kDa Fragments Research Articles

Granzyme B cleaves Tenascin-C to release its C-terminal domain in rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune disorders and is characterized by exacerbated joint inflammation that can lead to tissue remodeling and autoantigen generation. Despite the well-documented accumulation of the serine protease Granzyme B (GzmB) in the biospecimens of patients with RA, little is understood pertaining to its role in pathobiology. In the present study Tenascin-C (TN-C), a large extracellular matrix glycoprotein and an endogenous trigger of inflammation, was identified as a substrate for GzmB in RA. GzmB cleaves TN-C in vitro to generate three fragments: a 130 kDa fragment that remains anchored to the matrix, and two 70 and 30 kDa fragments that are released and solubilized. Mass spectrometry results seem to indicate that the 30 kDa fragment generated by GzmB most likely contains TN-C pro-inflammatory C-terminal fibrinogen-like domain. Soluble levels of GzmB and TN-C are also significantly elevated in the synovial fluids of RA patients compared to healthy controls, with two 70 kDa and 30 kDa soluble TN-C fragments detectable in the synovial fluids of RA patients. The molecular weights of these fragments coincide with those generated by GzmB in vitro, suggesting that GzmB also cleaves TN-C in RA patients. Granzyme K (GzmK), another member of the granzyme family, also cleaves TN-C in vitro. However, unlike GzmB, the molecular weights of TN-C fragments generated by GzmK in vitro do not correspond to fragments identified in patients. Altogether, our data supports the contribution of Granzyme B, but not Granzyme K, to RA through the cleavage of Tenascin-C.

Differences in Cardiac Troponin T Composition in Myocardial Infarction and End-Stage Renal Disease Patients: A Blood Tube Effect?

Cardiac troponin T (cTnT) is key in diagnosing myocardial infarction (MI) but is also elevated in end-stage renal disease (ESRD) patients. Specific larger cTnT proteoforms were identified for the acute phase of MI, while in serum of ESRD patients solely small cTnT fragments were found. However, others allocated this to a pre-analytic effect due to abundant thrombin generation in serum. Therefore, we investigated the effect of various anticoagulation methods on cTnT composition and concentration and compared the cTnT composition of MI and ESRD patients. The agreement of cTnT concentrations between simultaneously collected serum, lithium-heparin (LH) plasma, and ethylenediaminetetraacetic acid (EDTA) plasma was studied using the high-sensitivity (hs-)cTnT immunoassay. cTnT proteoform composition was investigated in a standardized time-dependent manner through spike experiments and in simultaneously collected blood matrixes of MI and ESRD patients. Excellent hs-cTnT concentration agreements were observed across all blood matrixes (slopes > 0.98; 95% CI, 0.96-1.04). Time-dependent degradation (40 kDa intact:29 kDa fragment:15 to 18 kDa fragments) was found in LH plasma and EDTA plasma, and serum in ratios (%) of 90:10:0, 0:5:95, and 0:0:100, respectively (48 h after blood collection). Moreover, gel filtration chromatography (GFC) profiles illustrated mainly larger cTnT proteoforms in MI patients, while in ESRD patients mainly 15 to 18 kDa fragments were found for all matrices. The extent of cTnT degradation in vitro is dependent on the (anti)coagulation method, without impacting hs-cTnT concentrations. Furthermore, mainly larger cTnT proteoforms were present in MI patients, while in ESRD patients mainly small 15 to 18 kDa cTnT fragments were found. These insights are essential when developing a novel hs-cTnT assay targeting larger cTnT proteoforms.

Monkeypox virus infection of human astrocytes causes gasdermin B cleavage and pyroptosis

Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50 kDa), evident by the appearance of cleaved N-terminal-GSDMB (30 kDa) and C-terminal- GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.

