Structural analysis of a U-superfamily conotoxin containing a mini-granulin fold: Insights into key features that distinguish between the ICK and granulin folds.

We are entering an exciting time in structural biology where artificial intelligence can be used to predict protein structures with greater accuracy than ever before. Extending this level of accuracy to the predictions of disulfide-rich peptide structures is likely to be more challenging, at least in the short term, given the tight packing of cysteine residues and the numerous ways that the disulfide bonds can potentially be linked. It has been previously shown in many cases that several disulfide bond connectivities can be accommodated by a single set of NMR-derived structural data without significant violations. Disulfide-rich peptides are prevalent throughout nature, and arguably the most well-known are those present in venoms from organisms such as cone snails. Here, we have determined the first three-dimensional structure and disulfide connectivity of a U-superfamily cone snail venom peptide, TxVIIB. TxVIIB has a VI/VII cysteine framework that is generally associated with an inhibitor cystine knot (ICK) fold; however, AlphaFold predicted that the peptide adopts a mini-granulin fold with a granulin disulfide connectivity. Our experimental studies using NMR spectroscopy and orthogonal protection of cysteine residues indicate that TxVIIB indeed adopts a mini-granulin fold but with the ICK disulfide connectivity. Our findings provide structural insight into the underlying features that govern formation of the mini-granulin fold rather than the ICK fold and will provide fundamental information for prediction algorithms, as the subtle complexity of disulfide isomers may be not adequately addressed by the current prediction algorithms.

Granulin in renal tubular epithelia is associated with interstitial inflammation and activates the TLR9-IFN-α pathway in lupus nephritis.

OBJECTIVE: This study aimed to investigate the possible role of granulin (GRN) in activating the TLR9-IFN-α pathway in renal tubular epithelial cells (RTECs) and explore clues that RTECs regulate the micro-environment of inflammatory response in lupus nephritis (LN). METHODS: Renal sections from 57 LN patients and 30 non-LN patients were sampled for histological study, and GRN overexpression RTECs were applied for cytological study. RESULTS: In the histological study, GRN is highly expressed in LN RTECs with tubulointerstitial inflammation (TII) and well co-localized with TLR9. ROC analysis suggested a potential relationship between GRN expression in RTECs and therapeutic response. Moreover, IFN-α also highly expressed in LN RTECs with TII, and the intensity of IFN-α is positively correlated with the co-localization intensity of GRN and TLR9. In the cytological study, LN serum, especially serum from LN with TII, activates the expression of TLR9 in RTECs, and GRN engages the interaction of TLR9 to activate the expression of IFN-α in RTECs. While TLR9 inhibitors can suppress the expression of IFN-α in RTECs, the degree of inhibition is dose-dependent. CONCLUSION: The expression of GRN in RTECs is associated with interstitial inflammation and therapeutic response. GRN may mediate the activation of the TLR9-IFN-α pathway in RTECs and involve in the micro-environment of inflammatory response in LN.

The progranulin cleavage product granulin 3 exerts a dominant negative effect on animal fitness.

Progranulin is an evolutionarily conserved protein that has been implicated in human neurodevelopmental and neurodegenerative diseases. Human progranulin is comprised of multiple cysteine-rich, biologically active granulin peptides. Granulin peptides accumulate with age and stress, however their functional contributions relative to full-length progranulin remain unclear. To address this, we generated C. elegans strains that produced quantifiable levels of both full-length progranulin/PGRN-1 protein and cleaved granulin peptide. Using these strains, we demonstrated that even in the presence of intact PGRN-1, granulin peptides suppressed the activity of the lysosomal aspartyl protease activity, ASP-3/CTSD. Granulin peptides were also dominant over PGRN-1 in compromising animal fitness as measured by progress through development and stress response. Finally, the degradation of human TDP-43 was impaired when the granulin to PGRN-1 ratio was increased, representing a disease-relevant downstream impact of impaired lysosomal function. In summary, these studies suggest that not only absolute progranulin levels, but also the balance between full-length progranulin and its cleavage products, is important in regulating lysosomal biology. Given its relevance in human disease, this suggests that the processing of progranulin into granulins should be considered as part of disease pathobiology and may represent a site of therapeutic intervention.

