A Convenient On-Site Oxidation Strategy for the N-hydroxylation of Melanostatin Neuropeptide using Cope Elimination

In this work, a convenient synthetic protocol for the unprecedented N-hydroxylation of proline residue in Melanostatin (MIF-1) neuropeptide is reported. This methodology is grounded on the incorporation of N-(cyanoethyl)prolyl residue followed by on-site oxidation by Cope elimination with m-chloroperbenzoic acid, exploring the unrecognized dual role of the cyanoethyl group as an effective N-protecting group under peptide synthesis conditions and as a suitable leaving group during the chemoselective on-site N-oxidation. Following this protocol N-hydroxy-MIF-1 is obtained with 78% global yield from N-(cyanoethyl)-L-proline. This synthetic approach opens a new avenue for access to N-hydroxylated Melanostatin analogs with direct application in neurochemistry and Parkinson’s research.

Identification of a proline-kinked amphipathic α-helix downstream from the methyltransferase domain of a potexvirus replicase and its role in virus replication and perinuclear complex formation

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus , the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. NMR analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a GFP-fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA co-located during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions and in vitro high molecular weight complexes were formed by both replication-competent and -incompetent viral replicons, and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales . Substitution of the proline residue but not the other residues necessary for replication allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that a perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

Puckering Transition of Proline Residue along the Pseudorotational Path: Revisited

Puckering transitions of the proline residue for Ac-Pro-X (X = OH, OMe, and NHMe) with trans and cis prolyl peptide bonds were explored along the pseudorotation phase angle using DFT...

The role of prolines and glycine in the transmembrane domain of LAT

ABSTRACTLAT is a critical regulator of T cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and superresolution imaging and flow cytometry we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics is further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of non-palmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

New Morphiceptin Peptidomimetic Incorporating (1S,2R,3S,4S,5R)-2-Amino-3,4,5-trihydroxycyclopen-tane-1-carboxylic acid: Synthesis and Structural Study

We present the synthesis and structural study of a new peptidomimetic of morphiceptin, which can formally be considered as the result of the replacement of the central proline residue of this natural analgesic drug with a subunit of (1S,2R,3S,4S,5R)-2-amino-3,4,5-trihydroxycyclopentane-1-carboxylic acid, previously obtained from L-idose. An optimized synthesis of this trihydroxylated cispentacin derivative is also reported. Molecular docking calculations on the target receptor support a favorable role of the hydroxy substituents of the non-natural β-amino acid incorporated into the peptidomimetic.

Single molecule tracking reveals the role of transitory dynamics of nucleoid-associated protein HU in organizing the bacterial chromosome

HU is the most conserved nucleoid-associated protein in eubacteria and has been implicated as a key player in global chromosome organization. The mechanism of HU-mediated nucleoid organization, however, remains poorly understood. Using single molecule tracking coupled with genetic manipulations, we characterized the dynamics of HU in live Escherichia coli cells. We found that native HU dimers bind and unbind chromosomal DNAs weakly and transitorily across the entire nucleoid volume but remain nucleoid-localized, reminiscent of random diffusion in a liquid phase-separated, membrane-less “macro-compartment” distinct from the remaining cytosol. Mutating three key surface lysine residues of HU nearly entirely abolished the weak and transitory interactions of HU with DNA and led to severe cell growth and DNA segregation defects, suggesting the importance of HU’s interactions with chromosomal DNA mediated by the positively charged surface. A conserved proline residue important for recognizing bent and cruciform DNAs such as that in recombination intermediates, similarly abolished HU’s rapid and transitory DNA interaction dynamics but had little impact on its apparent binding stability with nonspecific chromosomal DNAs. Interestingly, the proline residue appeared to be important for HUαβ dimer formation as mutating this residue makes HUαβ behave similarly to HUα2 dimers. Finally, we find that while prior evidence has found HU capable of depositing nucleoid-associated noncoding RNAs onto cruciform DNA structures, deletion of these specific naRNAs or inhibition of global transcription had a relatively minor effect on HU dynamics irrespective altered nucleoid compaction. Our results suggest a model of chromosome organization mediated by weak, transient interactions of HU, a substantial deviation from nucleoid-like proteins such as histones. Such collective sum of the numerous weak, transitory binding events of HU with nonspecific chromosome DNAs could generates a “force” to maintain a dynamic, fluid nucleoid with enough flexibility to rapidly facilitate global topological processes such as replication or nucleoid segregation.

A PQ-loop protein Ypq2 is involved in the exchange of arginine and histidine across the vacuolar membrane of Saccharomyces cerevisiae

In nutrient-rich conditions, basic amino acids are actively accumulated into the vacuoles by H+-coupled transporters in Saccharomyces cerevisiae. In addition to the H+-coupled systems, the existence of an exchanger for arginine and histidine was indicated by kinetic analysis using isolated vacuolar membrane vesicles; however, the gene(s) involved in the activity has not been identified. Here, we show that the uptake activity of arginine driven by an artificially imposed histidine gradient decreased significantly by the disruption of the gene encoding vacuolar PQ-loop protein Ypq2, but not by those of Ypq1 and Ypq3. The exchange activity was restored by the expression of YPQ2. Furthermore, the substitution of a conserved proline residue, Pro29, in Ypq2 greatly decreased the exchange activity. These results suggest that Ypq2 is responsible for the exchange activity of arginine and histidine across the vacuolar membrane, and the conserved proline residue in the PQ-loop motif is required for the activity.