The tetrapeptide sequence of IL-18 and IL-1β regulates their recruitment and activation by inflammatory caspases

Inflammasomes are multiprotein signaling complexes that activate the innate immune system. Canonical inflammasomes recruit and activate caspase-1, which then cleaves and activates IL-1β and IL-18, as well as gasdermin D (GSDMD) to induce pyroptosis. In contrast, non-canonical inflammasomes, caspases-4/-5 (CASP4/5) in humans and caspase-11 (CASP11) in mice, are known to cleave GSDMD, but their role in direct processing of other substrates besides GSDMD has remained unknown. Here, we show that CASP4/5 but not CASP11 can directly cleave and activate IL-18. However, CASP4/5/11 can all cleave IL-1β to generate a 27-kDa fragment that deactivates IL-1β signaling. Mechanistically, we demonstrate that the sequence identity of the tetrapeptide sequence adjacent to the caspase cleavage site regulates IL-18 and IL-1β recruitment and activation. Altogether, we have identified new substrates of the non-canonical inflammasomes and reveal key mechanistic details regulating inflammation that may aid in developing new therapeutics for immune-related disorders.

Correlations between different protein species of oral rinse MMP-8 and patient-related factors.

The aim of this study is to examine correlations between different oral rinse matrix metalloproteinase (MMP)-8 protein species in western blot (WB) analysis, quantitative MMP-8 measurements, and patient-related factors. Elevated activated MMP-8 (aMMP-8) associate with periodontitis and a diagnostic point-of-care technology has been developed based on aMMP-8. In WB, different MMP-8 protein species can be analyzed. Relative abundancyof fragmented 20-25 kDa forms in WB has been associated with and reflects MMP-8 activation and related fragmentation and elevated quantitative aMMP-8 measurements. A random sample of 192 participants from a periodontal disease screening study was used for this study. Oral rinse samples for biomarker analyses were collected before clinical periodontal examinations. aMMP-8 immunofluorometric (IFMA) and WB analysis (utilizing the same monoclonal antibody, 8708), polymorphonuclear leukocyte (PMN) elastase activity test and tissue inhibitor of metalloproteinases (TIMP)-1 ELISA levels were performed from the oral rinse samples. Distinct MMP-8 protein species were differentiated in the WB analysis. Principal component (PC) analysis was conducted to explore correlation patterns between the different species. Adjusted correlation analysis between the extracted PCs of WB and aMMP-8 IFMA levels and multilevel regression analysis were conducted to explore if the other periodontal disease-related biomarkers and clinical surrogate measures and patient-related factors are co-variating with the extracted components. Distinct correlation patterns between the MMP-8 protein species were observed. The first four PCs explained 89% of the whole variance in PC analysis. Statistically significant correlation (p < 0.05) were observed as follows: PC1 positively with 21 kDa (r = .69) and 25 kDa fragments (r = .55) and negatively with 150 kDa complexes (r = -.46). PC2 correlated with 45 (r = .70) and 55 kDa (r = .65) activated forms, PC3 with 70-80 kDa latent proforms (r = .63) and 90-100 kDa complexes (r = .67), and PC4 with 35 kDa fragments (r = .81). There were significant correlations between quantitative (IFMA) aMMP-8 measurements and PC1 (p < 0.001), PC2 (<0.05) and PC3 (<0.05) but not with PC4. In multilevel regression models age, PMN elastase activity, TIMP-1 levels, and a number of 4-5 mm periodontal pockets were associated with PC1, nonsmoking with PC2, ageand PMN elastase activity with PC3, and age and smoking with PC4. Relative abundancy of fragmented 21-25 kDa protein species was correlated with the quantitative aMMP-8 (IFMA) measurements, which is in line with previous results. Different patient-related factors (smoking, age, proteolytic activity) may modify the formation of different MMP-8 protein species in oral rinse samples and may cause variability in quantitative aMMP-8 measurement.

Cleavage fragments of the C-terminal tail of polycystin-1 are regulated by oxidative stress and induce mitochondrial dysfunction

