NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)

Human fibroblast growth factor (hFGF-1) is a ∼ 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu²⁺) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu²⁺ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu²⁺ appears to stabilize the protein bound to pS vesicles. Cu²⁺ and lipid binding interface mapped using 2D ¹H-¹⁵N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu²⁺ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu²⁺, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu²⁺-induced non-classical secretion of hFGF-1.     

Quantitative Profiling of Colorectal Cancer-Associated Bacteria Reveals Associations between Fusobacterium spp., Enterotoxigenic Bacteroides fragilis (ETBF) and Clinicopathological Features of Colorectal Cancer

Various studies have presented clinical or in vitro evidence linking bacteria to colorectal cancer, but these bacteria have not previously been concurrently quantified by qPCR in a single cohort. We quantify these bacteria (Fusobacterium spp., Streptococcus gallolyticus, Enterococcus faecalis, Enterotoxigenic Bacteroides fragilis (ETBF), Enteropathogenic Escherichia coli (EPEC), and afaC- or pks-positive E. coli) in paired tumour and normal tissue samples from 55 colorectal cancer patients. We further investigate the relationship between a) the presence and b) the level of colonisation of each bacterial species with site and stage of disease, age, gender, ethnicity and MSI-status. With the exception of S. gallolyticus, we detected all bacteria profiled here in both tumour and normal samples at varying frequencies. ETBF (FDR = 0.001 and 0.002 for normal and tumour samples) and afaC-positive E. coli (FDR = 0.03, normal samples) were significantly enriched in the colon compared to the rectum. ETBF (FDR = 0.04 and 0.002 for normal and tumour samples, respectively) and Fusobacterium spp. (FDR = 0.03 tumour samples) levels were significantly higher in late stage (III/IV) colorectal cancers. Fusobacterium was by far the most common bacteria detected, occurring in 82% and 81% of paired tumour and normal samples. Fusobacterium was also the only bacterium that was significantly higher in tumour compared to normal samples (p = 6e-5). We also identified significant associations between high-level colonisation by Fusobacterium and MSI-H (FDR = 0.05), age (FDR = 0.03) or pks-positive E. coli (FDR = 0.01). Furthermore, we exclusively identified atypical EPEC in our cohort, which has not been previously reported in association with colorectal cancer. By quantifying colorectal cancer-associated bacteria across a single cohort, we uncovered inter- and intra-individual patterns of colonization not previously recognized, as well as important associations with clinicopathological features, especially in the case of Fusobacterium and ETBF.     

Molecular cloning, overexpression and characterization of human interleukin 1alpha

Interleukin-1 alpha (IL-1alpha) regulates a wide range of important cellular processes. In this study for the first time, we report the cloning, expression, biophysical, and biological characterization of the human interleukin-1alpha. Human IL-1alpha has been expressed in Escherichia coli in high yields ( approximately 4mg per liter of the bacterial culture). The protein was purified to homogeneity ( approximately 98% purity) using affinity chromatography and size exclusion chromatography. Results of the steady-state fluorescence and 2D NMR experiments show that the recombinant IL-1alpha is in a folded conformation. Far-UV circular dichroism (CD) data suggest that IL-1alpha is an all beta-sheet protein with a beta-barrel architecture. Isothermal titration calorimetry (ITC) experiments show that the recombinant IL-1alpha binds strongly (K(d) approximately 5.6 x 10(-7) M) to S100A13, a calcium binding protein that chaperones the in vivo release of IL-1alpha into the extracellular compartment. Recombinant IL-1alpha was observed to exhibit strong cytostatic effect on human umbilical vascular endothelial cells. The findings of the present study not only pave way for an in-depth structural investigation of the molecular mechanism(s) underlying the non-classical release of IL-1alpha but also provide avenues for the rational design of potent inhibitors against IL-1alpha mediated pathogenesis.     