Patient with PSEN1 Glu318Gly and Other Possible Disease Risk Mutations, Diagnosed with Early Onset Alzheimer's Disease.

In this manuscript, we introduced a French EOAD patient in Korea who carried the presenilin-1 (PSEN1) Glu318Gly mutations with four possible risk variants, including sortilin-related receptor 1 (SORL1) Glu270Lys, ATP-binding cassette subfamily A member 7 (ABCA7) Val1946Met, translocase of outer mitochondrial membrane 40 (TOMM40) Arg239Trp, and granulin (GRN) Ala505Gly. The patient started to present memory decline and behavioral dysfunction in his early 60s. His brain imaging presented amyloid deposits by positron emission tomography (PET-CT). The multimer detection system (MDS) screening test for plasma for amyloid oligomers was also positive, which supported the AD diagnosis. It was verified that PSEN1 Glu318Gly itself may not impact amyloid production. However, additional variants were found in other AD and non-AD risk genes, as follows: SORL1 Glu270Lys was suggested as a risk mutation for AD and could increase amyloid peptide production and impair endosome functions. ABCA7 Val1946Met was a novel variant that was predicted to be damaging. The GRN Ala505Gly was a variant with uncertain significance; however, it may reduce the granulin levels in the plasma of dementia patients. Pathway analysis revealed that PSEN1 Glu318Gly may work as a risk factor along with the SORL1 and ABCA7 variants since pathway analysis revealed that PSEN1 could directly interact with them through amyloid-related and lipid metabolism pathways. TOMM40 and PSEN1 could have common mechanisms through mitochondrial dysfunction. It may be possible that PSEN1 Glu318Gly and GRN Ala505Gly would impact disease by impairing immune-related pathways, including microglia and astrocyte development, or NFkB-related pathways. Taken together, the five risk factors may contribute to disease-related pathways, including amyloid and lipid metabolism, or impair immune mechanisms.

A microglial activity state biomarker panel differentiates FTD-granulin and Alzheimer's disease patients from controls.

BACKGROUND: With the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states. METHODS: Using mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), genetically modified to yield the most opposite homeostatic (TREM2-knockout) and disease-associated (GRN-knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify proteomic changes in microglia and cerebrospinal fluid (CSF) of Grn- and Trem2-knockout mice. Additionally, we analyzed the proteome of GRN- and TREM2-knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort (GRN mutation carriers versus non-carriers), as well as the proteomic data set available from the EMIF-AD MBD study. RESULTS: We identified proteomic changes between the opposite activation states in mouse microglia and CSF, as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous GRN mutation carriers suffering from frontotemporal dementia (FTD). We identified a panel of six proteins (FABP3, MDH1, GDI1, CAPG, CD44, GPNMB) as potential indicators for microglial activation. Moreover, we confirmed three of these proteins (FABP3, GDI1, MDH1) to be significantly elevated in the CSF of Alzheimer's (AD) patients. Remarkably, each of these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals. CONCLUSIONS: The identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the Dominantly Inherited Alzheimer's Disease Network (DIAN) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates (FABP3, CD44, GPNMB) also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders.

Clonorchis sinensis granulin promotes malignant transformation of human intrahepatic biliary epithelial cells through interaction with M2 macrophages via regulation of STAT3 phosphorylation and the MEK/ERK pathway.