Mutations in the gene encoding polycystin-1 (PC1) are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). Cysts in ADPKD exhibit a Warburg-like metabolism characterized by dysfunctional mitochondria and aerobic glycolysis. PC1 is an integral membrane protein with a large extracellular domain, a short C-terminal cytoplasmic tail and shares structural and functional similarities with G protein-coupled receptors. Its exact function remains unclear. The C-terminal cytoplasmic tail of PC1 undergoes proteolytic cleavage, generating soluble fragments that are overexpressed in ADPKD kidneys. The regulation, localization, and function of these fragments is poorly understood. Here, we show that a ∼30kDa cleavage fragment (PC1-p30), comprising the entire C-terminal tail, undergoes rapid proteasomal degradation by a mechanism involving the von Hippel-Lindau tumor suppressor protein. PC1-p30 is stabilized by reactive oxygen species, and the subcellular localization is regulated by reactive oxygen species in a dose-dependent manner. We found that a second, ∼15kDa fragment (PC1-p15), is generated by caspase cleavage at a conserved site (Asp-4195) on the PC1 C-terminal tail. PC1-p15 is not subject to degradation and constitutively localizes to the mitochondrial matrix. Both cleavage fragments induce mitochondrial fragmentation, and PC1-p15 expression causes impaired fatty acid oxidation and increased lactate production, indicative of a Warburg-like phenotype. Endogenous PC1 tail fragments accumulate in renal cyst-lining cells in a mouse model of PKD. Collectively, these results identify novel mechanisms regarding the regulation and function of PC1 and suggest that C-terminal PC1 fragments may be involved in the mitochondrial and metabolic abnormalities observed in ADPKD.

TMEM2 is a bona fide hyaluronidase possessing intrinsic catalytic activity

Transmembrane protein 2 (TMEM2) was originally identified as a membrane-anchored protein of unknown function. We previously demonstrated that TMEM2 can degrade hyaluronan (HA). Furthermore, we showed that induced global knockout of Tmem2 in adult mice results in rapid accumulation of incompletely degraded HA in bodily fluids and organs, supporting the identity of TMEM2 as a cell surface hyaluronidase. In spite of these advances, no direct evidence has been presented to demonstrate the intrinsic hyaluronidase activity of TMEM2. Here, we directly establish the catalytic activity of TMEM2. The ectodomain of TMEM2 (TMEM2ECD) was expressed as a His-tagged soluble protein and purified by affinity and size-exclusion chromatography. Both human and mouse TMEM2ECD robustly degrade fluorescein-labeled HA into 5 to 10kDa fragments. TMEM2ECD exhibits this HA-degrading activity irrespective of the species of TMEM2 origin and the position of epitope tag insertion. The HA-degrading activity of TMEM2ECD is more potent than that of HYAL2, a hyaluronidase which, like TMEM2, has been implicated in cell surface HA degradation. Finally, we show that TMEM2ECD can degrade not only fluorescein-labeled HA but also native high-molecular weight HA. In addition to these core findings, our study reveals hitherto unrecognized confounding factors, such as the quality of reagents and the choice of assay systems, that could lead to erroneous conclusions regarding the catalytic activity of TMEM2. In conclusion, our results demonstrate that TMEM2 is a legitimate functional hyaluronidase. Our findings also raise cautions regarding the choice of reagents and methods for performing degradation assays for hyaluronidases.

Chopped! Newfound GSDMD cleavage facilitates tolerance to food allergens

In a recent article, He et al. report that, in response to dietary protein antigens, mouse intestinal epithelial cells (IECs) accumulate a newfound 13-kDa N terminus of gasdermin D (GSDMD-N13), cleaved by caspase-3/7. Unlike the pyroptotic 30-kDa fragment, GSDMD-N13 translocates to the nucleus, inducing CIITA and major histocompatibility complex class II (MHCII) expression to promote type 1 regulatory T (T1r) cell development, thus revealing its role in balancing immunity and food tolerance.

Mutations F352A and Y528A in human HSP90α reduce fibronectin association and fibrillogenesis in cell-derived matrices.

HSP90 is a ubiquitously expressed chaperone protein that regulates the maturation of numerous substrate proteins called ‘clients’. The glycoprotein fibronectin (FN) is an important protein of the extracellular matrix (ECM) and a client protein of HSP90. FN and HSP90 interact directly, and the FN ECM is regulated by exogenous HSP90 or HSP90 inhibitors. Here, we extend the analysis of the HSP90 – FN interaction. The importance of the N-terminal 70-kDa fragment of fibronectin (FN70) and FN type I repeat was demonstrated by competition for FN binding between HSP90 and the functional upstream domain (FUD) of the Streptococcus pyogenes F1 adhesin protein. Furthermore, His-HSP90α mutations F352A and Y528A (alone and in combination) reduced the association with full-length FN (FN-FL) and FN70 in vitro. Unlike wild type His-HSP90α, these HSP90 mutants did not enhance FN matrix assembly in the Hs578T cell line model when added exogenously. Interestingly, the HSP90 E353A mutation, which did not significantly reduce the HSP90 – FN interaction in vitro, dramatically blocked FN matrix assembly in Hs578T cell-derived matrices. Taken together, these data extend our understanding of the role of HSP90 in FN fibrillogenesis and suggest that promotion of FN ECM assembly by HSP90 is not solely regulated by the affinity of the direct interaction between HSP90 and FN.

