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Two latent and two hyperstable polymeric forms of human neuroserpin.

ABSTRACT: Human neuroserpin (hNS) is a serine protease inhibitor that belongs to the serpin superfamily and is expressed in nervous tissues. The serpin fold is generally characterized by a long exposed loop, termed the reactive center loop, that acts as bait for the target protease. Intramolecular insertion of the reactive center loop into the main serpin ?-sheet leads to the serpin latent form. As with other known serpins, hNS pathological mutants have been shown to accumulate as polymers composed of quasi-native protein molecules. Although hNS polymerization has been intensely studied, a general agreement about serpin polymer organization is still lacking. Here we report a biophysical characterization of native hNS that is shown to undergo two distinct conformational transitions, at 55°C and 85°C, both leading to distinct latent and polymeric species. The latent and polymer hNS forms obtained at 45°C and 85°C differ in their chemical and thermal stabilities; furthermore, the hNS polymers also differ in size and morphology. Finally, the 85°C polymer shows a higher content of intermolecular ?-sheet interactions than the 45°C polymer. Together, these results suggest a more complex conformational scenario than was previously envisioned, and, in a general context, may help reconcile the current contrasting views on serpin polymerization.

SUBMITTER: Ricagno S

PROVIDER: S-EPMC2980742 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Two latent and two hyperstable polymeric forms of human neuroserpin.

Ricagno Stefano S Pezzullo Margherita M Barbiroli Alberto A Manno Mauro M Levantino Matteo M Santangelo Maria Grazia MG Bonomi Francesco F Bolognesi Martino M

Biophysical journal 20101101 10

Human neuroserpin (hNS) is a serine protease inhibitor that belongs to the serpin superfamily and is expressed in nervous tissues. The serpin fold is generally characterized by a long exposed loop, termed the reactive center loop, that acts as bait for the target protease. Intramolecular insertion of the reactive center loop into the main serpin β-sheet leads to the serpin latent form. As with other known serpins, hNS pathological mutants have been shown to accumulate as polymers composed of qua ...[more]

PMID: 21081089

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Project description:BackgroundPulses of transcranial magnetic stimulation (TMS) with a predominantly anterior-posterior (AP) or posterior-anterior (PA) current direction over the primary motor cortex appear to activate distinct excitatory inputs to corticospinal neurons. In contrast, very few reports have examined whether the inhibitory neurons responsible for short-interval intracortical inhibition (SICI) are sensitive to TMS current direction.ObjectivesTo investigate whether SICI evaluated with AP and PA conditioning stimuli (CSPA and CSAP) activate different inhibitory pathways. SICI was always assessed using a PA-oriented test stimulus (TSPA).MethodsUsing two superimposed TMS coils, CSPA and CSAP were applied at interstimulus intervals (ISI) of 1-5 ms before a TSPA, and at a range of different intensities. Using a triple stimulation design, we then tested whether SICI at ISI of 3 ms using opposite directions of CS (SICICSPA3 and SICICSAP3) interacted differently with three other forms of inhibition, including SICI at ISI of 2 ms (SICICSPA2), cerebellum-motor cortex inhibition (CBI 5 ms) and short-latency afferent inhibition (SAI 22 ms). Finally, we compared the effect of tonic and phasic voluntary contraction on SICICSPA3 and SICICSAP3.ResultsCSAP produced little SICI at ISIs = 1 and 2 ms. However, at ISI = 3 ms, both CSAP and CSPA were equally effective at the same percent of maximum stimulator output. Despite this apparent similarity, combining SICICSPA3 or SICICSAP3 with other forms of inhibition led to quite different results: SICICSPA3 interacted in complex ways with CBI, SAI and SICICSPA2, whereas the effect of SICICSAP3 appeared to be quite independent of them. Although SICICSPA and SICICSAP were both reduced by the same amount during voluntary tonic contraction compared with rest, in a simple reaction time task SICICSAP was disinhibited much earlier following the imperative signal than SICICSPA.ConclusionsSICICSPA appears to activate a different inhibitory pathway to that activated by SICICSAP. The difference is behaviourally relevant since the pathways are controlled differently during volitional contraction. The results may explain some previous pathological data and open the possibility of testing whether these pathways are differentially recruited in a range of tasks.

Project description:AIM:This study analyzed microvesicles and exosomes, called as extracellular vesicles (EVs) excreted in serum and cerebrospinal fluid (CSF) from patients with cerebral or gestational toxoplasmosis. METHODS:Clinical samples from 83 individuals were divided into four groups. Group I, 20 sera from healthy individuals and pregnant women (seronegative for toxoplasmosis); group II, 21 sera from seropositive patients for toxoplasmosis (cerebral or gestational forms); group III, 26 CSF samples from patients with cerebral toxoplasmosis/HIV co-infection (CT/HIV) (seropositive for toxoplasmosis); and group IV, 16 CSF samples from seronegative patients for toxoplasmosis, but with HIV infection and other opportunistic infections (OI/HIV). Serum and CSF samples were ultracentrifuged to recover EVs. Next, vesicle size and concentration were characterized by Nanoparticle Tracking Analysis (NTA). RESULTS:Concentrations of serum-derived EVs from toxoplasmosis patients (mean: 2.4 x 1010 EVs/mL) were statically higher than of non-infected individuals (mean: 5.9 x 109 EVs/mL). Concentrations of CSF-derived EVs were almost similar in both groups. CT/HIV (mean: 2.9 x 109 EVs/mL) and OI/HIV (mean: 4.8 x 109 EVs/mL). Analyses by NTA confirmed that CSF-derived EVs and serum-derived EVs had size and shape similar to microvesicles and exosomes. The mean size of EVs was similar in serum and CSF. Thus, the concentration, and not size was able distinguish patients with toxoplasmosis than healthy individuals. Presence of exosomes was also confirmed by transmission electron microscopy and evidence of tetraspanins CD63 and CD9 in immunoblotting. Relative expressions of miR-146a-5p, miR-155-5p, miR-21-5p, miR-29c-3p and miR-125b-5p were estimated in exosomal miRNA extracted of EVs. Serum-derived EVs from group II (cerebral and gestational toxoplasmosis) up-expressed miR-125b-5p and miR-146a-5p. CSF-derived EVs from CT/HIV patients) up-expressed miR-155-5p and miR-21-5p and were unable to express miR-29c-3p. CONCLUSION:These data suggest the participation of EVs and exosomal miRNAs in unbalance of immune response as elevation of TNF-α, IL-6; and downregulation of IFN-γ in cerebral and gestational forms of toxoplasmosis.

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Two latent and two hyperstable polymeric forms of human neuroserpin.

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Two latent and two hyperstable polymeric forms of human neuroserpin.

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