Degradation Of Extracellular Proteins Research Articles

LYTACs: An Emerging Tool for the Degradation of Non-Cytosolic Proteins.

In the last two decades, targeted protein degradation has rapidly gained popularity as a technique to eliminate disease-causing undruggable proteins. Over the years, many tools have been devised to degrade proteins by exploiting natural protein homeostasis machinery available in our body, with LYTACs being the latest to come on board. LYTACs, or lysosome-targeting chimeras, make use of the lysosome degradation pathway by recruiting proteins to lysosome-shuttling receptors located at the cell surface. LYTACs are specifically meant for the degradation of membrane-bound and extracellular proteins, which account for the products of 40 % of all protein-encoding genes. In this highlight, we describe two studies that demonstrate the scope of LYTACs and its advantages over the other protein degradation platforms. In the first study, the LYTAC utilizes the cation-independent mannose-6-phosphate receptor (CI-M6PR), while the second study uses the asialoglycoprotein receptor (ASGPR) which is found only on the surface of liver cells.

Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins.

Targeted protein degradation (TPD) technology has drawn significant attention from researchers in both academia and industry. It is rapidly evolved as a new therapeutic modality and also a useful chemical tool in selectively depleting various protein targets. As most efforts focus on cytosolic proteins using PROteolysis TArgeting Chimera (PROTAC), LYsosome TArgeting Chimera (LYTAC) recently emerged as a promising technology to deliver extracellular protein targets to lysosome for degradation through the cation-independent mannose-6-phosphate receptor (CI-M6PR). In this study, we exploited the potential of the asialoglycoprotein receptor (ASGPR), a lysosomal targeting receptor specifically expressed on liver cells, for the degradation of extracellular proteins including membrane proteins. The ligand of ASGPR, triantennary N-acetylgalactosamine (tri-GalNAc), was conjugated to biotin, antibodies, or fragments of antibodies to generate a new class of degraders. We demonstrated that the extracellular protein targets could be successfully internalized and delivered into lysosome for degradation in liver cell lines specifically by these degraders. This work will add a new dimension to TPD with cell type specificity.

Role of autophagy in nonsegmental vitiligo Naguid and Rashed

Background Vitiligo is a skin and less common hair disease characterized by decline in melanocyte function and depigmentation, with a prevalence of 0.5–1% in most populations. Autophagy is the degradation of components of the cytoplasm within lysosomes. This is distinct from endocytosis-mediated lysosomal degradation of extracellular and plasma membrane proteins. Aim The aim was to detect biochemical parameter light chain 3 (LC3) to monitor autophagy in vitiligo skin of patients as compared with normal control persons to evaluate the role of autophagy in the vitiligo pathogenesis. Materials and methods This case–control study included 60 patients with vitiligo and 60 age-matched and sex-matched healthy controls. Herein, 4 mm punch skin biopsy was taken from every patient (vitiligo lesion) and control and kept in lysis solution for the stability of the studied parameters and was kept frozen at −80°C till analysis of autophagy protein LC3 by qRT PCR. Results The level of LC3 in lesional skin of vitiligo was significantly lower as compared with normal control persons.

Extracellular protein degradation on demand

A protein degradation platform enables the targeted depletion of extracellular and membrane-bound proteins.

Abstract MP154: Deletion of the Duffy Antigen Receptor for Chemokines (DARC) Confers Protection Against Abdominal Aortic Aneurysm (AAA) Formation

Introduction: Abdominal aortic aneurysms (AAA) are characterized by inflammation and matrix metalloproteinase (MMP)-mediated degradation of extra cellular matrix proteins leading to aortic dilation and potentially rupture. Inflammatory chemokines that promote AAA are modulated by binding to the Duffy antigen receptor for chemokines (DARC), a non-signaling receptor expressed primarily on erythrocytes. Interestingly, African Americans exhibit reduced frequency of AAA, and the majority of African descendent individuals do not express DARC on their erythrocytes. Here, we tested the hypothesis that DARC gene deletion protects against the development of AAA. Methods: The induction of AAA was performed using both angiotensin II (AngII) infusion and calcium chloride (CaCl 2 ) application models. Eight week old LDLR knockout (KO, control) and LDLR KO/DARC KO mice were infused with AngII via osmotic mini-pumps for four weeks. For the CaCl 2 application model, twelve week old DARC KO and WT control mice underwent laparotomy and 0.5 mol/L CaCl 2 was applied to the infrarenal aorta. Aortic dilation and AAA formation was assessed using ultrasound, mice were then euthanized and tissues were harvested for analysis. Results: The aortic diameter of LDLR KO/DARC KO mice was significantly lower relative to control mice after AngII infusion (P=0.02). There was no difference in the pressor response to AngII between groups. Furthermore, IL-6 levels were significantly reduced in the aortas of LDLR KO/DARC KO mice compared to control mice (P=0.001). Expression of MCP-1, which has strong affinity for DARC, tended to be higher in aortas from LDLR KO/DARC KO mice compared to control (P=0.063). In contrast, plasma IL-6 levels were similar in both LDLR KO/DARC KO and control mice, while MCP-1 tended to be lower in the plasma of LDLR KO/DARC KO (P=0.07). DARC KO mice likewise exhibited a trend toward reduced aortic dilation in the CaCl 2 application model compared to controls (P=0.09). Conclusions: DARC KO mice are protected against AAA formation, perhaps through differential regulation of aortic chemokine trafficking. Understanding the mechanisms by which loss of DARC confers protection from AAA formation may be relevant to ethnic differences in susceptibility to AAA.

