Heparin activation of protein Z-dependent protease inhibitor (ZPI) allosterically blocks protein Z activation through an extended heparin-binding site.

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action.

Plasma levels and urinary excretion of protein Z in patients with urolithiasis.

OBJECTIVES: Protein Z (PZ) is a γ-carboxyglutamic acid protein present in plasma that is involved in blood coagulation. Detailed analysis of urinary stones from patients with urolithiasis has revealed that PZ is often found in urinary stones composed of calcium oxalate monohydrate. In this study, we compared blood and urinary PZ concentrations between healthy individuals and patients with urolithiasis. METHODS: Plasma and urine were collected from healthy individuals and patients with urolithiasis who provided informed consent. PZ was detected as a urinary stone matrix protein in some of the patients. PZ was quantified by ELISA, creatinine was measured by the enzymatic method, and the total protein concentration was measured by the Bradford method. RESULTS: The plasma PZ level was 2.54 ± 1.02 µg/mL in healthy individuals and that in urolithiasis patients classified by stone history were from 1.16 ± 0.77 to 3.73 ± 1.09 µg/mL, which was not significantly different. The urinary excretion of PZ (PZ/creatinine) was also not different in patients with urolithiasis and in healthy individuals (from 54.1 ± 40.9 to 95.4 ± 69.4 ng/mg vs. 73.3 ± 36.0 ng/mg). A positive correlation was found between the plasma PZ level and creatinine-corrected urinary PZ concentration (r = 0.46). CONCLUSIONS: Both the plasma level and urinary excretion of PZ in urolithiasis patients were not significantly different with normal individuals. PZ detected in urinary stones as a matrix protein is thought to be incorporated into urinary stones regardless of blood and urine levels of PZ.

Protein Z and Protein Z-dependent protease inhibitor in patients with acute ischemic stroke: A prospective mechanistic study.

OBJECTIVES: Protein Z (PZ) /Protein Z-dependent protease inhibitor (ZPI) (PZ/ZPI) system is a new anticoagulant system discovered in recent years, which plays an important role in many diseases. We aimed to compare the plasma PZ/ZPI levels of acute ischemic stroke (AIS) patients and non-stroke control participants and the role of PZ/ZPI in the development of stroke was preliminarily analyzed. MATERIALS AND METHODS: Enzyme linked immunosorbent assay (ELISA) was used to detect and compare plasma PZ levels of 86 patients with acute AIS and 85 non-stroke control patients. Multivariable Logistic regression was used to analyze whether PZ was an independent risk factor for AIS. RESULTS: In the present study, plasma PZ is closely related to inflammatory response, coagulation process and platelet activation, and may participate in the development of AIS by inducing inflammatory responses and interfering with the coagulation process. CONCLUSIONS: Our results suggested that plasma PZ level is one of the independent risk factors of AIS, and plasma ZPI was closely related to coagulation and platelet parameter and may play a role in the coagulation process during AIS.

Plasma concentration of protein Z-dependent protease inhibitor and ZPI exon 3 mutations in preeclampsia.

