Purification and characterization of native human insulin-like growth factor binding protein-6

Insulin-like growth factor binding proteins (IGFBPs) are key regulators of insulin-like growth factor (IGF) mediated signal transduction and thereby can profoundly influence cellular phenotypes and cell fate. Whereas IGFBPs are extracellular proteins, intracellular activities were described for several IGFBP family members, such as IGFBP-3, which can be reinternalized by endocytosis and reaches the nucleus through routes that remain to be fully established. Within the family of IGFBPs, IGFBP-6 is unique for its specific binding to IGF-II. IGFBP-6 was described to possess additional IGF-independent activities, which have in part been attributed to its translocation to the nucleus; however, cellular uptake of IGFBP-6 was not described. To further explore IGFBP-6 functions, we developed a new method for the purification of native human IGFBP-6 from cell culture supernatants, involving a four-step affinity purification procedure, which yields highly enriched IGFBP-6. Whereas protein purified in this way retained the capacity to interact with IGF-II and modulate IGF-dependent signal transduction, our data suggest that, unlike IGFBP-3, human IGFBP-6 is not readily internalized by human tumor cells. To summarize, this work describes a novel and efficient method for the purification of native human insulin-like growth factor binding protein 6 (IGFBP-6) from human cell culture supernatants, applying a four-step chromatography procedure. Intactness of purified IGFBP-6 was confirmed by IGF ligand Western blot and ability to modulate IGF-dependent signal transduction. Cellular uptake studies were performed to further characterize the purified protein, showing no short-term uptake of IGFBP-6, in contrast to IGFBP-3

<i>fahd-1(tm5005) C</i>. <i>elegans</i> have a locomotion deficit.

<p>(A) 4-day old wild-type and <i>fahd-1(tm5005)</i> animals were placed on an empty NGM plate (without bacterial lawn) and the number of body bends was counted for one minute. <i>fahd-1(tm5005)</i> animals show fewer and more uncoordinated body bends compared to wild-type animals (28.53 ±SD 1.12 for wt vs. 11.57 ±SD 0.42 for <i>fahd-1(tm5005)</i>; p = 1.60E-05; N = 3 experiments (30 worms total)). (B) 4-day old wild type and <i>fahd-1(tm5005)</i> worms were put in the center of an NGM plate seeded with <i>E</i>. <i>coli</i> OP50. The radial dispersal rate (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134161#sec002" target="_blank">Materials and Methods</a>) was determined for three different time points (30 sec, 1 min, 2 min). The radial dispersal rate is decreased in <i>fahd-1(tm5005) C</i>. <i>elegans</i> compared to wild-type controls (3.41 ±SD 0.76 zones after two minutes for wt vs. 1.69 ±SD 0.11 zones after two minutes for <i>fahd-1(tm5005)</i>; p < 0.03 in all cases; N = 3 experiments (90 worms total)). (C) 4-day old wild-type and <i>fahd-1(tm5005)</i> hermaphrodites were screened for their swimming endurance performance in M9 buffer for up to 10 h. Wild-type worms are able to swim significantly longer than <i>fahd-1(tm5005)</i> worms (94.56% ±SD 4.72% swimming after 5 hours for wild-type vs. 23.72% ±SD 6.18 swimming after 10 hours for <i>fahd-1(tm5005)</i>; p = 0.001; N = 3 experiments (72 worms total)).</p

Expression and subcellular localization of FAHD-1 in nematodes.