BACKGROUND: Clonorchis sinensis granulin (CsGRN), a component of the excretory/secretory products of this species, is a multifunctional growth factor that can promote the metastasis of cholangiocarcinoma cells. However, the effect of CsGRN on human intrahepatic biliary epithelial cells (HIBECs) is unclear. Here, we investigated the effect of CsGRN on the malignant transformation of HIBECs and its possible underlying mechanism. METHODS: The malignant transformation phenotypes of HIBECs after CsGRN treatment were estimated by EdU-488 incorporation assay, colony formation assay, wound-healing assay, Transwell assay and western blot. The biliary damage of CsGRN-treated mice was detected by western blot, immunohistochemical staining and hematoxylin and eosin staining. The phenotypes of the macrophages [human monocytic leukemia cell line (THP-1)] were analyzed by flow cytometry, immunofluorescence and immunohistochemistry, both in vitro and in vivo. A co-culture system was developed to explore the interaction between THP-1 and HIBECs in CsGRN-containing medium. Enzyme-linked immunosorbent assay and western blot were used to detected the activation of interleukin 6 (IL-6), phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. An inhibitor of the MEK/ERK pathway, PD98059, was used to determine whether this pathway is involved in CsGRN-mediated cell interactions as well as in STAT3 phosphorylation and malignant transformation of HIBECs. RESULTS: Excessive hyperplasia and abnormal proliferation of HIBECs, enhanced secretion of hepatic pro-inflammatory cytokines and chemokines, as well as biliary damage, were observed in vitro and in vivo after treatment with CsGRN. The expression of the markers of M2 macrophages significantly increased in CsGRN-treated THP-1 cells and biliary duct tissues compared with the controls. Moreover, following treatment with CsGRN, the HIBECs underwent malignant transformation in the THP-1-HIBECs co-culture group. In addition, high expression of IL-6 was observed in the CsGRN-treated co-culture media, which activated the phosphorylation of STAT3, JAK2, MEK and ERK. However, treatment with an inhibitor of the MEK/ERK pathway, PD98059, decreased expression of p-STAT3 in CsGRN-treated HIBECs and further repressed the malignant transformation of HIBECs. CONCLUSIONS: Our results demonstrated that, by inducing the M2-type polarization of macrophages and activating the IL-6/JAK2/STAT3 and MEK/ERK pathways in HIBECs, CsGRN promoted the malignant transformation of the latter.

Granulin loss of function in human mature brain organoids implicates astrocytes in TDP-43 pathology.

Loss of function (LoF) of TAR-DNA binding protein 43 (TDP-43) and mis-localization, together with TDP-43-positive and hyperphosphorylated inclusions, are found in post-mortem tissue of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those carrying LoF variants in the progranulin gene (GRN). Modeling TDP-43 pathology has been challenging in vivo and in vitro. We present a three-dimensional induced pluripotent stem cell (iPSC)-derived paradigm-mature brain organoids (mbOrg)-composed of cortical-like-astrocytes (iA) and neurons. When devoid of GRN, mbOrgs spontaneously recapitulate TDP-43 mis-localization, hyperphosphorylation, and LoF phenotypes. Mixing and matching genotypes in mbOrgs showed that GRN-/- iA are drivers for TDP-43 pathology. Finally, we rescued TDP-43 LoF by adding exogenous progranulin, demonstrating a link between TDP-43 LoF and progranulin expression. In conclusion, we present an iPSC-derived platform that shows striking features of human TDP-43 proteinopathy and provides a tool for the mechanistic modeling of TDP-43 pathology and patient-tailored therapeutic screening for FTD and ALS.

Progranulin from different gliocytes in the nucleus accumbens exerts distinct roles in FTD- and neuroinflammation-induced depression-like behaviors.

