Proteomic analysis of fatty liver induced by starvation of medaka fish larvae.

When medaka fish (Oryzias latipes) larvae are grown in the absence of exogenous nutrition, the liver becomes dark and positive to Oil Red O staining from 7 days post-hatch (dph). We determined the mechanism of this starvation-induced development of fatty liver by proteomic analysis using livers obtained from larvae grown in the presence or absence of 2% glucose at 5 dph. Results showed that changes in the expression levels of enzymes involved in glycolysis or the tricarboxylic acid cycle were modest, whereas the expression levels of enzymes involved in amino acid catabolism or ß-oxidation of fatty acids were significantly elevated, suggesting that they become major energy sources under starvation conditions. Expression levels of enzymes for the uptake and ß-oxidation of fatty acids as well as synthesis of triacylglycerol were elevated, whereas those for the synthesis of cholesterol as well as export of cholesterol and triacylglycerol were decreased under starvation conditions, which explains the accumulation of triacylglycerol in the liver. Our results provide the basis for future research to understand how gene malfunction(s) affects the development of fatty liver, which can lead to nonalcoholic steatohepatitis and then to liver cirrhosis.Key words: amino acid catabolism, ß-oxidation, triacylglycerol, cholesterol, export.

Activation of XBP1 but not ATF6α rescues heart failure induced by persistent ER stress in medaka fish.

The unfolded protein response is triggered in vertebrates by ubiquitously expressed IRE1α/ß (although IRE1ß is gut-specific in mice), PERK, and ATF6α/ß, transmembrane-type sensor proteins in the ER, to cope with ER stress, the accumulation of unfolded and misfolded proteins in the ER. Here, we burdened medaka fish, a vertebrate model organism, with ER stress persistently from fertilization by knocking out the AXER gene encoding an ATP/ADP exchanger in the ER membrane, leading to decreased ATP concentration-mediated impairment of the activity of Hsp70- and Hsp90-type molecular chaperones in the ER lumen. ER stress and apoptosis were evoked from 4 and 6 dpf, respectively, leading to the death of all AXER-KO medaka by 12 dpf because of heart failure (medaka hatch at 7 dpf). Importantly, constitutive activation of IRE1α signaling-but not ATF6α signaling-rescued this heart failure and allowed AXER-KO medaka to survive 3 d longer, likely because of XBP1-mediated transcriptional induction of ER-associated degradation components. Thus, activation of a specific pathway of the unfolded protein response can cure defects in a particular organ.

Loss of ATF6α in a human carcinoma cell line is compensated not by its paralogue ATF6ß but by sustained activation of the IRE1 and PERK arms for tumor growth in nude mice.

To survive poor nutritional conditions, tumor cells activate the unfolded protein response, which is composed of the IRE1, PERK, and ATF6 arms, to maintain the homeostasis of the endoplasmic reticulum, where secretory and transmembrane proteins destined for the secretory pathway gain their correct three-dimensional structure. The requirement of the IRE1 and PERK arms for tumor growth in nude mice is established. Here we investigated the requirement for the ATF6 arm, which consists of ubiquitously expressed ATF6α and ATF6ß, by constructing ATF6α-knockout (KO), ATF6ß-KO, and ATF6α/ß-double KO (DKO) in HCT116 cells derived from human colorectal carcinoma. Results showed that these KO cells grew similarly to wild-type (WT) cells in nude mice, contrary to expectations from our analysis of ATF6α-KO, ATF6ß-KO, and ATF6α/ß-DKO mice. We then found that the loss of ATF6α in HCT116 cells resulted in sustained activation of the IRE1 and PERK arms in marked contrast to mouse embryonic fibroblasts, in which the loss of ATF6α is compensated for by ATF6ß. Although IRE1-KO in HCT116 cells unexpectedly did not affect tumor growth in nude mice, IRE1-KO HCT116 cells with ATF6α knockdown grew significantly more slowly than WT or IRE1-KO HCT116 cells. These results have unraveled the situation-dependent differential compensation strategies of ATF6α.

