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Early lung carcinoma development may be modulated by innate host cellular mechanisms that promote tumor growth and invasion. We recently identified how a loss-of-function mutation in the glycan sulfating enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in heparan sulfate biosynthesis) targeted to antigen presenting cells (APCs) may augment acquired anti-tumor T cell immune mechanisms. Crossing this mutation (Ndst1f/f CD11cCre+) onto a model of inducible spontaneous Kras mutant lung cancer [CCSP-rtTA; (tetO7) CMV-Kras-G12D] allowed us to examine how the APC mutation affects the formation and growth of early lung carcinoma. We examined early bronchocentric adenoma formation in the model, and the frequency of such events was significantly reduced on the mutant background. This was associated with significant reductions in tumor associated FOXP3+ cellular infiltration and CD163+ M2-type macrophage infiltration. The findings evolved prior to effector CD8+ T cell infiltration into tumors. The impact of this unique glycan under-sulfating mutation on inhibiting early Kras G12D mutant bronchocentric adenoma formation along with a cellular phenotype of inhibited tumor infiltration by cells involved in suppressive T-regulatory cell signaling (FOXP3+ cells) or tumor-permissive M2 macrophage functions (CD163+ cells) provides insight on how glycan targeting may modulate innate cellular mechanisms during early lung tumor development. The findings may also impact the future design of host-centered immunologic anti-tumor therapeutic strategies.

While recent research points to the importance of glycans in cancer immunity, knowledge on functional mechanisms is lacking. In lung carcinoma among other tumors, anti-tumor immunity is suppressed; and while some recent therapies boost T-cell mediated immunity by targeting immune-checkpoint pathways, robust responses are uncommon. Augmenting tumor antigen-specific immune responses by endogenous dendritic cells (DCs) is appealing from a specificity standpoint, but challenging. Here, we show that restricting a heparan sulfate (HS) loss-of-function mutation in the HS sulfating enzyme Ndst1 to predominantly conventional DCs (Ndst1f/f CD11cCre+ mutation) results in marked inhibition of Lewis lung carcinoma growth along with increased tumor-associated CD8+ T cells. In mice deficient in a major DC HS proteoglycan (syndecan-4), splenic CD8+ T cells showed increased anti-tumor cytotoxic responses relative to controls. Studies examining Ndst1f/f CD11cCre + mutants revealed that mutation was associated with an increase in anti-tumor cytolysis using either splenic CD8+ T cells or tumor-infiltrating (TIL) CD8+ T cells purified ex-vivo, and tested in pooled effector-to-target cytolytic assays against tumor cells from respective animals. On glycan compositional analysis, HS purified from Ndst1f/f CD11cCre + mutant DCs had reduced overall sulfation, including reduced sulfation of a tri-sulfated disaccharide species that was intriguingly abundant on wildtype DC HS. Interestingly, antigen presentation in the context of major histocompatibility complex class-I (MHC-I) was enhanced in mutant DCs, with more striking effects in the setting of HS under-sulfation, pointing to a likely regulatory role by sulfated glycans at the antigen/MHC-I - T-cell interface; and possibly future opportunities to improve antigen-specific T cell responses by immunologic targeting of HS proteoglycans in cancer.

Auto/paracrine factors secreted from cells affect differentiation of human pluripotent stem cells (hPSCs). However, the molecular mechanisms underlying the role of secreted factors are not well known. We previously showed that pattern formation in hPSCs induced by BMP4 could be reproduced by a simple reaction-diffusion of BMP and Noggin, a cell-secreted BMP4 inhibitor. However, the amount of Noggin secreted is unknown. In this study, we measured the concentration of Noggin secreted during the differentiation of hPSCs induced by BMP4. The Noggin concentration in the supernatant before and after differentiation was constant at approximately 0.69 ng/mL, which is approximately 50–200 times less than expected in the model. To explain the difference between the experiment and model, we assumed that macromolecules such as heparan sulfate proteoglycan on the cell surface act as a diffusion barrier structure, where the diffusion slows down to 1/400. The model with the diffusion barrier structure reduced the Noggin concentration required to suppress differentiation in the static culture model. The model also qualitatively reproduced the pattern formation, in which only the upstream but not the downstream hPSCs were differentiated in a one-directional perfusion culture chamber, with a small change in the amount of secreted Noggin resulting in a large change in the differentiation position. These results suggest that the diffusion barrier on the cell surface might enhance the auto/paracrine effects on monolayer hPSC culture., Highlights • Noggin was constantly secreted at about 0.69 ng/mL irrespective of cell state. • Noggin concentration was 1/145 than expected in the mere diffusion-reaction model. • Slow diffusion on the cell surface reduced the Noggin concentration in the medium. • The diffusion barrier reproduced pattern formation in the microchamber.