Structure of the transmembrane protein 2 (TMEM2) ectodomain and its apparent lack of hyaluronidase activity.

Background: Hyaluronic acid (HA) is a major polysaccharide component of the extracellular matrix. HA has essential functions in tissue architecture and the regulation of cell behaviour. HA turnover needs to be finely balanced. Increased HA degradation is associated with cancer, inflammation, and other pathological situations. Transmembrane protein 2 (TMEM2) is a cell surface protein that has been reported to degrade HA into ~5 kDa fragments and play an essential role in systemic HA turnover. Methods: We produced the soluble TMEM2 ectodomain (residues 106-1383; sTMEM2) in human embryonic kidney cells (HEK293) and determined its structure using X-ray crystallography. We tested sTMEM2 hyaluronidase activity using fluorescently labelled HA and size fractionation of reaction products. We tested HA binding in solution and using a glycan microarray. Results: Our crystal structure of sTMEM2 confirms a remarkably accurate prediction by AlphaFold. sTMEM2 contains a parallel β-helix typical of other polysaccharide-degrading enzymes, but an active site cannot be assigned with confidence. A lectin-like domain is inserted into the β-helix and predicted to be functional in carbohydrate binding. A second lectin-like domain at the C-terminus is unlikely to bind carbohydrates. We did not observe HA binding in two assay formats, suggesting a modest affinity at best. Unexpectedly, we were unable to observe any HA degradation by sTMEM2. Our negative results set an upper limit for k cat of approximately 10 -5 min -1. Conclusions: Although sTMEM2 contains domain types consistent with its suggested role in TMEM2 degradation, its hyaluronidase activity was undetectable. HA degradation by TMEM2 may require additional proteins and/or localisation at the cell surface.

Pharmacokinetics of Monoclonal Antibody and Antibody Fragments in The Mouse Eye Following Intravitreal Administration

Mice are rarely used in pharmacokinetic (PK) studies of ocular therapeutics due to the small size of their eyes and challenges in drug administration, tissue collection, and analysis of drug concentrations. Therefore, ocular PK of protein therapeutics in mouse eye following intravitreal (IVT) administration is not known. Here, we have presented the first of its kind investigation, to study the PK of 4 different size non-binding protein therapeutics in mouse plasma, cornea/ICB, vitreous humor, retina, and posterior cup (including choroid) following IVT administration. Administered proteins include trastuzumab (150 kDa) and F(ab)2 (100 kDa), Fab, and scFv (27 kDa) fragments of trastuzumab. An imaging and injection apparatus suitable for performing small (50 nL) IVT injections in mice was developed, and techniques for enucleation of the eye and dissection of ocular tissues were developed. Furthermore, a sensitive enzyme-linked immunosorbent assay (ELISA) for detection of proteins in very small amounts of ocular tissues were developed. It was observed that elimination from the vitreous chamber was the primary driver of PK in the cornea/ICB, retina, posterior cup, and plasma. Trastuzumab displays first-order kinetics in the vitreous humor with a half-life of 18.8 h. F(ab)2, Fab, and ScFv show biphasic PK profiles with distribution phases becoming more rapid as molecular weight decreases, and terminal elimination becoming longer as molecular weight decreases, with terminal half-lives of 16.3, 20.6, and 48.9 h, respectively. The mean residence times of trastuzumab, F(ab)2, Fab, and scFv in the vitreous humor were 26.0, 12.2, 10.7, and 8.16 h, respectively. It was found that the mean residence time in vitreous humor doubles with an increase in molecular weight of ∼69 kDa. Interestingly, the PK of proteins measured in the un-injected eye suggest the presence of a pathway for drug transfer between the eyes, which needs to be further validated. Overall, the findings presented here pave the way for drug discovery and development studies of protein therapeutics for ophthalmic indications in mice.

