Dependent Folding Research Articles

Leishmania donovani chaperonin TCP1γ subunit protects miltefosine induced oxidative damage

T-complex protein-1 (TCP1) is a chaperonin protein known to fold various proteins like actin and tubulin. In Leishmania donovani only one subunit of TCP1 that is gamma subunit (LdTCP1γ) has been functionally characterized. It not only performs ATP dependent protein folding but is also essential for survival and virulence. The present work demonstrates that LdTCP1γ also has a role in miltefosine resistance. Overexpression of LdTCP1γ in L. donovani promastigotes results in decreased sensitivity of parasites towards miltefosine, while single-allele replacement mutants exhibited increased sensitivity as compared to wild-type promastigotes. This response was specific to miltefosine with no cross-resistance to other drugs. The LdTCP1γ-mediated drug resistance was directly related to miltefosine-induced apoptotic death of the parasite, as was evidenced by 2 to 3-fold decrease in cell death parameters in overexpressing cells and >2-fold increase in single-allele replacement mutants. Further, deciphering the mechanism revealed that resistance of overexpressing cells was associated with efficient ROS neutralization due to increased levels of thiols and upregulation of cytosolic tryparedoxin peroxidase (cTxnPx). Further, modulation of LdTCP1γ expression in parasite also modulates the levels of proinflammatory cytokine (TNF-α) and anti-inflammatory cytokine (IL-10) of the host macrophages. The study provides evidence for the involvement of a chaperonin protein LdTCP1γ in the protection against miltefosine induced oxidative damage and reveals the fundamental role of LdTCP1γ in parasite biology.

N-Glycan-dependent protein folding and endoplasmic reticulum retention regulate GPI-anchor processing.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in the endoplasmic reticulum (ER). Soon after GPI is attached, an acyl chain on the GPI inositol is removed by post-GPI attachment to proteins 1 (PGAP1), a GPI-inositol deacylase. This is crucial for switching GPI-anchored proteins (GPI-APs) from protein folding to transport states. We performed haploid genetic screens to identify factors regulating GPI-inositol deacylation, identifying seven genes. In particular, calnexin cycle impairment caused inefficient GPI-inositol deacylation. Calnexin was specifically associated with GPI-APs, dependent on N-glycan and GPI moieties, and assisted efficient GPI-inositol deacylation by PGAP1. Under chronic ER stress caused by misfolded GPI-APs, inositol-acylated GPI-APs were exposed on the cell surface. These results indicated that N-glycans participate in quality control and temporal ER retention of GPI-APs, ensuring their correct folding and GPI processing before exiting from the ER. Once the system is disrupted by ER stress, unprocessed GPI-APs become exposed on the cell surface.

Probing the Ca(2+)-assisted π-π interaction during Ca(2+)-dependent protein folding.

Protein folding is governed by a balance of non-covalent interactions, of which cation-π and π-π play important roles. Theoretical calculations revealed a strong cooperativity between cation-π involving alkali and alkaline earth metal ions and π-π interactions, but however, no experimental evidence was provided in this regard. Here, we characterized a Ca(2+)-binding self-processing module (SPM), which mediates a highly-specific Ca(2+)-dependent autocatalytic processing of iron-regulated protein FrpC secreted by the pathogenic Gram-negative bacterium Neisseria meningitidis. The SPM undergoes a Ca(2+)-induced transition from an intrinsically unstructured conformation to the compact protein fold that is ultimately stabilized by the π-π interaction between two unique tryptophan residues arranged in the T-shaped orientation. Moreover, the pair of tryptophans is located in a close vicinity of a calcium-binding site, suggesting the involvement of a Ca(2+)-assisted π-π interaction in the stabilization of the tertiary structure of the SPM. This makes the SPM an excellent model for the investigation of the Ca(2+)-assisted π-π interaction during Ca(2+)-induced protein folding.

