The ER-Localized Transmembrane Protein EPG-3/VMP1 Regulates SERCA Activity to Control ER-Isolation Membrane Contacts for Autophagosome Formation.

During autophagosome formation in mammalian cells, isolation membranes (IMs; autophagosome precursors) dynamically contact the ER. Here, we demonstrated that the ER-localized metazoan-specific autophagy protein EPG-3/VMP1 controls ER-IM contacts. Loss of VMP1 causes stable association of IMs with the ER, thus blocking autophagosome formation. Interaction of WIPI2 with the ULK1/FIP200 complex and PI(3)P contributes to the formation of ER-IM contacts, and these interactions are enhanced by VMP1 depletion. VMP1 controls contact formation by promoting SERCA (sarco[endo]plasmic reticulum calcium ATPase) activity. VMP1 interacts with SERCA and prevents formation of the SERCA/PLN/SLN inhibitory complex. VMP1 also modulates ER contacts with lipid droplets, mitochondria, and endosomes. These ER contacts are greatly elevated by the SERCA inhibitor thapsigargin. Calmodulin acts as a sensor/effector to modulate the ER contacts mediated by VMP1/SERCA. Our study provides mechanistic insights into the establishment and disassociation of ER-IM contacts and reveals that VMP1 modulates SERCA activity to control ER contacts.

One amino acid drives the lipid droplet targeting sequence of a new noncoding RNA-encoded protein to mitochondrion.

Over the past decade, the majority of the mammalian genome considered to be noncoding has been revealed to be able to produce proteins. Many RNA molecules, mis-annotated as noncoding, actually are predicted to code for proteins. Some of those proteins have been identified and verified to play critical roles in multiple biological processes. The lipid droplet (LD) is a unique cellular organelle bound with a phospholipid monolayer membrane, and is closely associated with cellular lipid metabolism and metabolic disorders. However, it is still unclear how a protein targets to LDs. Here we identified a new protein on LDs, LDANP2, which is encoded by noncoding RNA, through a proteomics-based strategy. The key sequence for its localization on LDs, Truncation 3, is predicted to form an amphipathic helix. Surprisingly, the deletion of the first amino acid in Truncation 3 resulted in mitochondrial localization. How the types of amino acids would determine the LD or mitochondrial localizations of the protein was studied. The findings introduce a useful strategy to mine for new proteins and would provide clues to the understanding of how a protein would find its right organelle, with phospholipid monolayer or bilayer membrane.

Construction and application of artificial lipoproteins using adiposomes.

Lipoproteins are complex particles comprised of a neutral lipid core wrapped with a phospholipid monolayer membrane and apolipoproteins on the membrane, which is closely associated with metabolic diseases. To facilitate the elucidation of its formation and dynamics, as well as its applications, we developed an in vitro system in which adiposomes, consisting of a hydrophobic core encircled by a monolayer-phospholipid membrane, were engineered into artificial lipoproteins (ALPs) by recruiting one or more kinds of apolipoproteins, for example, apolipoprotein (Apo) A-I, ApoE, ApoA-IV, and ApoB. In vitro and in vivo studies demonstrated the stability and biological activity of ALPs derived from adiposomes, which resembles native lipoproteins. Of note, adiposomes bearing ApoE were internalized via clathrin-mediated endocytosis following LDLR binding and were delivered to lysosomes. On the other hand, adiposomes bearing ApoA-IV mimicked the existing form of endogenous ApoA-IV and exhibited significant improvement in glucose tolerance in mice. In addition, the construction process was simple, precise, reproducible, as well as easy to adjust for mass production. With this experimental system, different apolipoproteins can be recruited to build ALPs for some biological goals and potential applications in biomedicine.

Serum exosomes mediate delivery of arginase 1 as a novel mechanism for endothelial dysfunction in diabetes.