BACKGROUND: Neuroinflammation in the nucleus accumbens (NAc) is well known to influence the progression of depression. However, the molecular mechanisms triggering NAc neuroinflammation in depression have not been fully elucidated. Progranulin (PGRN) is a multifunctional growth factor that is linked to the innate immune response and inflammation, and PGRN plays a key role in neurodegenerative diseases such as frontotemporal dementia (FTD). Here, the purpose of this study was to validate whether PGRN was involved in the NAc neuroinflammation-promoted depressive-like phenotype. METHODS: A NAc neuroinflammation-relevant depression-like model was established using wild-type (WT) and PGRN-knockout (KO) mice after NAc injection with lipopolysaccharide (LPS), and various behavioral tests related to cognition, social recognition, depression and anxiety were performed with WT and PGRNKO mice with or without NAc immune challenge. RT‒PCR, ELISA, western blotting and immunofluorescence staining were used to determine the expression and function of PGRN in the neuroinflammatory reaction in the NAc after LPS challenge. The morphology of neurons in the NAc from WT and PGRNKO mice under conditions of NAc neuroinflammation was analyzed using Golgi-Cox staining, followed by Sholl analyses. The potential signaling pathways involved in NAc neuroinflammation in PGRNKO mice were investigated by western blotting. RESULTS: Under normal conditions, PGRN deficiency induced FTD-like behaviors in mice and astrocyte activation in the NAc, promoted the release of the inflammatory cytokines interleukin (IL)-6 and IL-10 and increased dendritic complexity and synaptic protein BDNF levels in the NAc. However, NAc neuroinflammation enhanced PGRN expression, which was located in astrocytes and microglia within the NAc, and PGRN deficiency in mice alleviated NAc neuroinflammation-elicited depression-like behaviors, seemingly inhibiting astrocyte- and microglia-related inflammatory reactions and neuroplasticity complexity in the NAc via the p38 and nuclear factor of kappa (NF-κB) signaling pathways present in the NAc after neuroinflammation. CONCLUSIONS: Our results suggest that PGRN exerts distinct function on different behaviors, showing protective roles in the FTD-like behavior and detrimental effects on the neuroinflammation-related depression-like behavior, resulting from mediating astrocyte and microglial functions from the NAc in different status.

Deficiency of the frontotemporal dementia gene GRN results in gangliosidosis.

Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN-mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases.

Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis.

Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

Progranulin (PGRN) is a glycoprotein implicated in several neurodegenerative diseases. It is highly expressed in microglia and macrophages and can be secreted or delivered to the lysosome compartment. PGRN comprises 7.5 granulin repeats and is processed into individual granulin peptides within the lysosome, but the functions of these peptides are largely unknown. Here, we identify CD68, a lysosome membrane protein mainly expressed in hematopoietic cells, as a binding partner of PGRN and PGRN-derived granulin E. Deletion analysis of CD68 showed that this interaction is mediated by the mucin-proline-rich domain of CD68. While CD68 deficiency does not affect the lysosomal localization of PGRN, it results in a specific decrease in the levels of granulin E but no other granulin peptides. On the other hand, the deficiency of PGRN, and its derivative granulin peptides, leads to a significant shift in the molecular weight of CD68, without altering CD68 localization within the cell. Our results support that granulin E and CD68 reciprocally regulate each other's protein homeostasis.

Granulin as an important immune molecule involved in lamprey tissue repair and regeneration by promoting cell proliferation and migration.

Progranulin (PGRN) is an autocrine growth factor that regulates cell proliferation, migration, wound healing, and tissue repair in mammals. Lamprey is the most primitive of the extant vertebrates and is regarded as the survivor of a once flourishing group of paleozoic vertebrates, with a history of more than 500 million years. To date, the evolutionary dynamics and the underlying function of the PGRNs remain largely unclear in lamprey. Here, we screened four genes encoding PGRNs from the genomes of Lethenteron reissneri and Petromyzon marinus, including one long form (named Lr-PGRN-L) and three short forms (named Lr-PGRN-S1, Lr-PGRN-S2, and Lr-PGRN-S3), and performed phylogenetic tree, functional domain, and synteny analyses to identify the evolutionary history of the four Lr-PGRNs. In addition, the expressions of the four Lr-pgrn family genes and the immune response against various pathogenic challenges were also investigated. We found that these genes were widely distributed in various tissues of lamprey and performed a variety of functions. Moreover, our results suggest that Lr-PGRN-S1 induces cell migration and proliferation, and is involved in repair after skin and spinal cord injury under appropriate conditions. Our findings are valuable because they improve the understanding of the evolutionary relationship of vertebrate pgrn genes, as well as providing new insights into the diverse and important roles of Lr-PGRNs.