A motor neuron disease-associated mutation produces non-glycosylated Seipin that induces ER stress and apoptosis by inactivating SERCA2b.

A causal relationship between endoplasmic reticulum (ER) stress and the development of neurodegenerative diseases remains controversial. Here, we focused on Seipinopathy, a dominant motor neuron disease, based on the finding that its causal gene product, Seipin, is a protein that spans the ER membrane twice. Gain-of-function mutations of Seipin produce non-glycosylated Seipin (ngSeipin), which was previously shown to induce ER stress and apoptosis at both cell and mouse levels albeit with no clarified mechanism. We found that aggregation-prone ngSeipin dominantly inactivated SERCA2b, the major calcium pump in the ER, and decreased the calcium concentration in the ER, leading to ER stress and apoptosis in human colorectal carcinoma-derived cells (HCT116). This inactivation required oligomerization of ngSeipin and direct interaction of the C-terminus of ngSeipin with SERCA2b, and was observed in Seipin-deficient neuroblastoma (SH-SY5Y) cells expressing ngSeipin at an endogenous protein level. Our results thus provide a new direction to the controversy noted above.

Drop-Dry Deposition of Ni(OH)2 Precursors for Fabrication of NiO Thin Films.

Drop-dry deposition (DDD) is a method of depositing thin films by heating and drying the deposition solution dropped on a substrate. We prepared Ni(OH)2 precursor thin films by DDD and annealed them in air to prepare NiO thin films. The appropriate deposition conditions were found by changing the number of drop-dry cycles and the concentrations of chemicals in the solution, and the Ni(OH)2 precursor film with a thickness of 0.3 µm and optical transmittance of more than 95% was successfully deposited. Raman and X-ray diffraction measurements were performed, and it was found that the NiO film was successfully fabricated after annealing at 400 °C. The p-type conductivity of the annealed film was confirmed by photoelectrochemical measurements. In addition, we prepared n-type ZnO by electrochemical deposition on NiO thin films. The current-voltage measurement results show that the ZnO/NiO heterojunction had rectification properties.

Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD.

Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the α1,6-linked mannosyl residue (M7A, M6, and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6, and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major α1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other α1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible.

Endoplasmic Reticulum-Associated Degradation Controls Virus Protein Homeostasis, Which Is Required for Flavivirus Propagation.

Many positive-stranded RNA viruses encode polyproteins from which viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though the required amounts for each protein are not the same. In this study, we found the extra membrane-anchored nonstructural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 is predominantly localized in the convoluted membrane (CM) of the viral replication organelle, and viral NS proteins are degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses. IMPORTANCE The results of this study reveal the cellular ERAD system controls the amount of each viral protein in virus-infected cells and that this "viral protein homeostasis" is critical for viral propagation. Furthermore, we clarified that the "convoluted membrane (CM)," which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/SEL1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.

Allosteric regulation accompanied by oligomeric state changes of Trypanosoma brucei GMP reductase through cystathionine-ß-synthase domain.

Guanosine 5'-monophosphate reductase (GMPR) is involved in the purine salvage pathway and is conserved throughout evolution. Nonetheless, the GMPR of Trypanosoma brucei (TbGMPR) includes a unique structure known as the cystathionine-ß-synthase (CBS) domain, though the role of this domain is not fully understood. Here, we show that guanine and adenine nucleotides exert positive and negative effects, respectively, on TbGMPR activity by binding allosterically to the CBS domain. The present structural analyses revealed that TbGMPR forms an octamer that shows a transition between relaxed and twisted conformations in the absence and presence of guanine nucleotides, respectively, whereas the TbGMPR octamer dissociates into two tetramers when ATP is available instead of guanine nucleotides. These findings demonstrate that the CBS domain plays a key role in the allosteric regulation of TbGMPR by facilitating the transition of its oligomeric state depending on ligand nucleotide availability.

EDEM2 stably disulfide-bonded to TXNDC11 catalyzes the first mannose trimming step in mammalian glycoprotein ERAD.