Exosomes, abundant in blood, deliver various molecules to recipient cells. Endothelial cells are directly exposed to circulating substances. However, how endothelial cells respond to serum exosomes (SExos) and the implications in diabetes-associated vasculopathy have never been explored. In the present study, we showed that SExos from diabetic db/db mice (db/db SExos) were taken up by aortic endothelial cells, which severely impaired endothelial function in nondiabetic db/m+ mice. The exosomal proteins, rather than RNAs, mostly account for db/db SExos-induced endothelial dysfunction. Comparative proteomics analysis showed significant increase of arginase 1 in db/db SExos. Silence or overexpression of arginase 1 confirmed its essential role in db/db SExos-induced endothelial dysfunction. This study is a demonstration that SExos deliver arginase 1 protein to endothelial cells, representing a cellular mechanism during development of diabetic endothelial dysfunction. The results expand the scope of blood-borne substances that monitor vascular homeostasis.

Biodegradable Zwitterion/PLGA Scaffold Enables Robust Healing of Rat Calvarial Defects with Ultralow Dose of rhBMP-2.

Designing smart scaffolds to reduce administration dosage under the premise of functional healing of bone defects to avoid the severe side effects associated with BMP-2 treatments is one of the essential goals in bone tissue engineering. Here, we report a novel biodegradable PLGA/PSBMA composite as the scaffold for bone tissue engineering. The introduction of zwitterionic PSBMA components can alter the intrinsic burst degradation behavior of PLGA and enable a sustained degradation of the scaffold over the time. The PLGA/PSBMA scaffold can sequester rhBMP-2 and enable a sustained release of the sequestered rhBMP-2 with preserved bioactivity. Furthermore, PLGA/PSBMA scaffolds were able to guide robust healing of critical-sized nonunion calvarial defects (5 mm) at an ultralow dose of 400 ng/scaffold, at which level successful healing of critical-sized bone defects has never been reported. These findings indicate the PLGA/PSBMA scaffolds as novel high-efficiency rhBMP-2 delivery vehicles for bone tissue engineering, and the concept of utilizing the material, which is capable of maintaining the bioactivity of the proteins in the preparation of scaffolds, may open a new avenue for the design of smart scaffolds/vehicles for high-efficiency protein/bioactive drug therapies.

The New Face of the Lipid Droplet: Lipid Droplet Proteins.

The lipid droplet (LD) is an organelle with vital functions found in nearly all organisms. LD proteomic research has provided fundamentally important insights into this organelle's functions. The review provides a summary of LD proteomic studies conducted across diverse organisms and cell and tissue types. The accumulated proteomic data are reviewed for evidence of a protein targeting mechanism for the organelle. The hypotheses for several specific localization mechanisms based on what is known about targeting mechanisms for other organelles and vesicles are provided. Although the nature of the mechanism is not known, the functional data demonstrate that the targeting mechanism and, indeed, the organelle itself, is conserved from prokaryotes to eukaryotes. It is hoped that the review will help inspire further research leading to novel discoveries in the field.

The Adrenal Lipid Droplet is a New Site for Steroid Hormone Metabolism.

Steroid hormones play essential roles for living organisms. It has been long and well established that the endoplasmic reticulum (ER) and mitochondria are essential sites for steroid hormone biosynthesis because several steroidogenic enzymes are located in these organelles. The adrenal gland lipid droplet (LD) proteomes from human, macaque monkey, and rodent are analyzed, revealing that steroidogenic enzymes are also present in abundance on LDs. The enzymes found include 3ß-hydroxysteroid dehydrogenase (HSD3B) and estradiol 17ß-dehydrogenase 11 (HSD17B11). Analyses by Western blot and subcellular localization consistently demonstrate that HSD3B2 is localized on LDs. Furthermore, in vitro experiments confirm that the isolated LDs from HeLa cell stably expressing HSD3B2 or from rat adrenal glands have the capacity to convert pregnenolone to progesterone. Collectively, these data suggest that LDs may be important sites of steroid hormone metabolism. These findings may bring novel insights into the biosynthesis and metabolism of steroid hormones and the development of treatments for adrenal disorders.

Lysine glycation of apolipoprotein A-I impairs its anti-inflammatory function in type 2 diabetes mellitus.