Protein Z-dependent protease inhibitor (ZPI) serves as a cofactor of inhibition of FXa and FXIa by protein Z. The levels of protein Z and polymorphisms have been shown in preeclampsia (PE) patients, but the plasma levels of ZPI and ZPI gene mutations were not reported yet. The principal aim of this study was to identify the concentration of ZPI and gene polymorphism in PE. ZPI levels were determined in 113 PE patients (age: 29.9 ± 3.9 years) and in 106 controls (normal pregnancy, age: 27.0 ± 2.8 years). ZPI was measured by enzyme-linked immunosorbent assay kit, and the gene polymorphism was determined by polymerase chain reaction and sequencing. The results showed ZPI antigen was found to be significantly lower in PE patients than in controls (ZPI, 1.24 ± 0.29 mg/L vs. 1.94 ± 0.35 mg/L, p < 0.05). The exon-3 missense mutations were distributed in patients and controls and there was no convincing correlation between these mutations and PE. It was of interest to observe a close relationship between the genotypes of the exon 3 polymorphisms 181 A > G and 481 A > T in the ZPI gene.Impact statementWhat is already known on this subject? The occurrence of PE is closely related to dysfunction of coagulation, and it is known that the decrease of PZ level can increase the occurrence probability of PE, while the polymorphism of PZ is not related to the occurrence of PE. As a cofactor of PZ, the content and polymorphism of ZPI which related to the occurrence of PE is worth further study.What the results of this study add? ZPI antigen was found to be significantly lower in PE patients than in controls, but there was no convincing correlation between exon-3 mutations and PE.What the implications are of these findings for clinical practice and/or further research? Our results support the view that ZPI plays a significant role in anticoagulant, and the genotype of the 181 gene polymorphism in exon-3 and 481 gene polymorphism in exon-3 are closely related. Other mutations like 435T > G(Phe145Leu), 972G > A(Trp324X), 1151A > G(Gln384Arg) are necessary to confirm the association between ZPI and prothrombotic state including PE.

Protein Z-dependent protease inhibitor (ZPI) is a physiologically significant inhibitor of prothrombinase function.

Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation. Here we provide evidence that, contrary to this view, the FXa-specific serpin inhibitor, protein Z-dependent protease inhibitor (ZPI), complexed with its cofactor, protein Z (PZ), functions as a physiologically significant inhibitor of prothrombinase-bound FXa during prothrombin activation. Kinetics studies showed that the rapid rate of inhibition of FXa by the ZPI-PZ complex on procoagulant membrane vesicles (ka(app) ∼107 m-1 s-1) was decreased ∼10-fold when FXa was bound to FVa in prothrombinase and a further ∼3-4-fold when plasma levels of S195A prothrombin were present (ka(app) 2 × 105 m-1 s-1). Nevertheless, the ZPI-PZ complex produced a major inhibition of thrombin generation during prothrombinase-catalyzed activation of prothrombin under physiologically relevant conditions. The importance of ZPI-PZ complex anticoagulant regulation of FXa both before and after incorporation into prothrombinase was supported by thrombin generation assays in plasma. These showed enhanced thrombin generation when the inhibitor was neutralized with a PZ-specific antibody and decreased thrombin generation when exogenous ZPI-PZ complex was added whether prothrombin was activated directly by FXa or through extrinsic or intrinsic pathway activators. Moreover, the PZ antibody enhanced thrombin generation both in the absence and presence of activated protein C (APC) anticoagulant activity. Taken together, these results suggest an important anticoagulant role for the ZPI-PZ complex in regulating both free FXa generated in the initiation phase of coagulation as well as prothrombinase-bound FXa in the propagation phase that complement prothrombinase regulation by APC.

A Proteomics Survey of Junín Virus Interactions with Human Proteins Reveals Host Factors Required for Arenavirus Replication.