Lysosome-targeting chimaeras for degradation of extracellular proteins.

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoproteinE4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.

Deletion of the Duffy Antigen Receptor for Chemokines (DARC) Confers Protection Against Abdominal Aortic Aneurysm (AAA) Formation

IntroductionAbdominal aortic aneurysms (AAA) are characterized by inflammation and matrix metalloproteinase (MMP)‐mediated degradation of extra cellular matrix proteins leading to aortic dilation and potentially rupture. Inflammatory chemokines that promote AAA are modulated by binding to the Duffy antigen receptor for chemokines (DARC), a non‐signaling receptor expressed primarily on erythrocytes. Interestingly, African Americans exhibit reduced frequency of AAA, and greater than 70% of individuals of African descent do not express DARC on their erythrocytes due to evolutionary pressure from malaria. Here, we tested the hypothesis that DARC gene deletion protects against the development of AAA.MethodsThe induction of AAA was performed using both angiotensin II (AngII) infusion and calcium chloride (CaCl2) application models. In brief, eight week old LDLR knockout (KO, control) and LDLR KO/DARC KO mice were infused with angiotensin II via osmotic minipumps for four weeks at a rate of 1,000 ng/kg/min. For the CaCl2 application model, 12 week old WT and DARC KO mice underwent laparotomy and 0.5 mol/L was applied to the infrarenal aorta. For both models, aortic dilation and AAA formation was assessed using ultrasound, mice were then euthanized and tissues were harvested for histology and biochemical analysis.ResultsLDLR KO/DARC KO mice were protected from the formation of AngII‐induced AAA, as only ~20% developed an aneurysm compared to ~80% in the control group. Concordantly, the aortic diameter of LDLR KO/DARC KO mice was significantly lower relative to control mice after AngII infusion (P=0.02). There was no difference in the pressor response to AngII between groups, but LDLR KO/DARC KO mice exhibited reduced macrophage infiltration, and aortic dilation compared to the control group. Furthermore, IL‐6 levels were significantly reduced in the aortas of LDLR KO/DARC KO mice compared to control mice (P=0.001). Interestingly, however, expression of MCP‐1, which has strong affinity for DARC, tended to be higher in aortas from LDLR KO/DARC KO mice compared to control (P=0.063). In contrast, plasma IL‐6 levels were similar in both LDLR KO/DARC KO and control mice, while MCP‐1 tended to be lower in the plasma of LDLR KO/DARC KO (P=0.07). Interestingly, DARC appears to be upregulated in the aortas of mice in response to AngII. Furthermore, DARC KO mice likewise exhibited reduced aortic dilation in the CaCl2 application model compared to controls.ConclusionsDARC KO mice are protected against AAA formation, perhaps through differential regulation of aortic chemokine trafficking. Understanding the mechanisms by which loss of DARC confers protection from AAA formation may be relevant to ethnic differences in susceptibility to AAA.Support or Funding InformationThis study was supported by the AHA [19PRE34400043 to T.W.B] and the National Institutes of Health [HL124097, HL126949, HL134354 and AR070029 to N.L.W].

Heparan sulfate and clusterin: Cleaning squad for extracellular protein degradation.

The mechanisms of quality control for extracellular proteins are still poorly understood. In this issue, Itakura et al. (2020. J. Cell. Biol.https://doi.org/10.1083/jcb.201911126) show that upon binding to misfolded proteins, the extracellular chaperone clusterin is internalized via the heparan sulfate receptor to undergo lysosomal degradation.