Spectroscopic and DFT studies on 6-Aminophenanthridine and its derivatives provide insights in their activity towards ribosomal RNA

6-Aminophenanthridine (6AP), a plant alkaloid possessing antiprion activity, inhibits ribosomal RNA dependent protein folding activity of the ribosome (referred as PFAR). We have compared 6AP and its three derivatives 6AP8Cl, 6AP8CF3 and 6APi for their activity in inhibition of PFAR. Since PFAR inhibition requires 6AP and its derivatives to bind to the ribosomal RNA (rRNA), we have measured the binding affinity of these molecules to domain V of 23S rRNA using fluorescence spectroscopy. Our results show that similar to the antiprion activity, both the inhibition of PFAR and the affinity towards rRNA follow the order 6AP8CF3>6AP8Cl>6AP, while 6APi is totally inactive. To have a molecular insight for the difference in activity despite similarities in structure, we have calculated the nucleus independent chemical shift using first principles density functional theory. The result suggests that the deviation of planarity in 6APi and steric hindrance from its bulky side chain are the probable reasons which prevent it from interacting with rRNA. Finally, we suggest a probable mode of action of 6AP, 6AP8CF3 and 6AP8Cl towards rRNA.

Disulfide Rearrangement Triggered by Translocon Assembly Controls Lipopolysaccharide Export

The presence of lipopolysaccharide (LPS) on the cell surface of Gram-negative bacteria is critical for viability. A conserved β-barrel membrane protein LptD (lipopolysaccharide transport protein D) translocates LPS from the periplasm across the outer membrane (OM). In Escherichia coli, this protein contains two disulfide bonds and forms the OM LPS translocon with the lipoprotein LptE. Here, we identified seven in vivo states on the oxidative-folding pathway of LptD. Proper assembly involved a nonfunctional intermediate containing non-native disulfides. Intermediate formation required the oxidase DsbA, and subsequent maturation to the active form with native disulfides was triggered by LptE. Thus, disulfide bond-dependent protein folding of LptD requires the proper assembly of a two-protein complex to promote disulfide bond rearrangement.

Impeded protein folding and function in active inflammatory bowel disease

The intestinal tract is covered by a total of 300 square metres of IECs (intestinal epithelial cells) that covers the entire intestinal mucosa. For protection against luminal xenobiotics, pathogens and commensal microbes, these IECs are equipped with membrane-bound transporters as well as the ability to secrete specific protective proteins. In patients with active IBD (inflammatory bowel disease), the expression of these proteins, e.g. ABC (ATP-binding cassette) transporters such as ABCG2 (ABC transporter G2) and defensins, is decreased, thereby limiting the protection against various luminal threats. Correct ER (endoplasmic reticulum)-dependent protein folding is essential for the localization and function of secreted and membrane-bound proteins. Inflammatory triggers, such as cytokines and nitric oxide, can impede protein folding, which causes the accumulation of unfolded proteins inside the ER. As a result, the unfolded protein response is activated which can lead to a cellular process named ER stress. The protein folding impairment affects the function and localization of several proteins, including those involved in protection against xenobiotics. In the present review, we discuss the possible inflammatory pathways affecting protein folding and eventually leading to IEC malfunction in patients with active IBD.

Metal-Coupled Folding of Cys2His2 Zinc-Finger

Zinc-fingers, which widely exist in eukaryotic cell and play crucial roles in life processes, depend on the binding of zinc ion for their proper folding. To computationally study the zinc-coupled folding of the zinc-fingers, charge transfer and metal induced protonation/deprotonation effects have to be considered. Here, by attempting to implicitly account for such effects in classical molecular dynamics and performing intensive simulations with explicit solvent for the peptides with and without zinc binding, we investigate the folding of the Cys2His2-type zinc-finger motif and the coupling between the peptide folding and zinc binding. We find that zinc ion not only stabilizes the native structure but also participates in the whole folding process. It binds to the peptide at an early stage of folding and directs or modulates the folding and stabilizations of the component beta-hairpin and alpha-helix. Such a crucial role of zinc binding is mediated by the packing of the conserved hydrophobic residues. We also find that the packing of the hydrophobic residues and the coordination of the native ligands are coupled. Meanwhile, the processes of zinc binding, mis-ligation, ligand exchange, and zinc induced secondary structure conversion as well as the water behavior due to the involvement of zinc ion are characterized. Our results are in good agreement with related experimental observations and provide significant insight into the general mechanisms of the metal cofactor dependent protein folding and other metal-induced conformational changes of biological importance.