Arenaviruses are negative-strand, enveloped RNA viruses that cause significant human disease. In particular, Junín mammarenavirus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. At present, little is known about the cellular proteins that the arenavirus matrix protein (Z) hijacks to accomplish its various functions, including driving the process of virus release. Furthermore, there is little knowledge regarding host proteins incorporated into arenavirus particles and their importance for virion function. To address these deficiencies, we used mass spectrometry to identify human proteins that (i) interact with the JUNV matrix protein inside cells or within virus-like particles (VLPs) and/or (ii) are incorporated into bona fide JUNV strain Candid#1 particles. Bioinformatics analyses revealed that multiple classes of human proteins were overrepresented in the data sets, including ribosomal proteins, Ras superfamily proteins, and endosomal sorting complex required for transport (ESCRT) proteins. Several of these proteins were required for the propagation of JUNV (ADP ribosylation factor 1 [ARF1], ATPase, H+ transporting, lysosomal 38-kDa, V0 subunit d1 [ATP6V0D1], and peroxiredoxin 3 [PRDX3]), lymphocytic choriomeningitis mammarenavirus (LCMV) (Rab5c), or both viruses (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide [ATP5B] and IMP dehydrogenase 2 [IMPDH2]). Furthermore, we show that the release of infectious JUNV particles, but not LCMV particles, requires a functional ESCRT pathway and that ATP5B and IMPDH2 are required for JUNV budding. In summary, we have provided a large-scale map of host machinery that associates with JUNV and identified key human proteins required for its propagation. This data set provides a resource for the field to guide antiviral target discovery and to better understand the biology of the arenavirus matrix protein and the importance of host proteins for virion function.IMPORTANCE Arenaviruses are deadly human pathogens for which there are no U.S. Food and Drug Administration-approved vaccines and only limited treatment options. Little is known about the host proteins that are incorporated into arenavirus particles or that associate with its multifunctional matrix protein. Using Junín mammarenavirus (JUNV), the causative agent of Argentine hemorrhagic fever, as a model organism, we mapped the human proteins that are incorporated into JUNV particles or that associate with the JUNV matrix protein. Functional analysis revealed host machinery that is required for JUNV propagation, including the cellular ESCRT pathway. This study improves our understanding of critical arenavirus-host interactions and provides a data set that will guide future studies to better understand arenavirus pathogenesis and identify novel host proteins that can be therapeutically targeted.

The use of novel epitope-tagged arenaviruses reveals that Rab5c-positive endosomal membranes are targeted by the LCMV matrix protein.

We report the development of recombinant New World (Junín; JUNV) and Old World (lymphocytic choriomeningitis virus; LCMV) mammarenaviruses that encode an HA-tagged matrix protein (Z). These viruses permit the robust affinity purification of Z from infected cells or virions, as well as the detection of Z by immunofluorescent microscopy. Importantly, the HA-tagged viruses grow with wild-type kinetics in a multi-cycle growth assay. Using these viruses, we report a novel description of JUNV Z localization in infected cells, as well as the first description of colocalization between LCMV Z and the GTPase Rab5c. This latter result, when combined with our previous findings that LCMV genome and glycoprotein also colocalize with Rab5c, suggest that LCMV may target Rab5c-positive membranes for preassembly of virus particles prior to budding. The recombinant viruses reported here will provide the field with new tools to better study Z protein functionality and identify key Z protein interactions with host machinery.

Low plasma protein Z levels are associated with an increased risk for perioperative bleedings.

BACKGROUND AND OBJECTIVES: Protein Z (PZ) deficiency has been implicated both in bleeding diatheses and in thrombophilia. Considering its ambiguous nature and the conflicting clinical data so far, we set out to evaluate the impact of low PZ on perioperative bleeding in patients who underwent surgical (ENT) interventions involving a high risk of bleeding. PATIENTS AND METHODS: After exclusion of other coagulation disorders, 154 Patients were stratified into quartiles according to PZ plasma concentrations to evaluate the relation between PZ and bleeding complications. RESULTS: Low PZ levels were associated with increased blood loss (P < .001), increased need for blood transfusions (P < .001), and a higher rate of surgical revisions (P = .009) in a concentration-dependent fashion. Low PZ caused earlier (within 24 hours) and repetitive bleedings (P = .005). The number of major bleeding episodes was significantly increased when low PZ was combined with bleeding history (P < .05). Finally, ROC analyses confirmed the predictive value of low PZ for bleeding complications and PZ-thresholds for clinical practice were determined. CONCLUSIONS: Low PZ appears to be an underestimated risk factor for perioperative bleeding. Determination of PZ plasma concentrations might be useful in the preoperative workup in patients with a bleeding history, when detailed clotting analyses remain inconclusive.

Protein Z and Endothelin-1 genetic polymorphisms in pediatric Egyptian sickle cell disease patients.