Apolipoprotein A-I (apoA-I), the major protein compontent of high-density lipoprotein (HDL), exerts many anti-atherogenic functions. This study aimed to reveal whether nonenzymatic glycation of specific sites of apoA-I impaired its anti-inflammatory effects in type 2 diabetes mellitus (T2DM). LC-MS/MS was used to analyze the specific sites and the extent of apoA-I glycation either modified by glucose in vitro or isolated from T2DM patients. Cytokine release in THP-1 monocyte-derived macrophages was tested by ELISA. Activation of NF-kappa B pathway was detected by western blot. The binding affinity of apoA-I to THP-1 cells was measured using 125I-labeled apoA-I. We identified seven specific lysine (Lys, K) residues of apoA-I (K12, K23, K40, K96, K106, K107 and K238) that were susceptible to be glycated either in vitro or in vivo. Glycation of apoA-I impaired its abilities to inhibit the release of TNF-α and IL-1ß against lipopolysaccharide (LPS) in THP-1 cells. Besides, the glycation levels of these seven K sites in apoA-I were inversely correlated with its anti-inflammatory abilities. Furthermore, glycated apoA-I had a lower affinity to THP-1 cells than native apoA-I had. We generated mutant apoA-I (K107E, M-apoA-I) with a substitution of glutamic acid (Glu, E) for lysine at the 107th site, and found that compared to wild type apoA-I (WT-apoA-I), M-apoA-I decreased its anti-inflammatory effects in THP-1 cells. We also modeled the location of these seven K residues on apoA-I which allowed us to infer the conformational alteration of glycated apoA-I and HDL. In summary, glycation of these seven K residues altered the conformation of apoA-I and consequently impaired the protective effects of apoA-I, which may partly account for the increased risk of cardiovascular disease (CVD) in diabetic subjects.

SILAC-based quantitative proteomic analysis of the livers of spontaneous obese and diabetic rhesus monkeys.

Type 2 diabetes mellitus (T2DM) is a severe metabolic disorder that affects more than 10% of the population worldwide. Obesity is a major cause of insulin resistance and contributes to the development of T2DM. Liver is an essential metabolic organ that plays crucial roles in the pathogenesis of obesity and diabetes. However, the underlying mechanisms of liver in the transition of obesity to diabetes are not fully understood. The nonhuman primate rhesus monkey is an appropriate animal for research of human diseases. Here, we first screened and selected three individuals of spontaneously diabetic rhesus monkeys. Interestingly, the diabetic monkeys were obese with a high body mass index at the beginning, but gradually lost their body weight during one year of observation. Furthermore, we performed stable isotope labeling with amino acids in cell culture-based quantitative proteomics to identify proteins and signaling pathways with altered expression in the liver of obese and diabetic monkeys. In total, 3,509 proteins were identified and quantified, of which 185 proteins displayed an altered expression level. Gene ontology analysis revealed that the expression of proteins involved in fatty acids ß-oxidation and galactose metabolism was increased in obese monkeys; while proteins involved in oxidative phosphorylation and branched chain amino acid (BCAA) degradation were upregulated in diabetic monkeys. In addition, we observed mild apoptosis in the liver of diabetic monkeys, suggesting liver injury at the late onset of diabetes. Taken together, our liver proteomics may reveal a distinct metabolic transition from fatty acids ß-oxidation in obese monkey to BCAA degradation in diabetic monkeys.

Lipid droplet proteins and metabolic diseases.

Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage which are composed of a neutral lipid core bounded by a protein decorated phospholipid monolayer. Although lipid storage is their most obvious function, LDs are far from inert as they participate in maintaining lipid homeostasis through lipid synthesis, metabolism, and transportation. Furthermore, they are involved in cell signaling and other molecular events closely associated with human disease such as dyslipidemia, obesity, lipodystrophy, diabetes, fatty liver, atherosclerosis, and others. The last decade has seen a great increase in the attention paid to LD biology. Regardless, many fundamental features of LD biology remain obscure. In this review, we will discuss key aspects of LD biology including their biogenesis, growth and regression. We will also summarize the current knowledge about the role LDs play in human disease, especially from the perspective of the dynamics of the associated proteins. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.