Rhomb-I, a P–I metalloproteinase from Lachesis muta rhombeata venom degrades vessel extra cellular matrix components and impairs platelet aggregation

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca2+ and Mg2+ ions, but inhibited by Co2+, Zn2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 °C. The amino acid sequence showed high homology with other snake venom metalloproteinases. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential basement membrane, extracellular matrix and plasma proteins. It preferentially cleaves the α-chains of fibrin (ogen). Rhomb-I inhibited convulxin- and von Willebrand factor (vWF)-induced aggregation on human platelets without significant effect on collagen-stimulated aggregation or other effectors. It digests vWF into a low-molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors glycoprotein (GP)Ibα and GPVI to release a 55-kDa soluble form. Both membrane glycoproteins GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding.

Abstract 423: β-Escin eradicates cancer stem-like population in HER2-positive breast cancer with trastuzumab resistance

Abstract Purpose: Trastuzumab resistance is a multifactorial phenomenon arising from the steric effects of p95HER2, activation of HER2 downstream signaling pathways, and the existence of cancer stem cells. We sought to examine the capacity of β-escin, an anti-inflammatory drug, to address trastuzumab resistance in HER2-positive breast cancer cells. Experimental designs: The effects of β-escin in trastuzumab-sensitive and -resistant cell lines were evaluated for apoptosis, HER2 expression, calpain activation and CSC-like properties. In vivo trastuzumab-resistant xenograft mice model was used to examine tumor growth, angiogenesis and organ toxicity of β-escin. Results: β-escin induced mitochondrial-mediated apoptosis accompanied by reactive oxygen species (ROS) production and increased active p18Bax fragmentation, leading to caspase-3/-7 activation. β-escin downregulated p95HER2, HER2 and HER3 as well as their phosphorylation. β-escin-induced HER2 degradation appears to be mediated by calpain activation. We observed that treatment with β-escin induced cleavage of HER2 and p95HER2 (at 75, 50 and 42 kDa fragments) in HER2 overexpressing MDA-MB-231 cells. Attenuation of CSC-related features by β-escin challenge was accompanied by marked reductions in CD44high/CD24low stem-like cells and aldehyde dehydrogenase 1 (ALDH1) activity as well as hindrance of mammosphere formation. β-escin administration also significantly retarded tumor growth and angiogenesis in a trastuzumab-resistant JIMT-1 xenograft model via downregulation of CSC-associated markers and intracellular domain HER2. Importantly, β-escin selectively inhibited malignant cells and was less toxic to normal mammary cells, and no toxic effects were found in liver and kidney function in animals. Conclusion: Herein, for the first time, we report the potent efficacy of β-escin, a drug repurposing candidate with an exceptional safety profile in addressing trastuzumab-resistant HER2-positive breast cancer. Citation Format: Soeun Park, Minsu Park, Dongmi Ko, Seongjae Kim, Juyeon Seo, Kee Dal Nam, Eunsun Jung, Yong koo Kang, Seojin Jang, So Ra Seock, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo, Jaeyoun Park. β-Escin eradicates cancer stem-like population in HER2-positive breast cancer with trastuzumab resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 423.

120-kDa FN-fragment Induces MCP-1 Production in Human Temporomandibular Joint Fibroblast-like Synoviocytes

Fibronectin is a component of the extracellular matrix in the synovium. Fibronectin fragments（FN-fragments）result from fibronectin breakdown by proteolytic enzymes released from an early inflammatory response. We examined the effect of a 120-kDa fibronectin fragment（120-kDa FN-fragment）in the inflammatory condition of temporomandibular joint（TMJ）synovitis. Fibroblast-like synoviocytes, which were isolated from the TMJ synovium using the outgrowth method, were treated with or without 120-kDa FN-fragment. The fibronectin fragment induced the expression of monocyte chemotactic protein（MCP）-1, -2, and -3 genes in fibroblast-like synoviocytes as seen with DNA microarray analysis and real-time PCR. Among 120-kDa FN-fragment response genes, MCP-1（also called CCL2）, which is the main MCP chemokine, was abundantly expressed. MCP-1 protein level was increased in the conditioned medium with fibroblast-like synoviocytes treated with 120-kDa FN-fragment using ELISA. Furthermore, experiments using inhibitors of signaling pathway revealed that 120-kDa FN-fragment up-regulated MCP-1 expression as a result of NF-κB activation. MCP-1 mainly induces monocyte/macrophage migration in inflammation caused by various diseases. These results suggest that production of the MCP chemokine by 120-kDa FN-fragment may be involved in promoting the inflammatory condition of TMJ.