BACKGROUND: Sickle cell disease (SCD) is a monogenic disease associated with multisystem morbidity. Vasculopathy caused by delicate imbalance between coagulation and endothelial systems plays a pivotal role in disease course. As Protein Z and Endothelin-1 genetic polymorphisms may increase the thrombotic risk, the aim of the current work was to verify the possible impact of Protein Z (PROZ G79A) and Endothelin-1 (EDN1 G5665T) polymorphisms on the clinic-laboratory features of the SCD in a cohort of Egyptian pediatric patients. METHODS: Genotyping of Protein Z G79A and Endothelin-1 G5665T was carried out by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) assay for 100 SCD patients and 100 controls. RESULTS: Protein -Z G79A polymorphism was not associated with vascular complications in the studied SCD patients. Endothelin-1 G5665T polymorphism was associated with pulmonary dysfunction (pulmonary artery hypertension and acute chest syndrome) and severe vaso-occlusive crises (VOC). CONCLUSION: Endothelin-1 G5665T polymorphism could be considered as a molecular predictor for pulmonary dysfunction and severe VOC in SCD. Further researches with larger cohorts are recommended to understand the pathophysiology of SCD and to explain the inter-patients' variability of disease severity.

Thermodynamic and kinetic characterization of the protein Z-dependent protease inhibitor (ZPI)-protein Z interaction reveals an unexpected role for ZPI Lys-239.

The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), circulates in blood as a tight complex with its cofactor, protein Z (PZ), enabling it to function as a rapid inhibitor of membrane-associated factor Xa. Here, we show that N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled K239C ZPI is a sensitive, moderately perturbing reporter of the ZPI-PZ interaction and utilize the labeled ZPI to characterize in-depth the thermodynamics and kinetics of wild-type and variant ZPI-PZ interactions. NBD-labeled K239C ZPI bound PZ with ∼3 nM KD and ∼400% fluorescence enhancement at physiologic pH and ionic strength. The NBD-ZPI-PZ interaction was markedly sensitive to ionic strength and pH but minimally affected by temperature, consistent with the importance of charged interactions. NBD-ZPI-PZ affinity was reduced ∼5-fold by physiologic calcium levels to resemble NBD-ZPI affinity for γ-carboxyglutamic acid/EGF1-domainless PZ. Competitive binding studies with ZPI variants revealed that in addition to previously identified Asp-293 and Tyr-240 hot spot residues, Met-71, Asp-74, and Asp-238 made significant contributions to PZ binding, whereas Lys-239 antagonized binding. Rapid kinetic studies indicated a multistep binding mechanism with diffusion-limited association and slow complex dissociation. ZPI complexation with factor Xa or cleavage decreased ZPI-PZ affinity 2-7-fold by increasing the rate of PZ dissociation. A catalytic role for PZ was supported by the correlation between a decreased rate of PZ dissociation from the K239A ZPI-PZ complex and an impaired ability of PZ to catalyze the K239A ZPI-factor Xa reaction. Together, these results reveal the energetic basis of the ZPI-PZ interaction and suggest an important role for ZPI Lys-239 in PZ catalytic action.

Postpartum Ischemic Stroke in Moyamoya Disease Associated with Protein Z Deficiency-A Case Report.

The authors describe herein the first adult case with moyamoya disease associated with protein Z deficiency. A 41-year-old woman was admitted to our hospital due to sudden onset of dysarthria and left extremity weakness 6 days after the delivery of her first baby. Previously, she repeated early fetal losses and was diagnosed with protein Z deficiency. Laboratory examination showed that the plasma concentration of protein Z was .73 µg/ml, being lower than the control. Radiological examination demonstrated typical findings of moyamoya disease with advanced stage on both sides. She successfully underwent surgical revascularization on both sides and was free from any cerebrovascular events during a follow-up period of 2 years. In addition to hemodynamic compromise, protein Z deficiency and hypercoagulation state after delivery might cause ischemic stroke in this case.

Characterization of the protein Z-dependent protease inhibitor interactive-sites of protein Z.