<p>(A) Young adult and gravid adult hermaphrodites of the transcriptional reporter N2;<i>Ex</i>[p_{<i>fahd-1</i>}GFP, pRF4] were monitored by confocal microscopy. <i>fahd-1</i> seems to be expressed in most tissues, including pharynx, canal cell, neurons, vulva, intestine, and stomato-intestinal muscle. Representative pictures are shown. Size bar = 100 μm. (B) Confocal images of young adult <i>C</i>. <i>elegans</i> of the transcriptional reporter N2;<i>Ex</i>[p_{<i>fahd-1</i>}GFP, pRF4]. Representative images are shown. Size bar = 10 μm. <i>Upper left panel</i>: Detailed picture of <i>fahd-1</i> expression in the peri-vulvar region; vulva and uterine muscles are stained positive. <i>Upper right panel</i>: Detailed picture of <i>fahd-1</i> expression in the pharyngeal region; buccal cavity, procorpus, metacorpus, isthmus, posterior bulb, and pharyngeal-intestinal valve, as well as sensory neurons in the head were stained positive. <i>Lower left panel</i>: Detailed picture of <i>fahd-1</i> expression in the mid-body region; body wall muscles, lateral ganglion, dorsal chord (DC), and ventral nerve chord (VNC) were stained positive. <i>Lower right panel</i>: Detailed picture of <i>fahd-1</i> expression in the anus region; the stomato-intestinal muscles, the anus and the intestine were stained positive. (C) Temporal expression pattern of <i>fahd-1</i>. The translational reporter N2;<i>Ex</i>[p_{<i>fahd-1</i>}FAHD-1::GFP, pRF4] was examined (and pictured) by confocal fluorescence microscopy from the time point the eggs were laid at gastrula stage to 10-day old post fertile adults. GFP fluorescence was overlaid with phase-contrast microscopy. The FAHD1-1::GFP fusion protein is present in all stages from gastrulation to old worms. Representative images are shown. Size bar = 50 μm. (D) Young adult hermaphrodites of the translational reporter N2;<i>Ex</i>[p_{<i>fahd-1</i>}FAHD-1::GFP, pRF4] expressing the FAHD-1::GFP fusion protein (shown in green) were stained with 100 nM MitoTracker Red (shown in red) overnight. Shown here are confocal pictures of the pharynx region of the worm recorded in the green and red channel, as well as the merged pictures. Regions of overlapping staining (shown in yellow) are indicated by arrows. Representative pictures are shown. Size bar = 10 μm.</p

Characterization of <i>fahd-1</i> deletion mutant animals.

<p>(A) <i>Left panel</i>: Single worm duplex PCR was performed on ten animals of the <i>fahd-1(tm5005)</i> mutant strain, to confirm the deletion on DNA level. Three primers were used, generating PCR products of 220 bp for the wild-type allele and 337 bp for the mutant allele. M = size marker; 1–10 = 10 different backcrossed <i>fahd-1(tm5005) C</i>. <i>elegans</i>; mut = mutant allele; wt = wild-type allele; b = <i>E</i>. <i>coli</i> OP50 (<i>C</i>. <i>elegans</i> bacterial food; to identify bands derived from bacterial contamination). <i>Right panel</i>: To confirm the deletion of the protein, protein lysates were prepared from wild-type and <i>fahd-1(tm5005)</i> mutant animals, as indicated. FAHD-1 protein was detected by Western blot using a peptide-specific rabbit polyclonal antibody against FAHD-1. β-actin served as a loading control. (B) Overall brood size was compared by counting progeny in wild-type and <i>fahd-1(tm5005) C</i>. <i>elegans</i> animals raised at 20°C and 25°C throughout the reproductive period. In <i>fahd-1(tm5005) C</i>. <i>elegans</i> the total brood size is reduced compared to wild-type controls when raised at 20°C (left panel; 281 ±SD 28.11 for wt vs. 238 ±SD 63.96 for <i>fahd-1(tm5005)</i>; p = 0.05; N = 9) and even more severe when raised at 25°C (right panel; 199 ±SD 71.30 for wt vs. 98.8 ±SD 49.23 for <i>fahd-1(tm5005)</i>; p = 0.0013; N = 10). (D) Egg laying patterns were determined for wild-type and <i>fahd-1(tm5005) C</i>. <i>elegans</i>. <i>Left panel</i>: 4-day old wild-type and <i>fahd-1(tm5005)</i> animals were kept in M9 buffer (without bacteria) for four hours and the number of eggs that were laid by each genotype during this time was counted. While wild-type worms do not lay their eggs under these unfavorable conditions, <i>fahd-1(tm5005)</i> worms ‘lose’ them over the course of time (2.08 ±SD 1.75 for wt vs. 10.26 ±SD 0.32; p = 0.0598 for 60 min, p < 0.002 in all other cases; N = 3 experiments (72 worms total)). <i>Right panel</i>: Micrographs were prepared from 4-day old, well-fed and unstressed hermaphrodites of the wild-type and <i>fahd-1(tm5005)</i> genotype. While wild-type worms carry between 18 and 20 eggs, <i>fahd-1(tm5005)</i> animals show great variety in the number of eggs, tending to accumulate them in the uterus. Representative pictures are shown.</p

FAH Domain Containing Protein 1 (FAHD-1) Is Required for Mitochondrial Function and Locomotion Activity in <i>C</i>. <i>elegans</i>