Abstract GS5-08: Soluble E-cadherin: a novel prognostic biomarker and driver of brain metastasis in inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer with rapid onset and a strong propensity to spread to distant organs. Five-year overall survival (OS) rates remain poor, in part because of the high risk of brain metastasis: 19% of patients with IBC have brain metastases within the first 2 years after diagnosis. Our hypothesis for this study was that soluble E-cadherin (sEcad), an 80-kDa extracellular proteolytic fragment of full-length E-cadherin - a tumor promoter in IBC, is crucial for driving brain metastasis in IBC. Methods: We analyzed serum sEcad levels in from 348 IBC patients by ELISA. Four IBC cell lines [ER–/HER2+ (MDA-IBC3; SUM190) and ER–/HER2– (SUM149; BCX010)], human brain microvascular endothelial cells, and immortalized human astrocytes were used in this study. Stable overexpression of sEcad in IBC cell lines was achieved using lentiviral vectors. Mass spectrometry and Bio-ID-based proteomics assays, and RNA sequencing were used to identify sEcad-interacting proteins and potential mechanisms. In vivo, we studied tumor growth and brain metastasis in mice by injecting IBC cells into the mammary fatpad or tail vein, respectively, of SCID/Beige mice. Results: In IBC patients, higher serum sEcad levels correlated with poorer OS (p=0.02), earlier development of metastasis (p=0.006), and development of brain metastasis (p=0.04). On multivariable analysis, sEcad independently predicted OS (hazard ratio [HR]=2.07 [95% CI 1.19-3.60], p=0.01). In vitro, sEcad overexpression in IBC cell lines promoted anchorage-independent growth, migration, invasion, and resistance to anoikis. In vivo, sEcad-overexpressing SUM149 cells promoted primary tumor growth (p=0.007). Mice injected with sEcad-overexpressing MDA-IBC3 cells also had higher incidence of brain metastasis (100% vs 50%, p=0.03), metastatic burden (p=0.02) and number of metastases per mouse (p=0.0009), and had worse OS (p=0.0016), and brain metastasis-free survival (p=0.04), relative to controls. We further found that sEcad increased cancer cell adhesion to brain endothelial cells (p=0.01) and promoted induction of reactive astrocytes (as identified by high glial fibrillary acidic protein levels) in vitro and in vivo. Mechanistically, mass spectrometry and Bio-ID assays identified X-linked inhibitor of apoptosis protein (XIAP), a potent inhibitor of apoptotic cell death, as a novel binding partner of sEcad, which was validated through co-immunoprecipitation. Further analysis showed that sEcad bound to the BIR2 domain of XIAP. XIAP is the most potent and best-defined anti-apoptotic IAP family member, and it could induce NF-κB activation to inhibit tumor cell apoptosis Gene set enrichment analysis of RNA-seq profiling data showed activation of NF-kB signaling and downregulation of apoptotic pathways in the sEcad-overexpressing SUM149 cells compared with controls. Immunoblotting revealed that sEcad enhanced XIAP expression, activated NF-κB signaling, and inhibited cleavage of caspase-3 in IBC cells. Conclusions: We found that higher serum sEcad correlates with development of brain metastases and independently predicts poor OS in patients with IBC. We further found that sEcad promotes tumor growth and brain metastasis, perhaps via activation of XIAP/NF-κB signaling in breast cancer cells and promotion of endothelial cell adhesion and reactive astrocytosis in the brain microenvironment. These findings uncover a novel and crucial role for sEcad in brain metastasis and provide new insights and potential therapeutic targets for patients with metastatic IBC. Citation Format: Xiaoding Hu, Yun Xiong, Emilly S. Villodre, Juhee Song, Ganiraju C Manyam, Moises J Tacam, Jing Wang, Chandra Bartholomeusz, Debu Tripathy, Naoto T. Ueno, Wendy Woodward, Savitri Krishnamurthy, Junjie Chen, Bisrat Debeb. Soluble E-cadherin: a novel prognostic biomarker and driver of brain metastasis in inflammatory breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS5-08.