BACKGROUND: Protein Z (PZ) has been reported to promote the inactivation of factor Xa (FXa) by PZ-dependent protease inhibitor (ZPI) by about three orders of magnitude. Previously, we prepared a chimeric PZ in which its C-terminal pseudo-catalytic domain was grafted on FX light-chain (Gla and EGF-like domains) (PZ/FX-LC). Characterization of PZ/FX-LC revealed that the ZPI interactive-site is primarily located within PZ pseudo-catalytic domain. Nevertheless, the cofactor function and apparent Kd of PZ/FX-LC for interaction with ZPI remained impaired ~6-7-fold, suggesting that PZ contains a ZPI interactive-site outside pseudo-catalytic domain. X-ray structural data indicates that Tyr-240 of ZPI interacts with EGF2-domain of PZ. Structural data further suggests that 3 other ZPI surface loops make salt-bridge interactions with PZ pseudo-catalytic domain. To identify ZPI interactive-sites on PZ, we grafted the N-terminal EGF2 subdomain of PZ onto PZ/FX-LC chimera (PZ-EGF2/FX-LC) and also generated two compensatory charge reversal mutants of PZ pseudo-catalytic domain (Glu-244 and Arg-212) and ZPI surface loops (Lys-239 and Asp-293). METHODS: PZ chimeras were expressed in mammalian cells and ZPI derivatives were expressed in Escherichia coli. RESULTS: The PZ EGF2 subdomain fusion restored the defective cofactor function of PZ/FX-LC. The activities of PZ and ZPI mutants were all impaired if assayed individually, but partially restored if the compensatory charge reversal mutants were used in the assay. CONCLUSIONS: PZ EGF2 subdomain constitutes an interactive-site for ZPI. Data with compensatory charge reversal mutants validates structural data that the identified residues are part of interactive-sites. GENERAL SIGNIFICANCE: Insight is provided into mechanisms through which specificity of ZPI-PZ-FXa complex formation is determined.

Prominent protein Z-induced thrombin inhibition in cirrhosis: a new functional assay for hypercoagulability assessment.

BACKGROUND AND AIMS: Protein Z (PZ) is an anticoagulant that accelerates the inhibitory effect of PZ-dependent protease inhibitor (ZPI) on coagulation factor Xa. We assessed functional status of PZ system in 158 patients with liver cirrhosis and 59 healthy controls. METHODS: Plasma PZ and ZPI levels were measured by enzyme immunoassay. Thrombin generation assays (TGA) were performed with and without thrombomodulin (TM) or PZ, and the ratios were calculated by dividing TGA values with TM or PZ by values without TM or PZ. RESULTS: PZ and ZPI levels were reduced and elevated in advanced cirrhosis, respectively. The lag time ratio-PZ was significantly higher in cirrhosis patients than controls and correlated with the model for end-stage liver disease score. The peak thrombin ratio-PZ and endogenous thrombin potential (ETP) ratio-PZ were significantly lower in cirrhosis patients than controls and correlated with the severity of liver cirrhosis. The peak thrombin ratio-PZ was dramatically reduced in advanced cirrhosis. Cirrhosis patients had a significantly higher ETP ratio-TM than the controls, although the ratio was not correlated with cirrhosis severity. The lag time ratio-PZ and peak time ratio-PZ were significantly correlated with the levels of all coagulation and anticoagulation factors. Interestingly, the lag time ratio-PZ and peak thrombin ratio-PZ were significantly associated with thrombotic events. CONCLUSION: The anticoagulant role of PZ is insufficient in advanced stages of cirrhosis. Our newly developed functional assay for measuring the PZ system is expected to reflect the ongoing hypercoagulability of cirrhosis.

Residues of the 39-loop restrict the plasma inhibitor specificity of factor IXa.