Charge and redox states modulate granulin-TDP-43 coacervation toward phase separation or aggregation.

Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. To study the factors that influence the dynamics between these cytoplasmic foci, we investigated the effects of cysteine-rich granulins (GRNs 1-7), which are the proteolytic products of progranulin, a protein implicated in FTLD, on TDP-43. We show that extracellular GRNs, typically generated during inflammation, internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show less likelihood of their presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Using charge patterning and conserved cysteines among the wild-type GRNs as guides, along with specifically engineered mutants, we discover that the negative charges on GRNs drive LLPS while the positive charges and the redox state of cysteines modulate these phase transitions. Furthermore, RNA and GRNs compete and expel one another from PrLD condensates, providing a basis for GRN's absence in SGs. Together, the results help uncover potential modulatory mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize and modulate cytoplasmic TDP-43 inclusions in proteinopathies.

Differential regulation of progranulin derived granulin peptides.

BACKGROUND: Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). PGRN is comprised of 7.5 granulin repeats and is processed into individual granulin peptides in the lysosome. However, very little is known about the levels and regulations of individual granulin peptides due to the lack of specific antibodies. RESULTS: Here we report the generation and characterization of antibodies specific to each granulin peptide. We found that the levels of granulins C, E and F are regulated differently  compared to granulins A and B in various tissues. The levels of PGRN and granulin peptides vary in different brain regions and the ratio between granulins and PGRN is highest in the cortical region in the adult male mouse brain. Granulin-A is localized in the lysosome in both neurons and microglia and its levels in microglia increase under pathological conditions. Interestingly,  the levels of granulin A in microglia change correspondingly with PGRN in response to stroke but not demyelination. Furthermore, deficiency of lysosomal proteases and the PGRN binding partner prosaposin leads to alterations in the ratios between individual granulin peptides. Granulins B, C and E are heavily glycosylated and the glycosylation patterns can be regulated. CONCLUSION: Our results support that the levels of individual granulin peptides are differentially regulated under physiological and pathological conditions and provide novel insights into how granulin peptides function in the lysosome.

Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration.

BACKGROUND: Progranulin loss-of-function mutations are linked to frontotemporal lobar degeneration with TDP-43 positive inclusions (FTLD-TDP-Pgrn). Progranulin (PGRN) is an intracellular and secreted pro-protein that is proteolytically cleaved into individual granulin peptides, which are increasingly thought to contribute to FTLD-TDP-Pgrn disease pathophysiology. Intracellular PGRN is processed into granulins in the endo-lysosomal compartments. Therefore, to better understand the conversion of intracellular PGRN into granulins, we systematically tested the ability of different classes of endo-lysosomal proteases to process PGRN at a range of pH setpoints. RESULTS: In vitro cleavage assays identified multiple enzymes that can process human PGRN into multi- and single-granulin fragments in a pH-dependent manner. We confirmed the role of cathepsin B and cathepsin L in PGRN processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, G, K, S and V) are able to process PGRN in distinctive, pH-dependent manners. In addition, we have demonstrated a new role for asparagine endopeptidase (AEP) in processing PGRN, with AEP having the unique ability to liberate granulin F from the pro-protein. Brain tissue from individuals with FTLD-TDP-Pgrn showed increased PGRN processing to granulin F and increased AEP activity in degenerating brain regions but not in regions unaffected by disease. CONCLUSIONS: This study demonstrates that multiple lysosomal proteases may work in concert to liberate multi-granulin fragments and granulins. It also implicates both AEP and granulin F in the neurobiology of FTLD-TDP-Pgrn. Modulating progranulin cleavage and granulin production may represent therapeutic strategies for FTLD-Pgrn and other progranulin-related diseases.

Granulin: An Invasive and Survival-Determining Marker in Colorectal Cancer Patients.