Structure of the transmembrane protein 2 (TMEM2) ectodomain and its lack of hyaluronidase activity

Background: Hyaluronic acid (HA) is a major polysaccharide component of the extracellular matrix. HA has essential functions in tissue architecture and the regulation of cell behaviour. HA turnover needs to be finely balanced. Increased HA degradation is associated with cancer, inflammation, and other pathological situations. Transmembrane protein 2 (TMEM2) is a cell surface protein that has been reported to degrade HA into ~5 kDa fragments and play an essential role in systemic HA turnover. Methods: We produced the soluble TMEM2 ectodomain (residues 106-1383; sTMEM2) in human embryonic kidney cells (HEK293) and determined its structure using X-ray crystallography. We tested sTMEM2 hyaluronidase activity using fluorescently labelled HA and size fractionation of reaction products. Results: Our crystal structure of sTMEM2 confirms a remarkably accurate prediction by AlphaFold. sTMEM2 contains a parallel β-helix typical of other polysaccharide-degrading enzymes, but an active site cannot be assigned with confidence. A lectin-like domain is inserted into the β-helix and predicted to be functional in carbohydrate binding. A second lectin-like domain at the C-terminus is unlikely to bind carbohydrates. Unexpectedly, we were unable to observe any HA degradation by sTMEM2. Our negative results set an upper limit for kcat of approximately 10-5 min-1. Conclusions: Although sTMEM2 contains domain types consistent with its suggested role in TMEM2 degradation, its hyaluronidase activity was undetectable. HA degradation by TMEM2 may require additional proteins and/or localisation at the cell surface.

Novel Small Molecule Positive Allosteric Modulator SPAM1 Triggers the Nuclear Translocation of PAC1-R to Exert Neuroprotective Effects through Neuron-Restrictive Silencer Factor.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts effective neuroprotective activity through its specific receptor, PAC1-R. We accidentally discovered that as a positive allosteric modulator (PAM) of PAC1-R, the small-molecule PAM (SPAM1) has a hydrazide-like structure, but different binding characteristics, from hydrazide for the N-terminal extracellular domain of PAC1-R (PAC1-R-EC1). SPAM1 had a significant neuroprotective effect against oxidative stress, both in a cell model treated with hydrogen peroxide (H2O2) and an aging mouse model induced by D-galactose (D-gal). SPAM1 was found to block the decrease in PACAP levels in brain tissues induced by D-gal and significantly induced the nuclear translocation of PAC1-R in PAC1R-CHO cells and mouse retinal ganglion cells. Nuclear PAC1-R was subjected to fragmentation and the nuclear 35 kDa, but not the 15 kDa fragments, of PAC1-R interacted with SP1 to upregulate the expression of Huntingtin (Htt), which then exerted a neuroprotective effect by attenuating the binding availability of the neuron-restrictive silencer factor (NRSF) to the neuron-restrictive silencer element (NRSE). This resulted in an upregulation of the expression of NRSF-related neuropeptides, including PACAP, the brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase (TH), and synapsin-1 (SYN1). The novel mechanism reported in this study indicates that SPAM1 has potential use as a drug, as it exerts a neuroprotective effect by regulating NRSF.

Pathological 25 kDa C-Terminal Fragments of TDP-43 Are Present in Lymphoblastoid Cell Lines and Extracellular Vesicles from Patients Affected by Frontotemporal Lobar Degeneration and Neuronal Ceroidolipofuscinosis Carrying a GRN Mutation.

Frontotemporal lobar degeneration (FTLD) is a complex disease, characterized by progressive degeneration of frontal and temporal lobes. Mutations in progranulin (GRN) gene have been found in up to 50% of patients with familial FTLD. Abnormal deposits of post-translationally-modified TAR DNA-binding protein of 43 kDa (TDP-43) represent one of the main hallmarks of the brain pathology. To investigate in peripheral cells the presence of the different TDP-43 forms, especially the toxic 25 kDa fragments, we analyzed lymphoblastoid cell lines (LCLs) and the derived extracellular vesicles (EVs) from patients carrying a GRN mutation, together with wild-type (WT) healthy controls. After characterizing EV sizes and concentrations by nanoparticle tracking analysis, we investigated the levels of different forms of the TDP-43 protein in LCLs and respective EVs by Western blot. Our results showed a trend of concentration decreasing in EVs derived from GRN-mutated LCLs, although not reaching statistical significance. A general increase in p-TDP-43 levels in GRN-mutated LCLs and EVs was observed. In particular, the toxic 25 kDa fragments of p-TDP-43 were only present in GRN-mutated LCLs and were absent in the WT controls. Furthermore, these fragments appeared to be more concentrated in EVs than in LCLs, suggesting a relevant role of EVs in spreading pathological molecules between cells.

