinal Caco-2 cell monolayers. Results revealed that RVPSL could be passively transported across Caco-2 cell monolayers. However, during the process of transport, 36.31% ± 1.22% of the initial RVPSL added to the apical side was degraded, but this degradation decreased to 23.49% ± 0.68% when the Caco-2 cell monolayers were preincubated with diprotin A (P < 0.001), suggesting that RVPSL had a low resistance to various brush border membrane peptidases. When transport from the apical side to the basolateral side was investigated, the apparent permeability coefficient (Papp) was (6.97 ± 1.11) × 10(-6) cm/s. The transport route of RVPSL appears to be the paracellular pathway via tight junctions, as only cytochalasin D, a disruptor of tight junctions (TJs), significantly increased the transport rate (P < 0.001). In addition, the relationship between the structure of RVPSL and transport across Caco-2 cell monolayers was studied by mutation of RVPSL. It was found that N-terminal Pro residues were more beneficial for transport of pentapeptides across Caco-2 cell monolayers than Arg and Val. Furthermore, RVPSL could be more easily transported as smaller peptides, especially in the form of dipeptides and tripeptides.

Screening and identification of DPP-IV inhibitory peptides from deer skin hydrolysates by an integrated approach of LC–MS/MS and in silico analysis

An integrated approach combining liquid chromatography/tandem mass spectrometry (LC–MS/MS) with in silico analysis was used to screen dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from deer skin hydrolysates. A total of 203, 244 and 60 peptides were identified from deer skin hydrolysates prepared with pepsin, pepsin + trypsin and pepsin + Alcalase, respectively, by LC–MS/MS. The percentages of peptides in the above mentioned hydrolysates containing a Pro residue at the penultimate position were 5.9, 20.9 and 20.0%, respectively. Five peptides with a Pro residue at the penultimate position were synthesized and assessed, and these five synthetic peptides possessed DPP-IV inhibitory activity with IC50 values from 83.3 to 1638.3 µΜ. One of the evaluated peptides contained an oxidized methionine. The effects of peptide modification and length on the DPP-IV inhibitory activity of the peptides were assessed. The results suggest that the integrated approach was efficient in identifying novel bioactive peptides from hydrolysates.

Specifically designed peptide structures effectively suppressed oxidative reactions in chemical and cellular systems

Peptides were designed based on the identified structural motif of the most potent antioxidant peptides derived from barley protein. Six peptides contained the effective pentapeptide QPYPQ with appended Gln and Pro residues and the other peptides possessed higher hydrophobicity. The peptides have the repetitive sequences of Q, P and Y to investigate the contribution of vicinal residues to their antioxidant capacity. Antioxidant activity of the synthetic peptides was evaluated in chemical and cellular models. Free radical scavenging assays demonstrated the positive role of QP and PY pairs in stabilizing the peptide radicals. Cellular models revealed that repetitive peptide sequences effectively inactivate lipid hydroperoxides and intracellular reactive oxygen species. Peptides with alternating residues were also found effective in inhibition of amyloid fibril formation. These results verified that in addition to the chemical structure of individual side chains, the combined effects of the vicinal residues are also important in antioxidant activity of the whole peptide.

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro(314)-Trp(316) turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro(314) of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.

The First Residue of the PWWP Motif Modulates HATH Domain Binding, Stability, and Protein-Protein Interaction.

Hepatoma-derived growth factor (hHDGF) and HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic structural motif, namely the PWWP motif. The HATH domain has attracted attention because of its ability to bind with heparin/heparan sulfate, DNA, and methylated histone peptide. Depending on the sequence of the PWWP motif, HRP HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro) forms. A-type HATH is highly unstable and tends to precipitate in solution. We replaced the Pro residue in P-type HATHHDGF with Ala and evaluated the influence on structure, dynamics, and ligand binding. Nuclear magnetic resonance (NMR) hydrogen/deuterium exchange and circular dichroism (CD) measurements revealed reduced stability. Analysis of NMR backbone (15)N relaxations (R1, R2, and nuclear Overhauser effect) revealed additional backbone dynamics in the interface between the β-barrel and the C-terminal helix bundle. The β1-β2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction through the loop. A-type HATH, therefore, shows a stronger tendency to aggregate when binding with heparin and DNA oligomers. This study defines the role of the first residue of the PWWP motif in modulating HATH domain stability and oligomer formation in binding.