Hydroxysteroid dehydrogenase family proteins on lipid droplets through bacteria, C. elegans, and mammals.

Lipid droplets (LDs) are the main fat storing sites in almost all species from bacteria to humans. The perilipin family has been found as LD proteins in mammals, Drosophila, and a couple of slime molds, but no bacterial LD proteins containing sequence conservation were identified. In this study, we reported that the hydroxysteroid dehydrogenase (HSD) family was found on LDs across all organisms by LD proteomic analysis. Imaging experiments confirmed LD targeting of three representative HSD proteins including ro01416 in RHA1, DHS-3 in C. elegans, and 17ß-HSD11 in human cells. In C. elegans, 17ß-HSD11 family proteins (DHS-3, DHS-4 and DHS-19) were localized on LDs in distinct tissues. In intestinal cells of C. elegans, DHS-3 targeted to cytoplasmic LDs, while DHS-9 labeled nuclear LDs. Furthermore, the N-terminal hydrophobic domains of 17ß-HSD11 family were necessary for their targeting to LDs. Last, 17ß-HSD11 family proteins induced LD aggregation, and deletion of DHS-3 in C. elegans caused lipid decrease. Independent of their presumptive catalytic sites, 17ß-HSD11 family proteins regulated LD dynamics and lipid metabolism through affecting the LD-associated ATGL, which was conserved between C. elegans and humans. Together, these findings for HSDs provide a new insight not only into the mechanistic studies of the dynamics and functions of LDs in multiple organisms, but also into understanding the evolutionary history of the organelle.

Secreted monocytic miR-150 enhances targeted endothelial cell migration.

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate target gene expression at the posttranscriptional level. Here, we report that secreted miRNAs can serve as signaling molecules mediating intercellular communication. In human blood cells and cultured THP-1 cells, miR-150 was selectively packaged into microvesicles (MVs) and actively secreted. THP-1-derived MVs can enter and deliver miR-150 into human HMEC-1 cells, and elevated exogenous miR-150 effectively reduced c-Myb expression and enhanced cell migration in HMEC-1 cells. In vivo studies confirmed that intravenous injection of THP-1 MVs significantly increased the level of miR-150 in mouse blood vessels. MVs isolated from the plasma of patients with atherosclerosis contained higher levels of miR-150, and they more effectively promoted HMEC-1 cell migration than MVs from healthy donors. These results demonstrate that cells can secrete miRNAs and deliver them into recipient cells where the exogenous miRNAs can regulate target gene expression and recipient cell function.

Ptrf transgenic mice exhibit obesity and fatty liver.

Polymerase I and transcript release factor (Ptrf, also known as Cavin1) is an essential component in the biogenesis and function of caveolae. Ptrf knockout mice or patients with PTRF mutations exhibit numerous pathologies including markedly aberrant fuel metabolism, lipodystrophy and muscular dystrophy. In this study, we generated Ptrf transgenic mice to explore its function in vivo. Compared with wild-type (WT) mice, we found that the Ptrf transgenic mice showed obesity with an increased level of ALT (alanine aminotransferase) and AST (aspartate transaminase). Ptrf transgenic mice exhibited severe fat degeneration and a higher degree of fat accumulation in the liver compared with WT mice. Consistently, we found that the expression of the fat synthesis gene, Fasn, was increased in the liver of Ptrf transgenic mice. Thus, Ptrf transgenic mice would be a good model for investigating the molecular mechanism and therapeutic targets of obesity and fatty liver associated diseases.

Lysine malonylation is elevated in type 2 diabetic mouse models and enriched in metabolic associated proteins.