The two plasma inhibitors, protein Z-dependent protease inhibitor (ZPI) and tissue factor pathway inhibitor (TFPI), effectively inhibit the activity of activated factor X (FXa); however, neither inhibitor exhibits any reactivity with the homologous protease activated factor IX (FIXa). In this study, we investigated the molecular basis for the lack of reactivity of FIXa with these plasma inhibitors and discovered that unique structural features within residues of the 39-loop are responsible for restricting the inhibitor specificity of FIXa. This loop in FXa is highly acidic and contains three Glu residues at positions 36, 37, and 39. On the other hand, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. We discovered that replacing residues of the 39-loop (residues 31-41) of FIXa with corresponding residues of FXa renders the FIXa chimera susceptible to inactivation by both ZPI and TFPI. Thus, the inactivation rate of the FIXa chimera by ZPI in the presence of protein Z (PZ), negatively charged membrane vesicles, and calcium ions approached the same diffusion-limited rate (>10(7) m(-1) s(-1)) that has been observed for the PZ-dependent inhibition of FXa by ZPI. Interestingly, sequence alignments indicated that, similar to FXa, residue 36 is a Glu in both mouse and bovine FIXa and that both proteases are also susceptible to inhibition by the PZ-ZPI complex. These results suggest that structural features within residues of the 39-loop contribute to the resistance of FIXa to inhibition by plasma inhibitors ZPI and TFPI.

C-terminal binding proteins are essential pro-survival factors that undergo caspase-dependent downregulation during neuronal apoptosis.

C-terminal binding proteins (CtBPs) are transcriptional co-repressors that are subject to proteasome-dependent downregulation during apoptosis. Alternative mechanisms that regulate CtBP expression are currently under investigation and the role of CtBPs in neuronal survival is largely unexplored. Here, we show that CtBPs are downregulated in cerebellar granule neurons (CGNs) induced to undergo apoptosis by a variety of stressors. Moreover, antisense-mediated downregulation of CtBP1 is sufficient to cause CGN apoptosis. Similarly, the CtBP inhibitor, 4-methylthio-2-oxobutyric acid, induces expression of the CtBP target Noxa and causes actinomycin-sensitive CGN apoptosis. Unexpectedly, we found that the mechanism of CtBP downregulation in CGNs undergoing apoptosis varies in a stimulus-specific manner involving either the proteasome or caspases. In the case of CGNs deprived of depolarizing potassium (5K apoptotic condition), caspases appear to play a dominant role in CtBP downregulation. However, incubation in 5K does not enhance the kinetics of CtBP1 degradation and recombinant CtBP1 is not cleaved in vitro by caspase-3. In addition, 5K has no significant effect on CtBP transcript expression. Finally, mouse embryonic stem cells display caspase-dependent downregulation of CtBP1 following exposure to staurosporine, an effect that is not observed in DGCR8 knockout cells which are deficient in miRNA processing. These data identify caspase-dependent downregulation of CtBPs as an alternative mechanism to the proteasome for regulation of these transcriptional co-repressors in neurons undergoing apoptosis. Moreover, caspases appear to regulate CtBP expression indirectly, at a post-transcriptional level, and via a mechanism that is dependent upon miRNA processing. We conclude that CtBPs are essential pro-survival proteins in neurons and their downregulation contributes significantly to neuronal apoptosis via the de-repression of pro-apoptotic genes.

Chicoric acid inserted in protein Z cavity exhibits higher stability and better wound healing effect under oxidative stress.

Oxidative stress is one of the limiting factors that inhibit wound healing. Phytochemicals especially chicoric acid have the potential to act as an antioxidant and scavenge reactive oxygen species, thereby promoting wound healing. However, most of the phytochemicals were easy to be degraded during storage or using due to the oxidative status in wound site. Herein, we introduce a high stable protein Z that can encapsulate chicoric acid during foaming. TEM results showed that the size of protein Z-chicoric acid is in the range of nanoscale (named PZ-CA nanocomposite), and protein Z encapsulation can significantly improve the stability of chicoric acid under oxidative stress. Moreover, PZ-CA nanocomposite exhibited favorable antioxidant properties, biocompatibility, and the ability to promote cell migration in vitro. The role of PZ-CA nanocomposite in skin regeneration was explored by a mice model. Results in vivo suggest that the PZ-CA nanocomposite promotes wound healing with a faster rate as compared with a commercial spray solution, mostly through attenuating the oxidative stress, promoting cell proliferation and collagen deposition. This work not only provides a delivery vector for bioactive molecules, but also develops a kind of nanocomposite with the property of promoting wound healing.