Relevance of partially structured states in the non-classical secretion of acidic fibroblast growth factor

Acidic fibroblast growth factor (aFGF) is a signal peptide-less protein that is secreted into the extracellular compartment as part of a multiprotein release complex, consisting of aFGF, S100A13 (a calcium binding protein), and a 40 kDa (p40) form of synaptotagmin (Syt1), a protein that participates in the docking of a variety of secretory vesicles. p40 Syt1, and specifically its C2A domain, is believed to play a major role in the non-classical secretion of the aFGF release complex mediated by the interaction of aFGF and p40 Syt1with the phospholipids of the cell membrane inner leaflet. In the present study, we investigate the structural characteristics of aFGF and the C2A domain of p40 Syt1 under acidic conditions, using a variety of biophysical techniques including multidimensional NMR spectroscopy. Urea-induced equilibrium unfolding (at pH 3.4) of both aFGF and the C2A domain are non-cooperative and proceed with the accumulation of stable intermediate states. 1-Anilino-8-napthalene sulfonate (ANS) binding and size-exclusion chromatography results suggest that both aFGF and the C2A domain exist as partially structured states under acidic conditions (pH 3.4). Limited trypsin digestion analysis and 1H-15N chemical shift perturbation data reveal that the flexibility of certain portions of the protein backbone is increased in the partially structured state(s) of aFGF. The residues that are perturbed in the partially structured state(s) in aFGF are mostly located at the N- and C-terminal ends of the protein. In marked contrast, most of the interactions stabilizing the native secondary structure are preserved in the partially structured state of the C2A domain. Isothermal titration calorimetry data indicate that the binding affinity between aFGF and the C2A domain is significantly enhanced at pH 3.4. In addition, both aFGF and the C2A domain exhibit much higher lipid binding affinity in their partially structured states. The translocation of the multiprotein FGF release complex across the membrane appears to be facilitated by the formation of partially structured states of aFGF and the C2A domain of p40 Syt1.     

Time-dependent changes in the denatured state(s) influence the folding mechanism of an all beta-sheet protein

Newt fibroblast growth factor (nFGF-1) is an approximately 15-kDa all beta-sheet protein devoid of disulfide bonds. Urea-induced equilibrium unfolding of nFGF-1, monitored by steady state fluorescence and far-UV circular dichroism spectroscopy, is cooperative with no detectable intermediate(s). Urea-induced unfolding of nFGF-1 is reversible, but the percentage of the protein recovered in the native state depends on the time of incubation of the protein in the denaturant. The yield of the protein in the native state decreases with the increase in time of incubation in the denaturant. The failure of the protein to refold to its native state is not due to trivial chemical reactions that could possibly occur upon prolonged incubation in the denaturant. (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra, limited proteolytic digestion, and fluorescence data suggest that the misfolded state(s) of nFGF-1 has structural features resembling that of the denatured state(s). GroEL, in the presence of ATP, is observed to rescue the protein from being trapped in the misfolded state(s). (1)H-(15)N HSQC data of nFGF-1, acquired in the denatured state(s) (in 8 m urea), suggest that the protein undergoes subtle time-dependent structural changes in the denaturant. To our knowledge, this report for the first time demonstrates that the commitment to adapt unproductive pathways leading to protein misfolding/aggregation occurs in the denatured state ensemble.     

S100A13-lipid interactions-role in the non-classical release of the acidic fibroblast growth factor

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles of l-phosphatidyl serine (pS). In comparison, Ca2+-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca2+ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.     

Secretion without Golgi

A growing number of proteins devoid of signal peptides have been demonstrated to be released through the non-classical pathways independent of endoplasmic reticulum and Golgi. Among them are two potent proangiogenic cytokines FGF1 and IL1alpha. Stress-induced transmembrane translocation of these proteins requires the assembly of copper-dependent multiprotein release complexes. It involves the interaction of exported proteins with the acidic phospholipids of the inner leaflet of the cell membrane and membrane destabilization. Not only stress, but also thrombin treatment and inhibition of Notch signaling stimulate the export of FGF1. Non-classical release of FGF1 and IL1alpha presents a promising target for treatment of cardiovascular, oncologic, and inflammatory disorders.     