Alzheimer's disease-associated U1 snRNP splicing dysfunction causes neuronal hyperexcitability and cognitive impairment.

Recent proteome and transcriptome profiling of Alzheimer's disease (AD) brains reveals RNA splicing dysfunction and U1 small nuclear ribonucleoprotein (snRNP) pathology containing U1-70K and its N-terminal 40-KDa fragment (N40K). Here we present a causative role of U1 snRNP dysfunction to neurodegeneration in primary neurons and transgenic mice (N40K-Tg), in which N40K expression exerts a dominant-negative effect to downregulate full-length U1-70K. N40K-Tg recapitulates N40K insolubility, erroneous splicing events, neuronal degeneration and cognitive impairment. Specifically, N40K-Tg shows the reduction of GABAergic synapse components (e.g., the GABA receptor subunit of GABRA2), and concomitant postsynaptic hyperexcitability that is rescued by a GABA receptor agonist. Crossing of N40K-Tg and the 5xFAD amyloidosis model indicates that the RNA splicing defect synergizes with the amyloid cascade to remodel the brain transcriptome and proteome, deregulate synaptic proteins, and accelerate cognitive decline. Thus, our results support the contribution of U1 snRNP-mediated splicing dysfunction to AD pathogenesis.

Cardiac troponin T degradation in end-stage renal disease patients appears to occur in vivo

Abstract Background Cardiac troponin T (cTnT) is a cornerstone biomarker for diagnosing myocardial infarction (MI). However, since the introduction of the high-sensitivity assays, cardiac troponins are also measured in pathologies with cardiac risk factors, such as end-stage renal disease (ESRD), making it more difficult for clinicians to diagnose MI. Previous studies of our group identified specific larger cTnT proteoforms for the acute phase of MI, while a study in serum of ESRD patients showed solely small cTnT fragments. However, others allocated the small cTnT fragments in serum of ESRD patients to a pre-analytic effect due to abundant thrombin generation in serum, as thrombin is known to cause fragmentation of cTnT. Purpose This study investigated the effect of multiple anticoagulation methods on cTnT proteoforms in vitro and in the blood of ESRD patients. Methods First, cTnT negative serum and lithium-heparinized (LH), ethylenediaminetetraacetic-acid (EDTA), and hirudin plasma tubes were spiked with human cTnT standard, incubated at 37°C till 48 hours, and analysed with Western Blotting using anti-cTnT Roche antibodies. Second, similar blood tubes were collected from ESRD patients (n=10) conform the Declaration of Helsinki, patient samples and spiked cTnT standards were separated using gel filtration chromatography (GFC) and cTnT was subsequently analysed using the Cobas 6000 analyser. Results After 48 hours of in vitro incubation of cTnT in hirudin plasma, no cTnT degradation was observed, while LH only showed minor degradation of 10% from intact 40 kDa cTnT to 29 kDa fragments (figure 1). Also, for both EDTA and serum, a time-dependent degradation from 40 kDa cTnT to 29 kDa fragments to 15–18 kDa fragments was observed. Moreover, in ESRD patients, GFC elution profiles of all blood tubes revealed that 85–98% of cTnT corresponded to small 15–18 kDa fragments (41–42 mL), while 2–15% were 29/40 kDa cTnT forms (27–28 mL). For comparison, standards of ternary T-I-C complex and intact cTnT eluted at 21 mL and 28 mL, respectively. Conclusions The extent of cTnT degradation in vitro is dependent on the anticoagulation method of the blood tubes, with absence of degradation exclusively in hirudin plasma. Since in ESRD patients for all blood tubes mainly small 15–18 kDa cTnT fragments were found, including hirudin plasma, cTnT degradation appears to occur in vivo. This observation supports the hypothesis that larger cTnT proteoforms might indeed be specific for the acute phase of MI, thereby supporting the opportunity for developing a more specific cTnT method. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ZonMw Veni grant