BACKGROUND: Granulin is a secreted, glycosylated peptide-originated by cleavage from a precursor protein-which is involved in cell growth, tumor invasion and angiogenesis. However, the specific prognostic impact of granulin in human colorectal cancer has only been studied to a limited extent. Thus, we wanted to assess the expression of granulin in colorectal cancer patients to evaluate its potential as a prognostic biomarker. METHODS: Expressional differences of granulin in colorectal carcinoma tissue (n = 94) and corresponding healthy colon mucosa were assessed using qRT-PCR. Immunohistochemistry was performed in colorectal cancer specimens (n = 97), corresponding healthy mucosa (n = 47) and colorectal adenomas (n = 19). Subsequently, the results were correlated with histopathological and clinical patients' data. HCT-116 cells were transfected with siRNA for invasion and migration assays. RESULTS: Immunohistochemistry and qRT-PCR revealed tumoral over expression of granulin in colorectal cancer specimens compared to corresponding healthy colon mucosa and adenomas. Tumoral overexpression of granulin was associated with a significantly impaired overall survival. Moreover, downregulation of granulin by siRNA significantly diminished the invasive capacities of HCT-116 cells in vitro. CONCLUSION: Expression of granulin differs in colorectal cancer tissue, adenomas and healthy colon mucosa. Furthermore, granulin features invasive and migrative capabilities and overexpression of granulin correlates with a dismal prognosis. This reveals its potential as a prognostic biomarker and granulin could be a worthwhile molecular target for individualized anticancer therapy.

Circular RNA circ_0008274 upregulates granulin to promote the progression of hepatocellular carcinoma via sponging microRNA -140-3p.

Circular RNA (circRNA) features prominently in the progression of hepatocellular carcinoma (HCC), of which the biological function and potential mechanism of circ_0008274 in HCC are obscure. The present study aims to explore circ_ 0008274's biological functions and underlying mechanisms in HCC. The expressions of circ_0008274, miR-140-3p and Granulin (GRN) mRNA in HCC tissues and cells were investigated by quantitative real-time polymerase chain reaction. Besides, GRN protein expression was measured by Western blot. Furthermore, chi-square test was used to probe the interrelation between circ_0008274 expression and clinicopathological parameters. In addition, cell counting kit-8 (CCK-8) and EdU assays were applied to detect cell proliferation. Moreover, transwell assay was used to detect cell migration and invasion. What's more, bioinformatics prediction, dual-luciferase reporter gene assay and RNA Immunoprecipitation experiments were used to corroborate the targeting interrelations among circ_0008274, miR-140-3p and GRN. Herein we reported that circ_0008274 was highly expressed in HCC, and its high expression enhanced the proliferation, migration, and invasion of HCC cells, while depleting circ_0008274 inhibited the malignant biological behaviors of HCC cells. Mechanistically, circ_0008274 upregulates GRN expressions via adsorbing miR-140-3p to expedite the progression of HCC.

Are granulins copper sequestering proteins?

Granulins (GRN 1-7) are short (~6 kDa), cysteine-rich proteins that are generated upon the proteolytic processing of progranulin (PGRN). These peptides, along with their precursor, have been implicated in multiple pathophysiological roles, especially in neurodegenerative diseases. Previously we showed that GRN-3 and GRN-5 are fully disordered in the reduced form implicating redox sensitive attributes to the proteins. Redox-based modulations are often carried out by metalloproteins in mitigating oxidative stress and maintaining metal-homeostasis within cells. To probe whether GRNs play a role in metal sequestration, we tested the metal binding propensity of the reduced forms of GRNs -3 and - 5 under neutral and acidic pH mimicking cytosolic and lysosomal conditions, respectively. We found, at neutral pH, both GRNs selectively bind Cu and no other divalent metal cations, with a greater specificity for Cu(I). Binding of Cu did not result in a disorder-to-order structural transition but partly triggered the multimerization of GRNs via uncoordinated cystines at both pH conditions. Overall, the results indicate that GRNs -3 and - 5 have surprisingly strong affinity for Cu in the pM range, comparable to other known copper sequestering proteins. The results also hint at a potential of GRNs to reduce Cu(II) to Cu(I), a process that has significance in mitigating Cu-induced ROS cytotoxicity in cells. Together, this report uncovers metal-coordinating property of GRNs for the first time, which may have profound significance in their structure and pathophysiological functions.