Novel antimicrobial peptide specifically active against Porphyromonas gingivalis.

Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including outer membrane vesicles, lipopolysaccharides, hemolysins and proteinases. Antimicrobial peptides (AMPs) including bacteriocins have been found to inhibit the growth of P. gingivalis; however, these peptides are relatively large molecules. Hence, it is difficult to synthesize them by a scale-up production. Therefore, this study aimed to synthesize a shorter AMP that was still active against P. gingivalis. A peptide that contained three cationic amino acids (Arg, His and Lys), two anionic amino acids (Glu and Asp), hydrophobic amino acids residues (Leu, Ile, Val, Ala and Pro) and hydrophilic residues (Ser and Gly) was obtained and named Pep-7. Its bioactivity and stability were tested after various treatments. The mechanism of action of Pep-7 and its toxicity to human red blood cells were investigated. The Pep-7 inhibited two pathogenic P. gingivalis ATCC 33277 and P. gingivalis ATCC 53978 (wp50) strains at a minimum bactericidal concentration (MBC) of 1.7 µM, but was ineffective against other oral microorganisms (P. intermedia, Tannerella forsythensis, Streptococcus salivarius and Streptococcus sanguinis). From transmission electron microscopy studies, Pep-7 caused pore formation at the poles of the cytoplasmic membranes of P. gingivalis. A concentration of Pep-7 at four times that of its MBC induced some hemolysis but only at 0.3%. The Pep-7 was heat stable under pressure (autoclave at 110 and 121 °C) and possessed activity over a pH range of 6.8-8.5. It was not toxic to periodontal cells over a range of 70.8-4.4 μM and did not induce toxic pro-inflammatory cytokines. The Pep-7 showed selective activity against Porphyromonas sp. by altering the permeability barriers of P. gingivalis. The Pep-7 was not mutagenic in vitro. This work highlighted the potential for the use of this synthetic Pep-7 against P. gingivalis.

⁸⁹Zr-Labeled Versus ¹²⁴I-Labeled αHER2 Fab with Optimized Plasma Half-Life for High-Contrast Tumor Imaging In Vivo.

Immuno-PET imaging of the tumor antigen HER2/neu allows for the noninvasive detection and monitoring of oncogene expression; such detection and monitoring are of prognostic value in patients with breast cancer. Compared with the full-size antibody trastuzumab, smaller protein tracers with more rapid blood clearance permit higher imaging contrast at earlier time points. Antigen-binding fragments (Fabs) of antibodies with moderately prolonged circulation achieved through the genetic fusion with a long, conformationally disordered chain of the natural amino acids Pro, Ala, and Ser (PASylation)-a biologic alternative to chemical conjugation with polyethylene glycol, PEG-offer a promising tracer format with improved pharmacokinetics for in vivo imaging. Recently, the transition metal radionuclide (89)Zr has attracted increasing interest for immuno-PET studies, complementing the conventional halogen radionuclide (124)I. To allow direct comparison of these 2 radioactive labels for the same protein tracer, the recombinant αHER2 Fab fused with 200 Pro, Ala, and Ser (PAS200) residues was either conjugated with (124)I via an iodination reagent or coupled with deferoxamine (Df) and complexed with (89)Zr. After confirmation of the stability of both radioconjugates and quality control in vitro, immuno-PET and biodistribution studies were performed with CD1-Foxn1(nu) mice bearing HER2-positive human tumor xenografts. (89)Zr⋅Df-Fab-PAS200 and (124)I-Fab-PAS200 showed specific tumor uptake of 11 and 2.3 percentage injected dose per gram 24 h after injection, respectively; both led to high tumor-to-blood (3.6 and 4.4, respectively) and tumor-to-muscle (20 and 43, respectively) ratios. With regard to off-target accumulation, overt (124)I activity was seen in the thyroid, as expected, whereas high kidney uptake was evident for (89)Zr; the latter was probably due to glomerular filtration and reabsorption of the protein tracer in proximal tubular cells. Both (89)Zr- and (124)I-labeled versions of αHER2 Fab-PAS200 allowed PET tumor imaging with high contrast. With its residualizing radiometal, the tracer (89)Zr⋅Df-Fab-PAS200 showed better in vivo stability and higher tumor uptake.