Sequential mannose trimming of N-glycan (Man9GlcNAc2 -> Man8GlcNAc2 -> Man7GlcNAc2) facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). Our gene knockout experiments in human HCT116 cells have revealed that EDEM2 is required for the first step. However, it was previously shown that purified EDEM2 exhibited no α1,2-mannosidase activity toward Man9GlcNAc2 in vitro. Here, we found that EDEM2 was stably disulfide-bonded to TXNDC11, an endoplasmic reticulum protein containing five thioredoxin (Trx)-like domains. C558 present outside of the mannosidase homology domain of EDEM2 was linked to C692 in Trx5, which solely contains the CXXC motif in TXNDC11. This covalent bonding was essential for mannose trimming and subsequent gpERAD in HCT116 cells. Furthermore, EDEM2-TXNDC11 complex purified from transfected HCT116 cells converted Man9GlcNAc2 to Man8GlcNAc2(isomerB) in vitro. Our results establish the role of EDEM2 as an initiator of gpERAD, and represent the first clear demonstration of in vitro mannosidase activity of EDEM family proteins.

Reinvestigation of Disulfide-bonded Oligomeric Forms of the Unfolded Protein Response Transducer ATF6.

ATF6α is an endoplasmic reticulum (ER)-embedded transcription factor which is rapidly activated by ER stress, and a major regulator of ER chaperone levels in vertebrates. We previously suggested that ATF6α occurs as a monomer, dimer and oligomer in the unstressed ER of Chinese hamster ovary cells due to the presence of two evolutionarily conserved cysteine residues in its luminal region (C467 and C618), and showed that ATF6α is reduced upon ER stress, such that only reduced monomer ATF6α is translocated to the Golgi apparatus for activation by proteolysis. However, mutagenesis analysis (C467A and C618A) revealed that the C618A mutant behaves in an unexpected manner (monomer and oligomer) during non-reducing SDS-PAGE, for reasons which remained unclear. Here, we used human colorectal carcinoma-derived HCT116 cells deficient in ATF6α and its relevant ATF6ß, and found that ATF6α dimer and oligomer are both dimers, which we designated C618-dimer and C467-dimer, respectively. We demonstrated that C467-dimer (previously considered an oligomer) behaved bigger than C618-dimer (previously considered a dimer) during non-reducing SDS-PAGE, based on their disulfide-bonded structures. Furthermore, ATF6α monomer physically associates with another ATF6α monomer in the absence of disulfide bonding, which renders two C467 residues in close proximity so that formation of C467-dimer is much easier than that of C618-dimer. In contrast, C618-dimer is more easily reduced upon ER stress. Thus, our analysis revealed that all forms of ATF6α, namely monomer, C618-dimer and C467-dimer, are activated by single reduction of a disulfide bond in response to ER stress, ensuring the rapidity of ATF6α activation.Key words: disulfide-bonded structure, endoplasmic reticulum, membrane-bound transcription factor, non-reducing SDS-PAGE, unfolded protein response.

Development of a Rapid in vivo Assay to Evaluate the Efficacy of IRE1-specific Inhibitors of the Unfolded Protein Response Using Medaka Fish.

Three types of transmembrane protein, IRE1α/IRE1ß, PERK, and ATF6α/ATF6ß, are expressed ubiquitously in vertebrates as transducers of the unfolded protein response (UPR), which maintains the homeostasis of the endoplasmic reticulum. IRE1 is highly conserved from yeast to mammals, and transmits a signal by a unique mechanism, namely splicing of mRNA encoding XBP1, the transcription factor downstream of IRE1 in metazoans. IRE1 contains a ribonuclease domain in its cytoplasmic region which initiates splicing reaction by direct cleavage of XBP1 mRNA at the two stem loop structures. As the UPR is considered to be involved in the development and progression of various diseases, as well as in the survival and growth of tumor cells, UPR inhibitors have been sought. To date, IRE1 inhibitors have been screened using cell-based reporter assays and fluorescent-based in vitro cleavage assays. Here, we used medaka fish to develop an in vivo assay for IRE1α inhibitors. IRE1α, IRE1ß, ATF6α and ATF6ß are ubiquitously expressed in medaka. We found that IRE1α/ATF6α-double knockout is lethal, similarly to IRE1α/IRE1ß- and ATF6α/ATF6ß-double knockout. Therefore, IRE1 inhibitors are expected to confer lethality to ATF6α-knockout medaka but not to wild-type medaka. One compound named K114 was obtained from 1,280 compounds using this phenotypic screening. K114 inhibited ER stress-induced splicing of XBP1 mRNA as well as reporter luciferase expression in HCT116 cells derived from human colorectal carcinoma, and inhibited ribonuclease activity of human IRE1α in vitro. Thus, this phenotypic assay can be used as a quick test for the efficacy of IRE1α inhibitors in vivo.Key words: endoplasmic reticulum, inhibitor screening, mRNA splicing, phenotypic assay, unfolded protein response.

UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage-specific manner.

The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (Sec23/24/13/31, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage.

SEL1L-dependent Substrates Require Derlin2/3 and Herp1/2 for Endoplasmic Reticulum-associated Degradation.

Accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). The ATF6 pathway is one of the three major pathways in vertebrates. Although ATF6, a transmembrane-type glycoprotein in the ER, functions as a UPR sensor/transducer, it is an unstable protein with a half-life of approximately 2 h and is constitutively subjected to the ER-associated degradation system with the location of the misfolded part in the ER lumen (ERAD-L). ERAD-L substrates are delivered to the cytosol through the retrotranslocon, which likely contains HRD1 (E3), gp78 (E3), SEL1L (a partner of HRD1), Derlin1/2/3 and Herp1/2. We previously showed that ATF6 represents a novel transmembrane-type ERAD-L substrate requiring both EDEM1/2/3-mediated mannose trimming and SEL1L. Here, by constructing and analyzing chicken DT40 cells deficient in various components of the retrotranslocon, we show that degradation of ATF6 requires Derlin2 or Derlin3 and that Derlin2 and Derlin3 are redundant for ERAD-L of ATF6. We further show that degradation of ATF6 requires Herp1 or Herp2 and that Herp1 and Herp2 are redundant for ERAD-L of ATF6. Furthermore, by investigating five more ERAD-L substrates, we show that SEL1L-dependent substrates require Derlin2/3 and Herp1/2 regardless of their soluble or transmembrane nature. Our results suggest that ERAD-L substrates take several routes to the cytosol. The HRD1-engaged route 1 requires SEL1L, Derlin2 or Derlin3, and Herp1 or Herp2, whereas the HRD1-engaged route 2 does not require them functionally. It remains to be determined whether the latter requires Derlin1 and whether these two routes are compositionally distinct.Key words: endoplasmic reticulum, proteasome, protein degradation, protein misfolding, ubiquitin.

Correction: A High-Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models.

[This corrects the article DOI: 10.1371/journal.pone.0161486.].

A High-Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models.

The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein folding. ER Ca2+ depletion and accumulation of unfolded proteins activate the molecular chaperone GRP78 (glucose-regulated protein 78) which in turn triggers the ER stress response (ERSR) pathway aimed to restore ER homeostasis. Failure to adapt to stress, however, results in apoptosis. We and others have shown that malignant cells are more susceptible to ERSR-induced apoptosis than their normal counterparts, implicating the ERSR as a potential target for cancer therapeutics. Predicated on these findings, we developed an assay that uses a GRP78 biosensor to identify small molecule activators of ERSR in glioma cells. We performed a quantitative high-throughput screen (qHTS) against a collection of ~425,000 compounds and a comprehensive panel of orthogonal secondary assays was formulated for stringent compound validation. We identified novel activators of ERSR, including a compound with a 2,9-diazaspiro[5.5]undecane core, which depletes intracellular Ca2+ stores and induces apoptosis-mediated cell death in several cancer cell lines, including patient-derived and 3D cultures of glioma cells. This study demonstrates that our screening platform enables the identification and profiling of ERSR inducers with cytotoxic activity and advocates for characterization of these compound in in vivo models.