Mitochondria and Ca2+ signaling: old guests, new functions

Mitochondria are ancient endosymbiotic guests that joined the cells in the evolution of complex life. While the unique ability of mitochondria to produce adenosine triphosphate (ATP) and their contribution to cellular nutrition metabolism received condign attention, our understanding of the organelle's contribution to Ca(2+) homeostasis was restricted to serve as passive Ca(2+) sinks that accumulate Ca(2+) along the organelle's negative membrane potential. This paradigm has changed radically. Nowadays, mitochondria are known to respond to environmental Ca(2+) and to contribute actively to the regulation of spatial and temporal patterns of intracellular Ca(2+) signaling. Accordingly, mitochondria contribute to many signal transduction pathways and are actively involved in the maintenance of capacitative Ca(2+) entry, the accomplishment of Ca(2+) refilling of the endoplasmic reticulum and Ca(2+)-dependent protein folding. Mitochondrial Ca(2+) homeostasis is complex and regulated by numerous, so far, genetically unidentified Ca(2+) channels, pumps and exchangers that concertedly accomplish the organelle's Ca(2+) demand. Notably, mitochondrial Ca(2+) homeostasis and functions are crucially influenced by the organelle's structural organization and motility that, in turn, is controlled by matrix/cytosolic Ca(2+). This review intends to provide a condensed overview on the molecular mechanisms of mitochondrial Ca(2+) homeostasis (uptake, buffering and storage, extrusion), its modulation by other ions, kinases and small molecules, and its contribution to cellular processes as fundamental basis for the organelle's contribution to signaling pathways. Hence, emphasis is given to the structure-to-function and mobility-to-function relationship of the mitochondria and, thereby, bridging our most recent knowledge on mitochondria with the best-established mitochondrial function: metabolism and ATP production.

Mitochondria maintain maturation and secretion of lipoprotein lipase in the endoplasmic reticulum

Considering the physiological Ca2+ dynamics within the ER (endoplasmic reticulum), it remains unclear how efficient protein folding is maintained in living cells. Thus, utilizing the strictly folding-dependent activity and secretion of LPL (lipoprotein lipase), we evaluated the impact of ER Ca2+ content and mitochondrial contribution to Ca2+-dependent protein folding. Exhaustive ER Ca2+ depletion by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases caused strong, but reversible, reduction of cell-associated and released activity of constitutive and adenovirus-encoded human LPL in CHO-K1 (Chinese-hamster ovary K1) and endothelial cells respectively, which was not due to decline of mRNA or intracellular protein levels. In contrast, stimulation with the IP3 (inositol 1,4,5-trisphosphate)-generating agonist histamine only moderately and transiently affected LPL maturation in endothelial cells that paralleled a basically preserved ER Ca2+ content. However, in the absence of extracellular Ca2+ or upon prevention of transmitochondrial Ca2+ flux, LPL maturation discontinued upon histamine stimulation. Collectively, these data indicate that Ca2+-dependent protein folding in the ER is predominantly controlled by intraluminal Ca2+ and is largely maintained during physiological cell stimulation owing to efficient ER Ca2+ refilling. Since Ca2+ entry and mitochondrial Ca2+ homoeostasis are crucial for continuous Ca2+-dependent protein maturation in the ER, their pathological alterations may result in dysfunctional protein folding.

Contact order dependent protein folding rates: kinetic consequences of a cooperative interplay between favorable nonlocal interactions and local conformational preferences.

Physical mechanisms underlying the empirical correlation between relative contact order (CO) and folding rate among naturally occurring small single-domain proteins are investigated by evaluating postulated interaction schemes for a set of three-dimensional 27mer lattice protein models with 97 different CO values. Many-body interactions are constructed such that contact energies become more favorable when short chain segments sequentially adjacent to the contacting residues adopt native-like conformations. At a given interaction strength, this scheme leads to folding rates that are logarithmically well correlated with CO (correlation coefficient r = 0.914) and span more than 2.5 orders of magnitude, whereas folding rates of the corresponding Gō models with additive contact energies have much less logarithmic correlation with CO and span only approximately one order of magnitude. The present protein chain models also exhibit calorimetric cooperativity and linear chevron plots similar to that observed experimentally for proteins with apparent simple two-state folding/unfolding kinetics. Thus, our findings suggest that CO-dependent folding rates of real proteins may arise partly from a significant positive coupling between nonlocal contact favorabilities and local conformational preferences.