Comparison of recombinant protein Z with natural protein Z derived from malt: From structure to functional properties.

Protein Z (PZ) is a prominent albumin found in the endosperm of barley seeds with a molecular weight of approximately 40 kDa. Its multifaceted functional attributes, including trypsin- and thrombin-inhibiting bioactivities and superior foaming properties, have garnered significant attention in research. Considering the post-translational modifications of PZ natural in barley malt, we tried to express recombinant protein Z (rPZ) in E. coli. The present study aims to undertake a comparative analysis between natural PZ and rPZ in order to elucidate their respective characteristics. After spectral analysis, there are significant differences in their secondary and tertiary structures. In addition, rPZ showed superior foamability and foam stability. As for the serpin-like activity, the inhibition rate of rPZ is much higher than that of PZ. In contrast with the inhibition activity, the digestability of rPZ is much lower than that of PZ. As for the cargo carrier properties, rPZ showed an excellent ability to stabilize astaxanthin at 37 °C. These results suggest that rPZ is more suitable as protein carrier, due to the high foamability, serpin-like activity and low digestive stability, which not only give a brief view of recombinant protein, but also give a direction for PZ in cargo delivery.

Amniotic Fluid Proteomics Analysis and In Vitro Validation to Identify Potential Biomarkers of Preterm Birth.

This study aimed to investigate the regulation of amniotic fibroblast (AFC) function by vitamin K-dependent protein Z (PROZ) during preterm birth (PTB) and its potential role in adverse pregnancy outcomes. Proteomic samples were collected from amniotic fluid in the second trimester, and AFC were isolated from the amniotic membrane and cultured in vitro. The expression of extracellular and intracellular PROZ in AFC was modulated, and their biological properties and functions were evaluated. Clinical analysis revealed a significant upregulation of PROZ expression in amniotic fluid from preterm pregnant women. In vitro experiments demonstrated that PROZ stimulated AFC migration, enhanced their proliferative capacity, and reduced collagen secretion. Overexpression of PROZ further enhanced cell migration and proliferation, while knockdown of PROZ had the opposite effect. PROZ plays a crucial role in promoting the proliferation and migration of amniotic membrane fibroblasts. Increased PROZ expression in amniotic fluid is associated with the occurrence of PTB. These findings shed light on the potential involvement of PROZ in adverse pregnancy outcomes and provide a basis for further research on its regulatory mechanisms during PTB.

Binding of curcumin to barley protein Z improves its solubility, stability and bioavailability.

Although excessive pharmaceutical activities of curcumin have been reported, the poor solubility, low stability and low bioavailability greatly limited its application. In this study, the interaction between protein Z (PZ) and curcumin, and the effects of PZ on the stability and bioavailability of curcumin were investigated. Fluorescence quenching results indicated that curcumin molecule binds PZ with a stoichiometry of 4:1, and the binding affinity is stronger than other reported protein carriers. Molecular dynamics simulation results suggested that curcumin binds in the hydrophobic region of PZ, and the interaction was maintained mainly by hydrogen-bond (Pro-287, Asn-340 and Tyr-234). PZ-curcumin complex possessed better encapsulation efficiency (64.10 %) and loading capacity (5.49 µg/mg) for curcumin. In addition, binding with PZ not only improved the thermal, light and digestive stability of curcumin significantly, but lowered its toxic effect on Caco-2 cells and improved relative bioavailability (305 %) compared with that of curcumin only.