Antinociceptive constituents from Saccharum officinarum L. juice.

This work aimed to investigate the main components of methanol fractions (MFSC and MFSCf) from Saccharum officinarum L. juice and their in vivo antinociceptive potential. After LC-ESI-MS and ESI-MS/MS analysis, phenolic compounds, such as dicaffeoylquinic acid, schaftoside, vicenin-2, stilbene glycoside and the major compound tricin-7-O-(2″- α-L-rhamnopyranosyl)-α-D-galacturonide (1), were identified. MFSC and MFSCf significantly inhibited nociceptive responses in classical mice pain models. The isolated flavone, 1, inhibited strongly the neurogenic phase in formalin test without interfering with the inflammatory one. The co-administration of the opioid antagonist, naloxone, significantly reversed the antinociceptive effects on the neurogenic phase of both methanol fractions and 1, demonstrating the involvement of the opioid system on the antinociceptive effect. This work describes for the first time the antinociceptive effect of flavonoids present in sugarcane juice, highlighting the isolation and the structural elucidation of tricin-7-O-(2″-α-L-rhamnopyranosyl)-α-D-galacturonide through ESI-MS/MS, 1D- and 2D-NMR.

Solution NMR investigation on the structure and function of the isolated J-domain from Sis1: Evidence of transient inter-domain interactions in the full-length protein.

J-domain/Hsp40 proteins cooperate in aiding with folding in the cell by binding partially folded client proteins and delivering them to be folded by Hsp70. The delivery occurs concomitantly to the stimulation of the ATPase activity of Hsp70 via the N-terminally located J-domain. Although several lines of investigation have been used to study J-domain proteins, the presence of highly flexible domains (G/F- and G/M-rich) hold up obtaining a detailed full-length structure. In this work, we present the high-resolution structure of the J-domain and the N-terminal part of the G/F domain of Sis1, solved by NMR, and used chemical-shift perturbation approaches to further study the structure/function relationship of the Sis1/Hsp70 interaction. When the J-domain was compared to the full-length protein and to a G/M domain deletion mutant, an internal interaction patch formed by hydrophobic and positively charged residues (V2, D9, R27, T39, F52 and R73) was identified. Curiously, the same patch is protected by internal contacts in the full-length protein and, in combination with the loop containing the conserved HPD motif, participates in the interaction with Hsp70. Combined, these results suggest that the J-domain in the full-length Sis1 is in a transient intermediate conformation, in which its interacting patch is protected and, at the same time, also in a favorable condition to bind Hsp70, facilitating the interaction between the two proteins. Finally, 1D NMR experiments showed that the addition of ATP is followed by the disruption of the J-domain/Hsp70 complex, a necessary step for aiding the folding of the client protein.

1H, 15N and 13C resonance assignments of the J-domain of co-chaperone Sis1 from Saccharomyces cerevisiae.

Protein folding in the cell is usually aided by molecular chaperones, from which the Hsp70 (Hsp = heat shock protein) family has many important roles, such as aiding nascent folding and participating in translocation. Hsp70 has ATPase activity which is stimulated by binding to the J-domain present in co-chaperones from the Hsp40 family. Hsp40s have many functions, as for instance the binding to partially folded proteins to be delivered to Hsp70. However, the presence of the J-domain characterizes Hsp40s or, by this reason, as J-proteins. The J-domain alone can stimulate Hsp70 ATPase activity. Apparently, it also maintains the same conformation as in the whole protein although structural information on full J-proteins is still missing. This work reports the 1H, 15N and 13C resonance assignments of the J-domain of a Hsp40 from Saccharomyces cerevisiae, named Sis1. Secondary structure and order parameter prediction from chemical shifts are also reported. Altogether, the data show that Sis1 J-domain is highly structured and predominantly formed by α-helices, results that are in very good agreement with those previously reported for the crystallographic structure.

¹H, ¹5N and ¹³C resonance assignments of PpdD, a type IV pilin from enterohemorrhagic Escherichia coli.

Bacterial type 4 pili (T4P) are long flexible fibers involved in adhesion, DNA uptake, phage transduction, aggregation and a flagella-independent movement called "twitching motility". T4P comprise thousands of copies of the major pilin subunit, which is initially inserted in the plasma membrane, processed and assembled into dynamic helical filaments. T4P are crucial for host colonization and virulence of many Gram-negative bacteria. In enterohemorrhagic Escherichia coli the T4P, called hemorrhagic coli pili (HCP) promote cell adhesion, motility, biofilm formation and signaling. To understand the mechanism of HCP assembly and function, we analyzed the structure of the major subunit prepilin peptidase-dependent protein D (PpdD) (also called HcpA), a 15 kDa pilin with two potential disulfide bonds. Here we present the (1)H, (15)N and (13)C backbone and side chain resonance assignments of the C-terminal globular domain of PpdD as a first step to its structural determination.