Novel Characteristics of Trypanosoma brucei Guanosine 5'-monophosphate Reductase Distinct from Host Animals.

The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5'-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5'-monophosphate (GMP) to inosine 5'-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present. Herein, we identified the GMPR in Trypanosoma brucei, a causative protozoan parasite of African trypanosomiasis, and found that the GMPR proteins were consistently localized to glycosomes in T. brucei bloodstream forms. We characterized its recombinant protein to investigate the enzymatic differences between GMPRs of T. brucei and its host animals. T. brucei GMPR was distinct in having an insertion of a tandem repeat of the cystathionine ß-synthase (CBS) domain, which was absent in mammalian and bacterial GMPRs. The recombinant protein of T. brucei GMPR catalyzed the conversion of GMP to IMP in the presence of NADPH, and showed apparent affinities for both GMP and NADPH different from those of its mammalian counterparts. Interestingly, the addition of monovalent cations such as K+ and NH4+ to the enzymatic reaction increased the GMPR activity of T. brucei, whereas none of the mammalian GMPR's was affected by these cations. The monophosphate form of the purine nucleoside analog ribavirin inhibited T. brucei GMPR activity, though mammalian GMPRs showed no or only a little inhibition by it. These results suggest that the mechanism of the GMPR reaction in T. brucei is distinct from that in the host organisms. Finally, we demonstrated the inhibitory effect of ribavirin on the proliferation of trypanosomes in a dose-dependent manner, suggesting the availability of ribavirin to develop a new therapeutic agent against African trypanosomiasis.

Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway.

Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ-ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ-ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER.

EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step.

Glycoproteins misfolded in the endoplasmic reticulum (ER) are subjected to ER-associated glycoprotein degradation (gpERAD) in which Htm1-mediated mannose trimming from the oligosaccharide Man8GlcNAc2 to Man7GlcNAc2 is the rate-limiting step in yeast. In contrast, the roles of the three Htm1 homologues (EDEM1/2/3) in mammalian gpERAD have remained elusive, with a key controversy being whether EDEMs function as mannosidases or as lectins. We therefore conducted transcription activator-like effector nuclease-mediated gene knockout analysis in human cell line and found that all endogenous EDEMs possess mannosidase activity. Mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 is performed mainly by EDEM3 and to a lesser extent by EDEM1. Most surprisingly, the upstream mannose trimming from Man9GlcNAc2 to Man8GlcNAc2 is conducted mainly by EDEM2, which was previously considered to lack enzymatic activity. Based on the presence of two rate-limiting steps in mammalian gpERAD, we propose that mammalian cells double check gpERAD substrates before destruction by evolving EDEM2, a novel-type Htm1 homologue that catalyzes the first mannose trimming step from Man9GlcNAc2.

Lipocalin-type prostaglandin D synthase scavenges biliverdin in the cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the second major protein in human cerebrospinal fluid (CSF) and belongs to the lipocalin superfamily composed of various secretory lipophilic ligand transporter proteins. However, the endogenous ligand of L-PGDS has not yet been elucidated. In this study, we purified L-PGDS from the CSF of aneurysmal subarachnoid hemorrhage (SAH) patients. Lipocalin-type PG D synthase showed absorbance spectra with major peaks at 280 and 392 nm and a minor peak at around 660 nm. The absorbance at 392 nm of L-PGDS increased from 1 to 9 days and almost disappeared at 2 months after SAH, whereas the L-PGDS activity decreased from 1 to 7 days and recovered to normal at 2 months after SAH. These results indicate that some chromophore had accumulated in the CSF after SAH and bound to L-PGDS, thus inactivating it. Matrix assisted laser desorption ionization time-of-flight mass spectrometry of L-PGDS after digestion of it with endoproteinase Lys-C revealed that L-PGDS had covalently bound biliverdin, a by-product of heme breakdown. These results suggest that L-PGDS acted as a scavenger of biliverdin, which is a molecule not found in normal CSF. This is the first report of identification of a pathophysiologically important endogenous ligand for this lipocalin superfamily protein in humans.