The extracellular matrix (ECM) is a three-dimensional network of macromolecules that provides a microenvironment capable of supporting and regulating cell functions. However, only a few research organisms are available for the systematic dissection of the composition and functions of the ECM, particularly during regeneration. We utilized the free-living flatworm Schmidtea mediterranea to develop an integrative approach consisting of decellularization, proteomics, and RNAi to characterize and investigate ECM functions during tissue homeostasis and regeneration. ECM-enriched samples were isolated from planarians, and their proteomes were characterized by LC–MS/MS. The functions of identified ECM components were interrogated using RNA interference. Using this approach, we found that heparan sulfate proteoglycan is essential for tissue regeneration. Our strategy provides an experimental approach for identifying both known and novel ECM components involved in regeneration., Graphical Abstract, Highlights • Decellularization method developed to isolate ECM from complete animals. • Optimization of decellularization method for protein MS compatibility. • Identification of known and novel ECM components for their function in tissue regeneration., In Brief The ECM is a three-dimensional network of macromolecules that supports and regulates cell functions. Yet, only a few research organisms are available for the systematic dissection of the composition and functions of the ECM, particularly during regeneration. In this study, we report an integrative approach involving whole animal decellularization, protein mass spectrometry (LC–MS/MS), and RNA interference-based loss-of-function assays to identify both known and novel ECM components involved in regeneration.

The recent emergence of coronavirus disease-2019 (COVID-19) as a global pandemic has prompted scientists to address an urgent need for defining mechanisms of disease pathology and treatment. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19, employs angiotensin converting enzyme 2 (ACE2) as its primary target for cell surface attachment and likely entry into the host cell. Thus, understanding factors that may regulate the expression and function of ACE2 in the healthy and diseased body is critical for clinical intervention. Over 66% of all adults in the United States are currently using a prescription drug and while earlier findings have focused on possible upregulation of ACE2 expression through the use of renin angiotensin system (RAS) inhibitors, mounting evidence suggests that various other widely administered drugs used in the treatment of hypertension, heart failure, diabetes mellitus, hyperlipidemias, coagulation disorders, and pulmonary disease may also present a varied risk for COVID-19. Specifically, we summarize mechanisms on how heparin, statins, steroids and phytochemicals, besides their established therapeutic effects, may also interfere with SARS-CoV-2 viral entry into cells. We also describe evidence on the effect of several vitamins, phytochemicals, and naturally occurring compounds on ACE2 expression and activity in various tissues and disease models. This comprehensive review aims to provide a timely compendium on the potential impact of commonly prescribed drugs and pharmacologically active compounds on COVID-19 pathology and risk through regulation of ACE2 and RAS signaling.

HIV-1 matrix protein p17 variants (vp17s) derived from non-Hodgkin's lymphoma (NHL) tissues of HIV-1–seropositive (HIV+) patients promote B-cell growth by activating the Akt signaling pathway. It is fundamental to understand the role played by vp17s in producing a microenvironment that fosters lymphoma development and progression. Therefore, we asked whether vp17s could be secreted from infected cells in their biologically active form. In this study, we show that two B-cell growth-promoting vp17s, NHL-a101 and NHL-a102, characterized by amino acid insertions at position 117 to 118 (Ala–Ala) or 125 to 126 (Gly–Asn), respectively, are secreted from HIV-1–infected Jurkat T cells during the active phase of viral replication. Secretion of biologically active vp17s also occurred in HeLa cells nucleofected with a plasmid expressing the entire Gag gene, following proteolytic cleavage of the Gag precursor polyprotein (Pr55Gag) by cellular aspartyl proteases. Binding of Pr55Gag to phosphatidylinositol-(4,5)-bisphosphate was indispensable for allowing the unconventional secretion of both wildtype p17 and vp17s. Indeed, here we demonstrate that inhibition of Pr55Gag binding to phosphatidylinositol-(4,5)-bisphosphate by using neomycin, or its enzymatic depletion achieved by overexpression of 5ptaseIV, significantly impair the secretion of p17s. We also demonstrated that heparan sulfate proteoglycans were involved in tethering p17s at the cell surface. This finding opens up an interesting way for investigating whether tethered p17s on the surface of HIV-1 reservoirs may represent a likely target for immune-mediated killing.