Local amino acid sequence patterns dominate the heterogeneous phenotype for the collagen connective tissue disease Osteogenesis Imperfecta resulting from Gly mutations.

Osteogenesis Imperfecta (OI), a hereditary connective tissue disease in collagen that arises from a single Gly → X mutation in the collagen chain, varies widely in phenotype from perinatal lethal to mild. It is unclear why there is such a large variation in the severity of the disease considering the repeating (Gly-X-Y)n sequence and the uniform rod-like structure of collagen. We systematically evaluate the effect of local (Gly-X-Y)n sequence around the mutation site on OI phenotype using integrated bio-statistical approaches, including odds ratio analysis and decision tree modeling. We show that different Gly → X mutations have different local sequence patterns that are correlated with lethal and nonlethal phenotypes providing a mechanism for understanding the sensitivity of local context in defining lethal and non-lethal OI. A number of important trends about which factors are related to OI phenotypes are revealed by the bio-statistical analyses; most striking is the complementary relationship between the placement of Pro residues and small residues and their correlation to OI phenotype. When Pro is present or small flexible residues are absent nearby a mutation site, the OI case tends to be lethal; when Pro is present or small flexible residues are absent further away from the mutation site, the OI case tends to be nonlethal. The analysis also reveals the dominant role of local sequence around mutation sites in the Major Ligand Binding Regions that are primarily responsible for collagen binding to its receptors and shows that non-lethal mutations are highly predicted by local sequence considerations alone whereas lethal mutations are not as easily predicted and may be a result of more complex interactions. Understanding the sequence determinants of OI mutations will enhance genetic counseling and help establish which steps in the collagen hierarchy to target for drug therapy.

Helix 8 and the i3 loop of the muscarinic M3 receptor are crucial sites for its regulation by the Gβ5-RGS7 complex.

The muscarinic M3 receptor (M3R) is a Gq-coupled receptor and is known to interact with many intracellular regulatory proteins. One of these molecules is Gβ5-RGS7, the permanently associated heterodimer of G protein β-subunit Gβ5 and RGS7, a regulator of G protein signaling. Gβ5-RGS7 can attenuate M3R-stimulated release of Ca2+ from intracellular stores or enhance the influx of Ca2+ across the plasma membrane. Here we show that deletion of amino acids 304–345 from the central portion of the i3 loop renders M3R insensitive to regulation by Gβ5-RGS7. In addition to the i3 loop, interaction of M3R with Gβ5-RGS7 requires helix 8. According to circular dichroism spectroscopy, the peptide corresponding to amino acids 548–567 in the C-terminus of M3R assumes an α-helical conformation. Substitution of Thr553 and Leu558 with Pro residues disrupts this α-helix and abolished binding to Gβ5-RGS7. Introduction of the double Pro substitution into full-length M3R (M3RTP/LP) prevents trafficking of the receptor to the cell surface. Using atropine or other antagonists as pharmacologic chaperones, we were able to increase the level of surface expression of the TP/LP mutant to levels comparable to that of wild-type M3R. However, M3R-stimulated calcium signaling is still severely compromised. These results show that the interaction of M3R with Gβ5-RGS7 requires helix 8 and the central portion of the i3 loop.

Understanding the importance of glycosylated threonine and stereospecific action of Drosocin, a Proline rich antimicrobial peptide

Glycosylation is an essential post-translational modification for few antimicrobial peptides of Proline rich class. In the present study we have shown the importance of Thr glycosylation over Ser glycosylation in Drosocin. Difference of a methyl group makes glycosylated-Thr preferred over glycosylated-Ser and renders higher activity to the peptide, probably due to the rigid conformation provided by the glycosylated-Thr. The structural rigidity provided by glycosylated-Thr to Drosocin backbone was mimicked by substituting glycosylated-Thr11, Ser7 and Ser12 with Pro residues. The designed non-glycosylated analogue, P7P11P12-Drosocin, exhibited functional and structural properties similar to that of the native monoglycosylated peptide. The functional importance of stereospecificity of amino acids and sugar was further explored. Interestingly, (all D) p7p11p12-Drosocin failed to exhibit antimicrobial activity but had comparable binding affinity to DnaK, one of the proposed targets for Proline rich class of antibacterial peptides, as that of its L counterpart. However, Drosocin containing either L or D enantiomeric sugar, displayed antimicrobial activity and binding affinity to bacterial heat shock protein, DnaK. The flow cytometry (FACS) experiments revealed the internalization of Drosocins bearing enantiomeric sugars and P7P11P12-Drosocin but not of its d-enantiomer into bacteria suggesting the importance of stereospecificity of amino acids for membrane entry. Once internalized both enantiomeric peptides may behave similarly. This assumption was corroborated by in vitro activity of (all D) p7p11p12-Drosocin in cell free assay where it abrogated transcription/translation pathway similar to l-enantiomer but could not inhibit the same in whole cell assay. These research findings provide insights into the mode of action of Proline rich class of antibacterial peptides and guidelines for designing functionally equivalent non-glycosylated analogues of glycosylated antibacterial peptides.

