Effect of Calcium Supplementation and TMEM16A Inhibition on Endoplasmic Reticulum Stress Induced by Dental Fluorosis in Mice.

BACKGROUND: Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis. METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis. RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium. CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.

Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the integrated stress response.

Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2αP) terminates signaling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a protein phosphatase 1 (PP1) catalytic subunit, the conserved C-terminally located ~70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential cofactor) efficiently dephosphorylate eIF2αP in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-ray crystallography, and biochemistry to uncover binding of the γ subunit of eIF2 to a short helical peptide repeated four times in the functionally important N terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an α-helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2γ in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2αP by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals.

Comprehensive Identification of Chemical Fingerprint and Screening of Potential Quality Markers of Aloe vera (L.) Burm. f. from Different Geographical Origins via Ultra-High-Performance Liquid Chromatography Hyphenated with Quadrupole-Orbitrap-High-Resolution Mass Spectrometry Combined with Chemometrics.

An integrated strategy was developed for the systematic chemical fingerprint and chemometrics analysis for the quality assessment of Aloe vera (L.) Burm. f. The ultra-performance liquid chromatography fingerprint was established, and all common peaks were tentatively identified by using ultra-high-performance liquid chromatography hyphenated with quadrupole-orbitrap-high-resolution mass spectrometry. Afterwards, the datasets of common peaks were subjected to hierarchical cluster analysis, principal component analysis and partial least squares discriminant analysis to holistically compare the differences. The results revealed that the samples were predicted to fall into four clusters, which were related to four different geographical locations. Using the proposed strategy, aloesin, aloin A, aloin B, aloeresin D and 7-O-methylaloeresin A were rapidly determined to be the potential characteristic quality markers. Finally, five screened compounds in 20 batches of samples were simultaneously quantified, and their total contents were ranked as follows: Sichuan province > Hainan province > Guangdong province > Guangxi province, which suggests that geographical origins may be an important factor affecting the quality of A. vera (L.) Burm. f. This new strategy can not only be used to explore possibly the latent active substance candidates for pharmacodynamic studies, but it is also an efficient analytical strategy for other complex traditional Chinese medicine systems.

Comprehensive chemical profile and quantitative analysis of the Shabyar tablet, a traditional ethnic medicine prescription, by ultra-high-performance liquid chromatography with quadrupole-orbitrap high-resolution mass spectrometry.

The Shabyar tablet is commonly used as a traditional ethnic medicine prescription for the treatment of night blindness, poor vision, and headaches. However, the chemical components of the Shabyar tablet have not been holistically explored, which seriously hinders the discovery of the activity. This study qualitatively and quantitatively investigated the overall chemical profile of the Shabyar tablet using ultra-high-performance liquid chromatography hyphenated with quadrupole-orbitrap high-resolution mass spectrometry. Altogether, 170 chemical components, including 59 flavonoids, 78 organic acids, 12 anthranones, three anthraquinones, one naphthalene, and 17 other compounds were tentatively identified and attributed, with 40 among these being unambiguously characterized in comparison with their corresponding authentic standards. To further determine the major representative constituents of the Shabyar tablet, a quantitative method was used for the simultaneous analysis of 33 characteristic components in Shabyar samples. The results were validated in terms of linearity, precision, repeatability, stability, and recovery. This newly developed approach could be successfully employed for evaluating the holistic quality of crude extracts and Chinese medicines in the Shabyar compound tablet and provide a solid chemical foundation for additional investigations on in vivo pharmacodynamics and therapeutic mechanisms to identify the potential effective components of traditional medicines.

CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

Higher-order phosphatase-substrate contacts terminate the integrated stress response.

Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally affecting flexible access of the phosphopeptide to the active site. However, catalytic efficiency of holophosphatases towards their phosphoprotein substrates remains unexplained. Here we present a cryo-EM structure of the tripartite PP1c-PPP1R15A-G-actin holophosphatase that terminates signaling in the mammalian integrated stress response (ISR) in the pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin, whose essential role in eIF2α dephosphorylation is supported crystallographically, biochemically and genetically, aligns the catalytic and regulatory subunits, creating a composite surface that engages the N-terminal domain of eIF2α to position the distant phosphoserine-51 at the active site. Substrate residues that mediate affinity for the holophosphatase also make critical contacts with eIF2α kinases. Thus, a convergent process of higher-order substrate recognition specifies functionally antagonistic phosphorylation and dephosphorylation in the ISR.

Calcium depletion challenges endoplasmic reticulum proteostasis by destabilising BiP-substrate complexes.