Integrative Genomic Analysis Reveals Four Protein Biomarkers for Platelet Traits.

RATIONALE: Mean platelet volume (MPV) and platelet count (PLT) are platelet measures that have been linked to cardiovascular disease (CVD) and mortality risk. Identifying protein biomarkers for these measures may yield insights into CVD mechanisms. OBJECTIVE: We aimed to identify causal protein biomarkers for MPV and PLT among 71 CVD-related plasma proteins measured in FHS (Framingham Heart Study) participants. METHODS AND RESULTS: We conducted integrative analyses of genetic variants associated with PLT/MPV with protein quantitative trait locus variants associated with plasma proteins followed by Mendelian randomization to infer causal relations of proteins for PLT/MPV. We also tested protein-PLT/MPV association in FHS participants. Using induced pluripotent stem cell-derived megakaryocyte clones that produce functional platelets, we conducted RNA-sequencing and analyzed expression differences between low- and high-platelet producing clones. We then performed small interfering RNA gene knockdown experiments targeting genes encoding proteins with putatively causal platelet effects in megakaryocyte clones to examine effects on platelet production. In protein-trait association analyses, ten proteins were associated with MPV and 31 with PLT. Mendelian randomization identified 4 putatively causal proteins for MPV and 4 for PLT. GP-5 (Glycoprotein V), GRN (granulin), and MCAM (melanoma cell adhesion molecule) were associated with PLT, while MPO (myeloperoxidase) showed significant association with MPV in both analyses. RNA-sequencing analysis results were directionally concordant with observed and Mendelian randomization-inferred associations for GP-5, GRN, and MCAM. In siRNA gene knockdown experiments, silencing GP-5, GRN, and MPO decreased PLTs. Genome-wide association study results suggest several of these may be linked to CVD risk. CONCLUSIONS: We identified 4 proteins that are causally linked to PLTs. These proteins may also have roles in the pathogenesis of CVD via a platelet/blood coagulation-based mechanism.

Liver fluke granulin promotes extracellular vesicle-mediated crosstalk and cellular microenvironment conducive to cholangiocarcinoma.

Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with the liver fluke is a major risk factor for cholangiocarcinoma (CCA). The liver fluke Opisthorchis viverrini secretes a growth factor termed liver fluke granulin, a homologue of the human progranulin, which contributes significantly to biliary tract fibrosis and morbidity. Here, extracellular vesicle (EV)-mediated transfer of mRNAs from human cholangiocytes to naïve recipient cells was investigated following exposure to liver fluke granulin. To minimize the influence of endogenous progranulin, its cognate gene was inactivated using CRISPR/Cas9-based gene knock-out. Several progranulin-depleted cell lines, termed ΔhuPGRN-H69, were established. These lines exhibited >80% reductions in levels of specific transcript and progranulin, both in gene-edited cells and within EVs released by these cells. Profiles of extracellular vesicle RNAs (evRNA) from ΔhuPGRN-H69 for CCA-associated characteristics revealed a paucity of transcripts for estrogen- and Wnt-signaling pathways, peptidase inhibitors and tyrosine phosphatase related to cellular processes including oncogenic transformation. Several CCA-specific evRNAs including MAPK/AKT pathway members were induced by exposure to liver fluke granulin. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 cells exposed to liver fluke granulin. Of these, CCA-associated evRNAs modified the CCA microenvironment in naïve cells co-cultured with EVs from ΔhuPGRN-H69 cells exposed to liver fluke granulin, and induced translation of MAPK phosphorylation related-protein in naïve recipient cells in comparison with control recipient cells. Exosome-mediated crosstalk in response to liver fluke granulin promoted a CCA-specific program through MAPK pathway which, in turn, established a CCA-conducive disposition.