Chiral recognition of diketopiperazines cyclo(Pro-Gly) using (−)-epigallocatechin-3-O-gallate

Upon a formation of complexes of EGCg and cyclo(l-Pro-Gly), cyclo(d-Pro-Gly) in D2O, a chirality of cyclo(Pro-Gly) was recognized by difference of the chemical shift of 1H NMR signal for H7α, H7β,8α of the Pro residue. Judging from the crystal structures of the 2:2 complexes of EGCg and cyclo(l-Pro-Gly), cyclo(d-Pro-Gly), such a difference of the chemical shift might be due to magnetic anisotropic shielding effect by the ring current from the B ring of EGCg.

Crystal structure of IMP-2 metallo-β-lactamase from Acinetobacter spp.: comparison of active-site loop structures between IMP-1 and IMP-2.

IMP-2, a subclass B1 metallo-β-lactamase (MBL), is a Zn(II)-containing hydrolase. This hydrolase, involved in antibiotic resistance, catalyzes the hydrolysis of the C-N bond of the β-lactam ring in β-lactam antibiotics such as benzylpenicillin and imipenem. The crystal structure of IMP-2 MBL from Acinetobacter spp. was determined at 2.3 Å resolution. This structure is analogous to that of subclass B1 MBLs such as IMP-1 and VIM-2. Comparison of the structures of IMP-1 and IMP-2, which have an 85% amino acid identity, suggests that the amino acid substitution at position 68 on a β-strand (β3) (Pro in IMP-1 versus Ser in IMP-2) may be a staple factor affecting the flexibility of loop 1 (comprising residues at positions 60-66; EVNGWGV). In the IMP-1 structure, loop 1 adopts an open, disordered conformation. On the other hand, loop 1 of IMP-2 forms a closed conformation in which the side chain of Trp64, involved in substrate binding, is oriented so as to cover the active site, even though there is an acetate ion in the active site of both IMP-1 and IMP-2. Loop 1 of IMP-2 has a more flexible structure in comparison to IMP-1 due to having a Ser residue instead of the Pro residue at position 68, indicating that this difference in sequence may be a trigger to induce a more flexible conformation in loop 1.

On the split personality of penultimate proline.

The influence of the position of the amino acid proline in polypeptide sequences is examined by a combination of ion mobility spectrometry-mass spectrometry (IMS-MS), amino acid substitutions, and molecular modeling. The results suggest that when proline exists as the second residue from the N-terminus (i.e., penultimate proline), two families of conformers are formed. We demonstrate the existence of these families by a study of a series of truncated and mutated peptides derived from the 11-residue peptide Ser(1)-Pro(2)-Glu(3)-Leu(4)-Pro(5)-Ser(6)-Pro(7)-Gln(8)-Ala(9)-Glu(10)-Lys(11). We find that every peptide from this sequence with a penultimate proline residue has multiple conformations. Substitution of Ala for Pro residues indicates that multiple conformers arise from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds as Xaa-Ala peptide bonds are unlikely to adopt the cis isomer, and examination of spectra from a library of 58 peptides indicates that ~80% of sequences show this effect. A simple mechanism suggesting that the barrier between the cis- and trans-proline forms is lowered because of low steric impedance is proposed. This observation may have interesting biological implications as well, and we note that a number of biologically active peptides have penultimate proline residues.

Exploring the Genomic Roadmap and Molecular Phylogenetics Associated with MODY Cascades Using Computational Biology.