The Membrane-binding Motif of the Chloroplast Signal Recognition Particle Receptor (cpFtsY) Regulates GTPase Activity

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane.Proper compartmentalization of proteins relies on the ability of protein localization pathways to transport proteins efficiently from their sites of synthesis to their sites of function. The signal recognition particle (SRP)² and its receptor function in every kingdom of life to target proteins to the endoplasmic reticulum (eukaryotes), cytoplasmic membrane (prokaryotes), and thylakoid membrane (chloroplasts) (1). The targeting function of SRP relies on a conserved 54-kDa SRP subunit (SRP54; Ffh in Escherichia coli and cpSRP54 in chloroplasts) as well as a conserved SRP receptor (SRα; FtsY in E. coli and cpFtsY in chloroplasts). For cytosolic SRPs (SRP54 and Ffh), interactions with a substrate signal sequence and an SRP RNA moiety are prerequisite for interaction with the SRP receptor (SRα and FtsY) (2). GTP binding and hydrolysis by both SRP54 and SRα coordinate substrate release from SRP to the translocon and release of SRP from SRα. In chloroplasts, cpFtsY functions along with a unique SRP (cpSRP) to post-translationally target nuclear encoded proteins to thylakoid membranes (3). Light-harvesting chlorophyll a/b-binding proteins (LHCPs) imported into the chloroplast stroma are bound by cpSRP to form a soluble targeting complex, which directs the LHCP substrate to the thylakoid membrane translocon Alb3 (Albino3) in a GTP- and cpFtsY-dependent manner (14, 36). Although many general steps of SRP protein targeting seem largely conserved across evolutionary boundaries, the nature and dynamics of the receptor appear to have diverged.In eukaryotic systems, SRα is peripherally bound to the membrane through association with the integral membrane subunit SRβ. In contrast, no chloroplast or bacterial homolog of SRβ has been identified. cpFtsY and E. coli FtsY (EcFtsY) are found partitioned between the membrane and the stroma or cytosol, respectively. The membrane-binding capacity of EcFtsY serves to stimulate GTPase activity and appears critical in that only membrane-associated EcFtsY supports the release of nascent chains from SRP to the translocon (4, 5). However, the partitioning activity is not strictly required because EcFtsY tethered to the membrane is functional in vivo (37). Given the conserved nature of partitioning among bacterial and chloroplast SRP receptors, partitioning may play an, as of yet, unidentified role in protein targeting by SRP. Nevertheless, differences in lipid composition between bacterial and thylakoid membranes make it interesting to speculate that there are mechanistic differences in membrane partitioning.Like many prokaryotic FtsY homologs (e.g. Thermus aquaticus), cpFtsY lacks the N-terminal acidic domain (A domain) implicated in EcFtsY membrane binding (6). Although the highly conserved FtsY GTPase domain (NG domain) of EcFtsY (EcFtsY_NG) fails to support protein targeting, the addition of the last A domain residue, Phe-196 of a conserved double-Phe motif (EcFtsY_NG+1), restores protein targeting in vivo (7). In vitro studies also show that EcFtsY_NG+1 retains the capacity to bind membranes and support integration of SRP-dependent substrates, although at significantly reduced levels compared with full-length EcFtsY (8). A resolved structure of EcFtsY_NG+1 suggests that the amphipathic nature of the region containing Phe-196 plays a critical role in membrane association (9). Furthermore, it has been demonstrated that liposomes stimulate GTP hydrolysis rates of SRP with EcFtsY_NG+1, but not with EcFtsY_NG, supporting the idea that the A domain in its entirety is not strictly required.For cpFtsY, the necessity and functional role(s) of partitioning between a thylakoid-bound and a soluble phase, as well as the role of N-terminal residues in these functions, remain unknown. In addition, both the conformational state of membrane-bound cpFtsY and EcFtsY and the mechanism responsible for controlling membrane partitioning and altered GTPase activity remain unclear. Because of the gain of function exhibited by EcFtsY_NG+1 and the conserved nature of the surrounding motif (9), it seems likely that this conserved region is necessary to support membrane binding and corresponding functions not only in EcFtsY but also in FtsY homologs.To examine the functional role of the N-terminal region of cpFtsY, we have utilized deletion and point mutants in assays that reconstitute cpFtsY activities, including the cpSRP-dependent integration of LHCP. Together, our data indicate that the conserved lipid-binding motif identified in bacterial FtsY homologs is present in cpFtsY and is both necessary and sufficient for thylakoid binding and critical for LHCP targeting.