Protein lysine malonylation, a newly identified protein post-translational modification (PTM), has been proved to be evolutionarily conserved and is present in both eukaryotic and prokaryotic cells. However, its potential roles associated with human diseases remain largely unknown. In the present study, we observed an elevated lysine malonylation in a screening of seven lysine acylations in liver tissues of db/db mice, which is a typical model of type 2 diabetes. We also detected an elevated lysine malonylation in ob/ob mice, which is another model of type 2 diabetes. We then performed affinity enrichment coupled with proteomic analysis on liver tissues of both wild-type (wt) and db/db mice and identified a total of 573 malonylated lysine sites from 268 proteins. There were more malonylated lysine sites and proteins in db/db than in wt mice. Five proteins with elevated malonylation were verified by immunoprecipitation coupled with Western blot analysis. Bioinformatic analysis of the proteomic results revealed the enrichment of malonylated proteins in metabolic pathways, especially those involved in glucose and fatty acid metabolism. In addition, the biological role of lysine malonylation was validated in an enzyme of the glycolysis pathway. Together, our findings support a potential role of protein lysine malonylation in type 2 diabetes with possible implications for its therapy in the future.

Comparative proteomic study reveals 17ß-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is characterized by a massive accumulation of lipid droplets (LDs). The aim of this study was to determine the function of 17ß-hydroxysteroid dehydrogenase-13 (17ß-HSD13), one of our newly identified LD-associated proteins in human subjects with normal liver histology and simple steatosis, in NAFLD development. LDs were isolated from 21 human liver biopsies, including 9 cases with normal liver histology (group 1) and 12 cases with simple steatosis (group 2). A complete set of LD-associated proteins from three liver samples of group 1 or group 2 were determined by 2D LC-MS/MS. By comparing the LD-associated protein profiles between subjects with or without NAFLD, 54 up-regulated and 35 down-regulated LD-associated proteins were found in NAFLD patients. Among them, 17ß-HSD13 represents a previously unidentified LD-associated protein with a significant up-regulation in NAFLD. Because the 17ß-HSD family plays an important role in lipid metabolism, 17ß-HSD13 was selected for validating the proteomic findings and exploring its role in the pathogenesis of NAFLD. Increased hepatic 17ß-HSD13 and its LD surface location were confirmed in db/db (diabetic) and high-fat diet-fed mice. Adenovirus-mediated hepatic overexpression of human 17ß-HSD13 induced a fatty liver phenotype in C57BL/6 mice, with a significant increase in mature sterol regulatory element-binding protein 1 and fatty acid synthase levels. The present study reports an extensive set of human liver LD proteins and an array of proteins differentially expressed in human NAFLD. We also identified 17ß-HSD13 as a pathogenic protein in the development of NAFLD.

Lipid droplet remodeling and interaction with mitochondria in mouse brown adipose tissue during cold treatment.

Brown adipose tissue (BAT) maintains animal body temperature by non-shivering thermogenesis, which is through uncoupling protein 1 (UCP1) that uncouples oxidative phosphorylation and utilizes ß-oxidation of fatty acids released from triacylglycerol (TAG) in lipid droplets (LDs). Increasing BAT activity and "browning" other tissues such as white adipose tissue (WAT) can enhance the expenditure of excess stored energy, and in turn reduce prevalence of metabolic diseases. Although many studies have characterized the biology of BAT and brown adipocytes, BAT LDs especially their activation induced by cold exposure remain to be explored. We have isolated LDs from mouse interscapular BAT and characterized the full proteome using mass spectrometry. Both morphological and biochemical experiments showed that the LDs could tightly associate with mitochondria. Under cold treatment mouse BAT started expressing LD structure protein PLIN-2/ADRP and increased expression of PLIN1. Both hormone sensitive lipase (HSL) and adipose TAG lipase (ATGL) were increased in LDs. In addition, isolated BAT LDs showed increased levels of the mitochondrial protein UCP1, and prolonged cold exposure could stimulate BAT mitochondrial cristae biogenesis. These changes were in agreement with the data from transcriptional analysis. Our results provide the BAT LD proteome for the first time and show that BAT LDs facilitate heat production by coupling increasing TAG hydrolysis through recruitment of ATGL and HSL to the organelle and expression of another LD resident protein PLIN2/ADRP, as well as by tightly associating with activated mitochondria. These findings will benefit the study of BAT activation and the interaction between LDs and mitochondria.