Targeted Degradation of Extracellular Proteins with ENDTACs

Key words protein degradation - receptor internalization - extracellular proteins

Identification of a factor controlling lysosomal homeostasis using a novel lysosomal trafficking probe

Lysosomes are largely responsible for significant degradation of intracellular and extracellular proteins via the secretory pathway, autophagy, and endocytosis. Therefore, dysregulation of lysosomal homeostasis influences diverse cellular functions. However, a straightforward and quantitative method to measure the integrity of the lysosomal pathway has not been developed. Here, we report the plasmid-based lysosomal-METRIQ (MEasurement of protein Transporting integrity by RatIo Quantification) probe that enables simple quantification of lysosomal integrity by lysosomal green and cytosolic red fluorescent proteins using a flow cytometer. In cultured cells, the lysosomal-METRIQ probe detected not only suppression of the lysosomal pathway but also upregulation of lysosomal activity such as lysosomal biogenesis. To identify factors involved in lysosomal homeostasis, we carried out compound screening and found that the cyclin-dependent kinase (CDK) inhibitors kenpaullone and purvalanol A induce synthesis of cathepsin D and an increase in the number of lysosomes. Subsequent studies revealed that CDK5 maintains lysosomal homeostasis independently of cell cycle arrest. Our results suggest that the lysosomal-METRIQ probe is an effective and efficient tool for measuring lysosomal activity in mammalian cells.

Wnt canonical pathway activates macropinocytosis and lysosomal degradation of extracellular proteins

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Wnt treatment markedly increased the endocytosis and degradation in lysosomes of BSA. In this study, we report that in addition to receptor-mediated endocytosis, Wnt also triggers the intake of large amounts of extracellular fluid by macropinocytosis, a nonreceptor-mediated actin-driven process. Macropinocytosis induction is rapid and independent of protein synthesis. In the presence of Wnt, large amounts of nutrient-rich packages such as proteins and glycoproteins were channeled into lysosomes after fusing with smaller receptor-mediated vesicles containing glycogen synthase kinase 3 (GSK3) and protein arginine ethyltransferase 1 (PRMT1), an enzyme required for canonical Wnt signaling. Addition of Wnt3a, as well as overexpression of Disheveled (Dvl), Frizzled (Fz8), or dominant-negative Axin induced endocytosis. Depletion of the tumor suppressors adenomatous polyposis coli (APC) or Axin dramatically increased macropinocytosis, defined by incorporation of the high molecular weight marker tetramethylrhodamine (TMR)-dextran and its blockage by the Na+/H+ exchanger ethylisopropyl amiloride (EIPA). Macropinocytosis was blocked by dominant-negative vacuolar protein sorting 4 (Vps4), indicating that the Wnt pathway is dependent on multivesicular body formation, a process called microautophagy. SW480 colorectal cancer cells displayed constitutive macropinocytosis and increased extracellular protein degradation in lysosomes, which were suppressed by restoring full-length APC. Accumulation of the transcriptional activator β-catenin in the nucleus of SW480 cells was inhibited by methyltransferase inhibition, EIPA, or the diuretic amiloride. The results indicate that Wnt signaling switches metabolism toward nutrient acquisition by engulfment of extracellular fluids and suggest possible treatments for Wnt-driven cancer progression.

Multiplex Matrix Metalloproteinases Analysis in the Cerebrospinal Fluid Reveals Potential Specific Patterns in Multiple Sclerosis Patients.

Background: Matrix metalloproteinases (MMPs) are pleiotropic enzymes involved in extracellular protein degradation and turnover. MMPs are implicated in the pathogenesis of many neurological diseases, including multiple sclerosis (MS).Objective: To search the level of MMPs in the cerebrospinal fluid (CSF) of MS patients and detect possible disease-specific patterns.Methods: CSF samples from 32 MS patients and, from 15 control subjects with other inflammatory neurological diseases (OIND) were analyzed. The Bio-Plex Pro Human MMP 9-Plex Panel (Bio-Rad) was used for the quantification of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, and MMP-13.Results: CSF MMP-1 and MMP-12 levels were significantly reduced in MS as compared with OIND. In MS patients' CSF: (i) MMP-1 levels were significantly higher in women vs. men; (ii) MMP-10 concentrations were higher in patients with CSF-restricted IgG oligoclonal bands, and (iii) MMP-7 levels were increased in patients with longer disease duration. In the OIND group MMP-7 and MMP-12 levels significantly and directly correlated with age.Conclusions: Our study contributes to investigating the role of MMPs in MS, with regard to CSF immunological features and disease duration. Sex-specific differences were also detected in MMPs CSF levels.