<div><p>The fumarylacetoacetate hydrolase (FAH) protein superfamily of metabolic enzymes comprises a diverse set of enzymatic functions, including ß-diketone hydrolases, decarboxylases, and isomerases. Of note, the FAH superfamily includes many prokaryotic members with very distinct functions that lack homologs in eukaryotes. A prokaryotic member of the FAH superfamily, referred to as Cg1458, was shown to encode a soluble oxaloacetate decarboxylase (ODx). Based on sequence homologies to Cg1458, we recently identified human FAH domain containing protein-1 (FAHD1) as the first eukaryotic oxaloacetate decarboxylase. The physiological functions of ODx in eukaryotes remain unclear. Here we have probed the function of <i>fahd-1</i>, the nematode homolog of FAHD1, in the context of an intact organism. We found that mutation of <i>fahd-1</i> resulted in reduced brood size, a deregulation of the egg laying process and a severe locomotion deficit, characterized by a reduced frequency of body bends, reduced exploratory movements and reduced performance in an endurance exercise test. Notably, mitochondrial function was altered in the <i>fahd-1(tm5005)</i> mutant strain, as shown by a reduction of mitochondrial membrane potential and a reduced oxygen consumption of <i>fahd-1(tm5005)</i> animals. Mitochondrial dysfunction was accompanied by lifespan extension in worms grown at elevated temperature; however, unlike in mutant worms with a defect in the electron transport chain, the mitochondrial unfolded protein response was not upregulated in worms upon inactivation of <i>fahd-1</i>. Together these data establish a role of <i>fahd-1</i> to maintain mitochondrial function and consequently physical activity in nematodes.</p></div

Mitochondrial dysfunction in <i>fahd-1(tm5005) C</i>. <i>elegans</i>.

<p>(A) 3-day old <i>fahd-1(tm5005)</i> and wild-type <i>C</i>. <i>elegans</i> were incubated with 100 nM TMRE overnight. <i>Left panel</i>: <i>fahd-1(tm5005)</i> animals exhibit a decreased mitochondrial membrane potential as shown by fluorescence microscopy. Representative images are shown. Size bar = 100 μm. <i>Right panel</i>: For quantification of the pixel intensities, fluorescence pictures were analyzed using ImageJ (25.93 ±SD 7.61 for wt vs. 17.69 ±SD 9.87 for <i>fahd-1(tm5005)</i>; p = 0.0005; N = 30). (B) Oxygen consumption rate was determined in wild-type and <i>fahd-1(tm5005)</i> animals by Oxygraph 2K measurement and the basic respiration rate was determined. In <i>fahd-1(tm5005)</i> animals this parameter is significantly reduced (0.0659 ±SD 0.0026 pmol sec^-1 worm^-1 for wt vs. 0.0.0571 ±SD 0.0062 pmol sec^-1 worm^-1 for <i>fahd-1(tm5005)</i>; p = 0.0475; N = 4).</p

Lifespan of <i>fahd-1(tm5005)</i> animals is increased at elevated temperature.

<p>(A) Lifespan analysis of <i>fahd-1(tm5005) C</i>. <i>elegans</i> compared to wild-type <i>C</i>. <i>elegans</i> maintained at 20°C. There is no difference in the lifespans between the two strains (median survival: 16 days for both groups; p = 0.6773; N > 200). (B) Lifespan analysis of <i>fahd-1(tm5005) C</i>. <i>elegans</i> compared to wild-type <i>C</i>. <i>elegans</i> raised at 25°C. The lifespan of <i>fahd-1(tm5005) C</i>. <i>elegans</i> is significantly increased (median survival: 8 days for wt vs. 10 days for <i>fahd-1(tm5005)</i>; p < 0.0001; N > 200). (C) <i>Left panel</i>: FAHD-1 knock-down by ingested RNAi in wild-type worms also leads to an extension of the median lifespan when performed at 25°C (median survival: 12 days for RNAi control vs. 15 days for RNAi <i>fahd-1</i>; p < 0.0001; N > 200). <i>Right panel</i>: The efficiency of the knock-down was tested by Western blot using a peptide-specific rabbit polyclonal antibody against FAHD-1. β-actin served as a loading control. The lifespan assays were performed at least three times. One exemplary experiment is shown. (D) Representative fluorescence images (left panel) and quantitation of fluorescence intensities (right panel) of 3-day old worms of the strain SJ4100: N2;<i>Is</i>[p_<i>hsp-6</i>GFP] that were fed with control bacteria or bacteria expressing dsRNA directed against <i>fahd-1</i> or <i>cco-1</i>. FAHD-1 knockdown by ingested RNAi does not activate the UPR^mt at 25°C. In control experiments, UPR^mt was induced by knock-down of <i>cco-1</i>. Size bar = 500 μm.</p