Identification of lipid droplet structure-like/resident proteins in Caenorhabditis elegans.

The lipid droplet (LD) is a cellular organelle that stores neutral lipids in cells and has been linked with metabolic disorders. Caenorhabditis elegans has many characteristics which make it an excellent animal model for studying LDs. However, unlike in mammalian cells, no LD structure-like/resident proteins have been identified in C. elegans, which has limited the utility of this model for the study of lipid storage and metabolism. Herein based on three lines of evidence, we identified that MDT-28 and DHS-3 previously identified in C. elegans LD proteome were two LD structure-like/resident proteins. First, MDT-28 and DHS-3 were found to be the two most abundant LD proteins in the worm. Second, the proteins were specifically localized to LDs and we identified the domains responsible for this targeting in both proteins. Third and most importantly, the depletion of MDT-28 induced LD clustering while DHS-3 deletion reduced triacylglycerol content (TAG). We further characterized the proteins finding that MDT-28 was ubiquitously expressed in the intestine, muscle, hypodermis, and embryos, whereas DHS-3 was expressed mainly in intestinal cells. Together, these two LD structure-like/resident proteins provide a basis for future mechanistic studies into the dynamics and functions of LDs in C. elegans.

Dynamics of the lipid droplet proteome of the Oleaginous yeast rhodosporidium toruloides.

Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.

Integrated omics study delineates the dynamics of lipid droplets in Rhodococcus opacus PD630.

Rhodococcus opacus strain PD630 (R. opacus PD630), is an oleaginous bacterium, and also is one of few prokaryotic organisms that contain lipid droplets (LDs). LD is an important organelle for lipid storage but also intercellular communication regarding energy metabolism, and yet is a poorly understood cellular organelle. To understand the dynamics of LD using a simple model organism, we conducted a series of comprehensive omics studies of R. opacus PD630 including complete genome, transcriptome and proteome analysis. The genome of R. opacus PD630 encodes 8947 genes that are significantly enriched in the lipid transport, synthesis and metabolic, indicating a super ability of carbon source biosynthesis and catabolism. The comparative transcriptome analysis from three culture conditions revealed the landscape of gene-altered expressions responsible for lipid accumulation. The LD proteomes further identified the proteins that mediate lipid synthesis, storage and other biological functions. Integrating these three omics uncovered 177 proteins that may be involved in lipid metabolism and LD dynamics. A LD structure-like protein LPD06283 was further verified to affect the LD morphology. Our omics studies provide not only a first integrated omics study of prokaryotic LD organelle, but also a systematic platform for facilitating further prokaryotic LD research and biofuel development.

Identification of SERPINB1 as a physiological inhibitor of human granzyme H.

The granzyme/perforin pathway is a major mechanism for cytotoxic lymphocytes to eliminate virus-infected and tumor cells. The balance between activation and inhibition of the proteolytic cascade must be tightly controlled to avoid self damage. Granzyme H (GzmH) is constitutively expressed in NK cells and induces target cell death; however, how GzmH activity is regulated remains elusive. We reported earlier the crystal structures of inactive D102N-GzmH alone and in complex with its synthetic substrate and inhibitor, as well as defined the mechanisms of substrate recognition and enzymatic activation. In this study, we identified SERPINB1 as a potent intracellular inhibitor for GzmH. Upon cleavage of the reactive center loop at Phe(343), SERPINB1 forms an SDS-stable covalent complex with GzmH. SERPINB1 overexpression suppresses GzmH- or LAK cell-mediated cytotoxicity. We determined the crystal structures of active GzmH and SERPINB1 (LM-DD mutant) in the native conformation to 3.0- and 2.9-Å resolution, respectively. Molecular modeling reveals the possible conformational changes in GzmH for the suicide inhibition. Our findings provide new insights into the inhibitory mechanism of SERPINB1 against human GzmH.