Integrated comparison of the miRNAome and mRNAome in muscles of dermatomyositis and polymyositis reveals common and specific miRNA-mRNAs.

Dermatomyositis (DM) and polymyositis (PM) are refractory systemic autoimmune diseases with unknown pathogenesis. miRNAs is an important epigenetic mechanism to regulate gene expression. We performed whole miRNAs analysis, transcription analysis and the association between miRNAome and mRNAome. For transcription and miRNAs analysis, there were common and specific mRNAs and miRNAs in the muscles of DM and PM. Among them, the expression levels of miR-196a-5p and CPM were negatively correlated in PM, miR-193b-3p and NECAP2 were negatively correlated in DM and PM. Protein carboxypeptidase M (CPM) plays roles in the degradation of extracellular proteins and in the migration and invasion of cancer cells, and protein NECAP2plays roles in adaptor protein AP-1-mediated fast recycling from early endosomes. The functions of them in the pathogenesis of DM/PM need further studies. Our study identified and confirmed differentially miRNAs and mRNAs in DM and PM. Our observations have laid the groundwork for further diagnostic and mechanistic studies of DM and PM.

Transcription Factor Eb Is Required for Macropinocytosis-Mediated Growth Recovery of Nutrient-Deprived Kras-Mutant Cells.

Macropinocytosis is a regulated form of endocytosis that mediates the nonselective uptake of nutrients to support growth under nutrient-deprived conditions. KRAS-mutant cancer cells upregulate macropinocytosis to import extracellular proteins, which subsequently undergo proteolytic degradation in the lysosome. Although transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and function, its role in the degradation of extracellular protein from macropinocytosis in KRAS-mutant cells has not previously been elucidated. In this study, we investigated the role of TFEB in the recovery of macropinocytosis-mediated mTORC1 activity and cell growth under nutrient depletion. Mouse embryonic fibroblasts (MEFs) expressing KrasG12D and KRAS-mutant human cancer cells took up markedly higher levels of tetramethylrhodamine (TMR)-dextran than the corresponding wild-type cells. siRNA-mediated inhibition of TFEB did not influence extracellular TMR-dextran uptake, but significantly attenuated lysosomal degradation of extracellular protein. Bovine serum albumin (BSA) treatment restored p-S6K levels and cell proliferation suppressed by leucine deprivation, and these effects were blocked by siTFEB. Collectively, our results show that TFEB plays a role in macropinocytosis-mediated KRAS-mutant cell growth under nutrient deprivation by promoting lysosomal degradation of extracellular proteins.

Comparative Genomics Reveals Evidence of Genome Reduction and High Extracellular Protein Degradation Potential in Kangiella.

The genus Kangiella has recently been proposed within the family Kangiellaceae, belonging to order Oceanospirillales. Here, we report the complete genome sequence of a novel strain, Kangiella profundi FT102, which is the only Kangiella species isolated from a deep sea sediment sample. Furthermore, gaps in the publicly available genome scaffold of K. aquimarina DSM 16071 (NCBI Reference Sequence: NZ_ARFE00000000.1) were also filled using polymerase chain reaction (PCR) and Sanger sequencing. A comparative genomic analysis of five Kangiella and 18 non-Kangiella strains revealed insights into their metabolic potential. It was shown that low genomic redundancy and Kangiella-lineage-specific gene loss are the key reasons behind the genome reduction in Kangiella compared to that in any other free-living Oceanospirillales strain. The occurrence of relatively diverse and more frequent extracellular protease-coding genes along with the incomplete carbohydrate metabolic pathways in the genome suggests that Kangiella has high extracellular protein degradation potential. Growth of Kangiella strains has been observed using amino acids as the only carbon and nitrogen source and tends to increase with additional tryptone. Here, we propose that extracellular protein degradation and amino acid utilization are significant and prominent features of Kangiella. Our study provides more insight into the genomic traits and proteolytic metabolic capabilities of Kangiella.

Urinary phthalate metabolite concentrations in relation to levels of circulating matrix metalloproteinases in pregnant women

Phthalate exposure has been shown to be associated with adverse pregnancy outcomes. However, human studies informing relevant mechanistic pathways are lacking. Experimental studies have reported that matrix metalloproteinases (MMPs), which are responsible for extracellular protein degradation, may be upregulated in response to phthalate exposure. In this exploratory study we measured urinary phthalate metabolite concentrations, plasma MMP levels, and relevant covariates among 134 pregnant women. There were statistically significant or suggestive positive relationships between several phthalates, particularly between metabolites of di-(2-ethylhexyl) phthalate, with MMP-1 and MMP-9 levels. Further research is needed to confirm these results and how they may inform the mechanisms involved between phthalate exposure and adverse pregnancy outcomes.