The metazoan endoplasmic reticulum (ER) serves both as a hub for maturation of secreted proteins and as an intracellular calcium storage compartment, facilitating calcium-release-dependent cellular processes. ER calcium depletion robustly activates the unfolded protein response (UPR). However, it is unclear how fluctuations in ER calcium impact organellar proteostasis. Here, we report that calcium selectively affects the dynamics of the abundant metazoan ER Hsp70 chaperone BiP, by enhancing its affinity for ADP. In the calcium-replete ER, ADP rebinding to post-ATP hydrolysis BiP-substrate complexes competes with ATP binding during both spontaneous and co-chaperone-assisted nucleotide exchange, favouring substrate retention. Conversely, in the calcium-depleted ER, relative acceleration of ADP-to-ATP exchange favours substrate release. These findings explain the rapid dissociation of certain substrates from BiP observed in the calcium-depleted ER and suggest a mechanism for tuning ER quality control and coupling UPR activity to signals that mobilise ER calcium in secretory cells.

Overexpression of p62 Induces Autophagy and Promotes Proliferation, Migration and Invasion of Nasopharyngeal Carcinoma Cells through Promoting ERK Signaling Pathway.

BACKGROUND: Increasing evidence has shown that p62 plays an important role in tumorigenesis. However, relatively little is known about the association between p62 and tumor invasion and metastasis; in addition, its role in NPC (nasopharyngeal carcinoma, NPC) has been rarely investigated. OBJECTIVE: To investigate the effect of p62 on tumorigenesis and metastasis in nasopharyngeal carcinoma. METHODS: Western blotting, immunofluorescent staining and immunohistochemistry were used to evaluate p62 protein expression. Subsequently, cell viability, colony formation, migration, invasion and autophagy assays were performed. anti-p62 autoantibodies in sera were detected by ELISA. These data were correlated with clinicopathological parameters. RESULTS: We confirmed that p62 was significantly up-regulated in NPC tissues. Furthermore, high expression of p62 was observed in NPC cell lines, and especially in the highly metastatic 5-8F cells. In vitro, down-regulation of p62 inhibited proliferation, clone forming ability, autophagy, migration, and invasion in 5-8F cells, whereas p62 overexpression resulted in the opposite effects in 6-10B cells. Moreover, we confirmed that p62 promotes NPC cell proliferation, migration, and invasion by activating ERK (extracellular signal-regulated kinase, ERK). Clinical analysis indicated that high p62 expression correlates with lymph node and distant metastasis (P<0.05). Serum anti-p62 autoantibodies were increased in NPC patients and levels were associated with metastasis. CONCLUSION: Our data establish p62 targeting ERK as potential determinant in the NPC, which supplies a new pathway to treat NPC. Furthermore, p62 is a potential biomarker which might be closely related to the tumorigenesis and metastasis in NPC.

Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR.

Coupling of endoplasmic reticulum (ER) stress to dimerisation-dependent activation of the UPR transducer IRE1 is incompletely understood. Whilst the luminal co-chaperone ERdj4 promotes a complex between the Hsp70 BiP and IRE1's stress-sensing luminal domain (IRE1LD) that favours the latter's monomeric inactive state and loss of ERdj4 de-represses IRE1, evidence linking these cellular and in vitro observations is presently lacking. We report that enforced loading of endogenous BiP onto endogenous IRE1α repressed UPR signalling in CHO cells and deletions in the IRE1α locus that de-repressed the UPR in cells, encode flexible regions of IRE1LD that mediated BiP-induced monomerisation in vitro. Changes in the hydrogen exchange mass spectrometry profile of IRE1LD induced by ERdj4 and BiP confirmed monomerisation and were consistent with active destabilisation of the IRE1LD dimer. Together, these observations support a competition model whereby waning ER stress passively partitions ERdj4 and BiP to IRE1LD to initiate active repression of UPR signalling.

An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP.

AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide-binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.

Potential of the integration of coagulation and ozonation as a pretreatment of reverse osmosis concentrate from coal gasification wastewater reclamation.

Combination of coagulation and ozonation was used to treat brine derived from a three-stage reverse osmosis (RO) process during coal gasification wastewater reclamation. Effects of operating parameters on the removals of total organic carbon (TOC), color and UV absorbance at 254 nm (A254) were investigated during coagulation and ozonation. All the removal efficiencies of TOC, A254 and color of FeCl3 coagulant are about twice those of AlCl3 coagulant at the same molar dose since almost all the molecular weight fractions of RO concentrate (ROC) could be removed effectively by FeCl3 coagulant while only the fractions of molecular weight > 3 k Da could be removed effectively by AlCl3 coagulant. The TOC removal increases with the increasing of ozone dose and reaction temperature during ozonation of ROC after coagulation pretreatment. TOC and color of ROC after pretreated by coagulation could be further removed effectively during ozonation since ozonation can significant reduce the fluorescence response of all the fractions of effluent organic matter in ROC. It is unexpectedly found that the increase of A254 is observed after ozonation, this is because the intensity of absorbance at 254 nm by the low molecular weight transformation products (<2 k Da) increases significantly with the reaction time after 30 min. The coagulation coupling with ozonation is efficient in the removals of both TOC and color of ROC.

MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP.

Despite its known role as a secreted neuroprotectant, much of the mesencephalic astrocyte-derived neurotrophic factor (MANF) is retained in the endoplasmic reticulum (ER) of producer cells. There, by unknown mechanisms, MANF plays a role in protein folding homeostasis in complex with the ER-localized Hsp70 chaperone BiP. Here we report that the SAF-A/B, Acinus, and PIAS (SAP) domain of MANF selectively associates with the nucleotide binding domain (NBD) of ADP-bound BiP. In crystal structures the SAP domain engages the cleft between NBD subdomains Ia and IIa, stabilizing the ADP-bound conformation and clashing with the interdomain linker that occupies this site in ATP-bound BiP. MANF inhibits both ADP release from BiP and ATP binding to BiP, and thereby client release. Cells lacking MANF have fewer ER stress-induced BiP-containing high molecular weight complexes. These findings suggest that MANF contributes to protein folding homeostasis as a nucleotide exchange inhibitor that stabilizes certain BiP-client complexes.

Measurement of the total angiotensinogen and its reduced and oxidised forms in human plasma using targeted LC-MS/MS.

Angiotensinogen (AGT) is a critical protein in the renin-angiotensin-aldosterone system and may have an important role in the pathogenesis of pre-eclampsia. The disulphide linkage between cysteines 18 and 138 has a key role in the redox switch of AGT which modulates the release of angiotensin I with consequential effects on blood pressure. In this paper, we report a quantitative targeted LC-MS/MS method for the reliable measurement of the total AGT and its reduced and oxidised forms in human plasma. AGT was selectively enriched from human plasma using two-dimensional chromatography employing concanavalin A lectin affinity and reversed phase steps and then deglycosylated using PNGase F. A differential alkylation approach was coupled with targeted LC-MS/MS method to identify the two AGT forms in the plasma chymotryptic digest. An additional AGT proteolytic marker peptide was identified and used to measure total AGT levels. The developed MS workflow enabled the reproducible detection of total AGT and its two distinct forms in human plasma with analytical precision of ≤ 15%. The LC-MS/MS assay for total AGT in plasma showed a linear response (R2 = 0.992) with a limit of quantification in the low nanomolar range. The method gave suitable validation characteristics for biomedical application to the quantification of the oxidation level and the total level of AGT in plasma samples collected from normal and pre-eclamptic patients.

Structural basis for the specificity of renin-mediated angiotensinogen cleavage.

The renin-angiotensin cascade is a hormone system that regulates blood pressure and fluid balance. Renin-mediated cleavage of the angiotensin I peptide from the N terminus of angiotensinogen (AGT) is the rate-limiting step of this cascade; however, the detailed molecular mechanism underlying this step is unclear. Here, we solved the crystal structures of glycosylated human AGT (2.30 Å resolution), its encounter complex with renin (2.55 Å), AGT cleaved in its reactive center loop (RCL; 2.97 Å), and spent AGT from which the N-terminal angiotensin peptide was removed (2.63 Å). These structures revealed that AGT undergoes profound conformational changes and binds renin through a tail-into-mouth allosteric mechanism that inserts the N terminus into a pocket equivalent to a hormone-binding site on other serpins. These changes fully extended the N-terminal tail, with the scissile bond for angiotensin release docked in renin's active site. Insertion of the N terminus into this pocket accompanied a complete unwinding of helix H of AGT, which, in turn, formed key interactions with renin in the complementary binding interface. Mutagenesis and kinetic analyses confirmed that renin-mediated production of angiotensin I is controlled by interactions of amino acid residues and glycan components outside renin's active-site cleft. Our findings indicate that AGT adapts unique serpin features for hormone delivery and binds renin through concerted movements in the N-terminal tail and in its main body to modulate angiotensin release. These insights provide a structural basis for the development of agents that attenuate angiotensin release by targeting AGT's hormone binding pocket.

iTRAQ-based quantitative proteomic analysis of differentially expressed proteins in chemoresistant nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a highly prevalent disease in Southeast Asia. The disease is typically diagnosed in the later stages, and chemotherapy resistance often causes treatment failure. To investigate the underlying mechanisms of drug resistance, we searched for chemoresistant-associated proteins in NPC and drug-resistant NPC cell lines using isobaric tags for relative and absolute quantitation combined with nano liquid chromatography-tandem mass spectrometry. The chemoresistant NPC cell lines CNE1DDP and CNE2DDP were resistant to 1 mg/L cisplatin, had resistant indexes of 4.58 and 2.63, respectively, and clearly grew more slowly than the NPC cell lines CNE1 and CNE2. Using three technical replicates, we identified 690 nonredundant proteins, 56 of which were differentially expressed in both groups of cell lines (CNE1 vs. CNE1DDP and CNE2 vs. CNE2DDP). Gene Ontology, KEGG pathway, and miRNA analyses and protein-protein interactions of differentially expressed proteins showed that proteins TRIM29, HSPB1, CLIC1, ANXA1, and STMN1, among others, may play a role in the mechanisms of chemoresistance in clinical therapy. The chemotherapy-resistant proteomic profiles obtained may allow the identification of novel biomarkers for early detection of chemoresistance in NPC and other cancers.

AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP contributes to protein folding homeostasis by engaging unfolded client proteins in a process that is tightly coupled to ATP binding and hydrolysis. The inverse correlation between BiP AMPylation and the burden of unfolded ER proteins suggests a post-translational mechanism for adjusting BiP's activity to changing levels of ER stress, but the underlying molecular details are unexplored. We present biochemical and crystallographic studies indicating that irrespective of the identity of the bound nucleotide AMPylation biases BiP towards a conformation normally attained by the ATP-bound chaperone. AMPylation does not affect the interaction between BiP and J-protein co-factors but appears to allosterically impair J protein-stimulated ATP-hydrolysis, resulting in the inability of modified BiP to attain high affinity for its substrates. These findings suggest a molecular mechanism by which AMPylation serves as a switch to inactivate BiP, limiting its interactions with substrates whilst conserving ATP.

PDLIM5 links kidney anion exchanger 1 (kAE1) to ILK and is required for membrane targeting of kAE1.

Anion exchanger 1 (AE1) mediates Cl-/HCO3- exchange in erythrocytes and kidney intercalated cells where it functions to maintain normal bodily acid-base homeostasis. AE1's C-terminal tail (AE1C) contains multiple potential membrane targeting/retention determinants, including a predicted PDZ binding motif, which are critical for its normal membrane residency. Here we identify PDLIM5 as a direct binding partner for AE1 in human kidney, via PDLIM5's PDZ domain and the PDZ binding motif in AE1C. Kidney AE1 (kAE1), PDLIM5 and integrin-linked kinase (ILK) form a multiprotein complex in which PDLIM5 provides a bridge between ILK and AE1C. Depletion of PDLIM5 resulted in significant reduction in kAE1 at the cell membrane, whereas over-expression of kAE1 was accompanied by increased PDLIM5 levels, underscoring the functional importance of PDLIM5 for proper kAE1 membrane residency, as a crucial linker between kAE1 and actin cytoskeleton-associated proteins in polarized cells.

G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases.

Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrate-selective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex.

Characteristic size of a collimated Gaussian beam in turbulent atmosphere.

The characteristic size of a collimated Gaussian beam propagating through 1-13 km atmospheric paths is investigated by simulating phase screens using the fast Fourier transform method. Taking a threshold into account, a method to derive a modified centroid and corresponding characteristic radii of the short-term spots is proposed. Effective radius, robust radius, sharpness radius, and maximum radius are analyzed by probability statistics. Furthermore, several parameters representing the energy content of the spots within each radius and the energy duty cycle of the maximum radius are studied. The study shows that, when the modified centroid is taken as a center, the effective radius is more suitable for application after a long propagation path, while the maximum radius is more effective for a short distance. However, when all effective subspots of a short-term image are investigated, the maximum radius is usually utilized, and the energy duty cycle represents the effect probability.

Structural basis for catalytic activation of protein Z-dependent protease inhibitor (ZPI) by protein Z.

The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), is catalytically activated by its cofactor, protein Z (PZ), to regulate the function of blood coagulation factor Xa on membrane surfaces. The X-ray structure of the ZPI-PZ complex has shown that PZ binds to a unique site on ZPI centered on helix G. In the present study, we show by Ala-scanning mutagenesis of the ZPI-binding interface, together with native PAGE and kinetic analyses of PZ binding to ZPI, that Tyr240 and Asp293 of ZPI are crucial hot spots for PZ binding. Complementary studies with protein Z-protein C chimeras show the importance of both pseudocatalytic and EGF2 domains of PZ for the critical ZPI interactions. To understand how PZ acts catalytically, we analyzed the interaction of reactive loop-cleaved ZPI (cZPI) with PZ and determined the cZPI X-ray structure. The cZPI structure revealed changes in helices A and G of the PZ-binding site relative to native ZPI that rationalized an observed 6-fold loss in PZ affinity and PZ catalytic action. These findings identify the key determinants of catalytic activation of ZPI by PZ and suggest novel strategies for ameliorating hemophilic states through drugs that disrupt the ZPI-PZ interaction.