Maturity onset diabetes of the young (MODY) is a metabolic and genetic disorder. It is different from type 1 and type 2 diabetes with low occurrence level (1-2%) among all diabetes. This disorder is a consequence of β-cell dysfunction. Till date, 11 subtypes of MODY have been identified, and all of them can cause gene mutations. However, very little is known about the gene mapping, molecular phylogenetics, and co-expression among MODY genes and networking between cascades. This study has used latest servers and software such as VarioWatch, ClustalW, MUSCLE, G Blocks, Phylogeny.fr, iTOL, WebLogo, STRING, and KEGG PATHWAY to perform comprehensive analyses of gene mapping, multiple sequences alignment, molecular phylogenetics, protein-protein network design, co-expression analysis of MODY genes, and pathway development. The MODY genes are located in chromosomes-2, 7, 8, 9, 11, 12, 13, 17, and 20. Highly aligned block shows Pro, Gly, Leu, Arg, and Pro residues are highly aligned in the positions of 296, 386, 437, 455, 456 and 598, respectively. Alignment scores inform us that HNF1A and HNF1B proteins have shown high sequence similarity among MODY proteins. Protein-protein network design shows that HNF1A, HNF1B, HNF4A, NEUROD1, PDX1, PAX4, INS, and GCK are strongly connected, and the co-expression analyses between MODY genes also show distinct association between HNF1A and HNF4A genes. This study has used latest tools of bioinformatics to develop a rapid method to assess the evolutionary relationship, the network development, and the associations among eleven MODY genes and cascades. The prediction of sequence conservation, molecular phylogenetics, protein-protein network and the association between the MODY cascades enhances opportunities to get more insights into the less-known MODY disease.

Aib-containing peptide analogs: Cellular uptake and utilization in oligonucleotide delivery

α-Aminoisobutyric acid (Aib)-containing peptide analogs derived from TV-XIIa, a cell-penetrating peptide (CPP), were synthesized to explore structure-activity relationships. The replacement of Aib at position 1, 5, or 9 in the TV-XIIa amino acid sequence with alanine (Ala) suppressed the cellular uptake, whereas the simultaneous substitution of the two proline (Pro) residues at positions 6 and 10 with Aib (P-IV) considerably increased the cellular uptake. In order to explore the potential use of the Aib-containing peptide analogs for the cellular delivery of oligonucleotides (ODNs), we synthesized a covalent conjugate (P-IV-AON) of a 15-mer antisense ODN, which is complementary to luciferase gene, with P-IV, and the antisense effect of the P-IV-AON conjugate on luciferase expression in A549 cells was examined. Luciferase expression was decreased in the presence of the conjugate upon treatment with the reaction buffer at the concentrations of 5 and 10μM.

Distinct Biochemical and Functional Properties of Two Rab5 Homologs from the Rice Blast Fungus Magnaporthe oryzae

Rab5 is a key regulator of early endocytosis by promoting early endosomal fusion and motility. In this study, we have unexpectedly found distinct properties of the two Rab5 homologs (MoRab5A and MoRab5B) from Magnaporthe oryzae, a pathogenic fungus in plants whose infection causes rice blast disease. Like mammalian Rab5, MoRab5A and MoRab5B can bind to several Rab5 effectors in a GTP-dependent manner, including EEA1, Rabenosyn-5, and Rabaptin-5. However, MoRab5A shows distinct binding characteristics in the sense that both the wild-type and the GTP hydrolysis-defective constitutively active mutant bind the effectors equally well in GST pull-down assays, suggesting that MoRab5A is defective in GTP hydrolysis and mostly in the GTP-bound conformation in the cell. Indeed, GTP hydrolysis assays indicate that MoRab5A GTPase activity is dramatically lower than MoRab5B and human Rab5 and is insensitive to RabGAP5 stimulation. We have further identified a Pro residue in the switch I region largely responsible for the distinct MoRab5A properties by characterization of MoRab5A and MoRab5B chimeras and mutagenesis. The differences between MoRab5A and MoRab5B extend to their functions in the cell. Although they both target to early endosomes, only MoRab5B closely resembles human Rab5 in promoting early endosome fusion and stimulating fluid phase endocytosis. In contrast, MoRab5A correlates with another related early endosomal Rab, Rab22, in terms of the presence of the switch I Pro residue and the blocked GTPase activity. Our data thus identify MoRab5B as the Rab5 ortholog and suggest that MoRab5A specializes to perform a non-redundant function in endosomal sorting.