SNAT7 is the primary lysosomal glutamine exporter required for extracellular protein-dependent growth of cancer cells

Lysosomes degrade cellular components sequestered by autophagy or extracellular material internalized by endocytosis and phagocytosis. The macromolecule building blocks released by lysosomal hydrolysis are then exported to the cytosol by lysosomal transporters, which remain undercharacterized. In this study, we designed an in situ assay of lysosomal amino acid export based on the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis that detects lysosomal storage. This assay was used to screen candidate lysosomal transporters, leading to the identification of sodium-coupled neutral amino acid transporter 7 (SNAT7), encoded by the SLC38A7 gene, as a lysosomal transporter highly selective for glutamine and asparagine. Cell fractionation confirmed the lysosomal localization of SNAT7, and flux measurements confirmed its substrate selectivity and showed a strong activation by the lysosomal pH gradient. Interestingly, gene silencing or editing experiments revealed that SNAT7 is the primary permeation pathway for glutamine across the lysosomal membrane and it is required for growth of cancer cells in a low free-glutamine environment, when macropinocytosis and lysosomal degradation of extracellular proteins are used as an alternative source of amino acids. SNAT7 may, thus, represent a novel target for glutamine-related anticancer therapies.

Matrix metalloproteinases outside vertebrates

The matrix metalloproteinase (MMP) family belongs to the metzincin clan of zinc-dependent metallopeptidases. Due to their enormous implications in physiology and disease, MMPs have mainly been studied in vertebrates. They are engaged in extracellular protein processing and degradation, and present extensive paralogy, with 23 forms in humans. One characteristic of MMPs is a ~165-residue catalytic domain (CD), which has been structurally studied for 14 MMPs from human, mouse, rat, pig and the oral-microbiome bacterium Tannerella forsythia. These studies revealed close overall coincidence and characteristic structural features, which distinguish MMPs from other metzincins and give rise to a sequence pattern for their identification. Here, we reviewed the literature available on MMPs outside vertebrates and performed database searches for potential MMP CDs in invertebrates, plants, fungi, viruses, protists, archaea and bacteria. These and previous results revealed that MMPs are widely present in several copies in Eumetazoa and higher plants (Tracheophyta), but have just token presence in eukaryotic algae. A few dozen sequences were found in Ascomycota (within fungi) and in double-stranded DNA viruses infecting invertebrates (within viruses). In contrast, a few hundred sequences were found in archaea and >1000 in bacteria, with several copies for some species. Most of the archaeal and bacterial phyla containing potential MMPs are present in human oral and gut microbiomes. Overall, MMP-like sequences are present across all kingdoms of life, but their asymmetric distribution contradicts the vertical descent model from a eubacterial or archaeal ancestor. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Pancreatic Tumors Exhibit Enhanced Extracellular Protein Catabolism

Abstract PDACs gain necessary amino acids via macropinocytosis and degradation of extracellular proteins.

Genomic reconstruction of multiple lineages of uncultured benthic archaea suggests distinct biogeochemical roles and ecological niches.

Genomic bins belonging to multiple archaeal lineages were recovered from distinct redox regimes in sediments of the White Oak River estuary. The reconstructed archaeal genomes were identified as belonging to the rice cluster subgroups III and V (RC-III, RC-V), the Marine Benthic Group D (MBG-D), and a newly described archaeal class, the Theionarchaea. The metabolic capabilities of these uncultured archaea were inferred and indicated a common capability for extracellular protein degradation, supplemented by other pathways. The multiple genomic bins within the MBG-D archaea shared a nearly complete reductive acetyl-CoA pathway suggesting acetogenic capabilities. In contrast, the RC-III metabolism appeared centered on the degradation of detrital proteins and production of H2, whereas the RC-V archaea lacked capabilities for protein degradation and uptake, and appeared to be specialized on carbohydrate fermentation. The Theionarchaea appeared as complex metabolic hybrids; encoding a complete tricarboxylic acid cycle permitting carbon (acetyl-CoA) oxidation, together with a complete reductive acetyl-CoA pathway and sulfur reduction by a sulfhydrogenase. The differentiated inferred capabilities of these uncultured archaeal lineages indicated lineage-specific linkages with the nitrogen, carbon and sulfur cycles. The predicted metabolisms of these archaea suggest preferences for distinct geochemical niches within the estuarine sedimentary environment.