High plasma triglyceride (TG) levels and low HDL-C levels are risk factors for atherosclerosis and cardiovascular disease. Both plasma TG and HDL-C levels are regulated in part by the circulating inhibitor, angiopoietin-like 3 (ANGPTL3). ANGPTL3 inhibits the phospholipase, endothelial lipase (EL), which hydrolyzes the phospholipids of HDL, thus decreasing plasma HDL levels. ANGPTL3 also inhibits LPL, the lipase primarily responsible for the clearance of TGs from the circulation. Previous studies have shown that ANGPTL3 requires complex formation with the related ANGPTL protein, angiopoietin-like 8 (ANGPTL8), to efficiently inhibit LPL, but the role of ANGPTL8 in EL inhibition is not known. In this study, we characterized inhibition and binding of EL by ANGPTL3 and investigated the role of ANGPTL8 in EL inhibition. We found that inhibition of EL by ANGPTL3 was dose dependent and temperature dependent. Interestingly, this inhibition was diminished when EL was bound to endothelial cells or in the presence of heparin. Unlike previous findings with LPL, we found that ANGPTL8 did not significantly alter the binding or the inhibition of EL by ANGPTL3. In addition, we found that a common ANGPTL8 variant, which encodes an R59W mutation, altered the ability of ANGPTL3 to bind and inhibit LPL but not EL. Together, our data indicate that ANGPTL8 is not necessary for EL inhibition. We conclude that ANGPTL8 is specific for the regulation of TG-rich lipoproteins through the LPL pathway and that therapeutically targeting ANGPTL8 for the treatment of hypertriglyceridemia or cardiovascular disease may have different outcomes than targeting ANGPTL3.

Tauopathies, such as Alzheimer's disease (AD), are neurodegenerative disorders characterized by the deposition of hyperphosphorylated tau aggregates. Proteopathic tau seeds spread through the brain in a temporospatial pattern, indicative of transsynaptic propagation. It is hypothesized that reducing the uptake of tau seeds and subsequent induction of tau aggregation could be a potential approach for abrogating disease progression in AD. Here, we studied to what extent different endosomal routes play a role in the neuronal uptake of preformed tau seeds. Using pharmacological and genetic tools, we identified dynamin-1, actin, and Rac1 as key players. Furthermore, inhibition of PIKfyve, a protein downstream of Rac1, reduced both the trafficking of tau seeds into lysosomes and the induction of tau aggregation. Our work shows that tau aggregates are internalized by a specific endocytic mechanism and that their fate once internalized can be pharmacologically modulated to reduce tau seeding in neurons.

Type XV collagen is a non-fibrillar collagen that is associated with basement membranes and belongs to the multiplexin subset of the collagen superfamily. Collagen XV was initially studied because of its sequence homology with collagen XVIII/endostatin whose anti-angiogenic and anti-tumorigenic properties were subjects of wide interest in the past years. But during the last fifteen years, collagen XV has gained growing attention with increasing number of studies that have attributed new functions to this widely distributed collagen/proteoglycan hybrid molecule. Despite the cumulative evidence of its functional pleiotropy and its evolutionary conserved function, no review compiling the current state of the art about collagen XV is currently available. Here, we thus provide the first comprehensive view of the knowledge gathered so far on the molecular structure, tissue distribution and functions of collagen XV in development, tissue homeostasis and disease with an evolutionary perspective. We hope that our review will open new roads for promising research on collagen XV in the coming years., Highlights • Type XV collagen belongs to the multiplexin subset of the collagen superfamily. • It is evolutionarily conserved collagen and associated with basement membranes. • This collagen/proteoglycan hybrid molecule contains an anti-angiogenic restin domain. • It has important functions in the cardiovascular and the neuromuscular systems. • Its expression is dysregulated in various diseases including cancers.

In Alzheimer's disease (AD), pathological forms of tau are transferred from cell to cell and "seed" aggregation of cytoplasmic tau. Phosphorylation of tau plays a key role in neurodegenerative tauopathies. In addition, apolipoprotein E (apoE), a major component of lipoproteins in the brain, is a genetic risk determinant for AD. The identification of the apoE receptor, low-density lipoprotein receptor-related protein 1 (LRP1), as an endocytic receptor for tau raises several questions about the role of LRP1 in tauopathies: is internalized tau, like other LRP1 ligands, delivered to lysosomes for degradation, and does LRP1 internalize pathological tau leading to cytosolic seeding? We found that LRP1 rapidly internalizes 125I-labeled tau, which is then efficiently degraded in lysosomal compartments. Surface plasmon resonance experiments confirm high affinity binding of tau and the tau microtubule-binding domain to LRP1. Interestingly, phosphorylated forms of recombinant tau bind weakly to LRP1 and are less efficiently internalized by LRP1. LRP1-mediated uptake of tau is inhibited by apoE, with the apoE4 isoform being the most potent inhibitor, likely because of its higher affinity for LRP1. Employing post-translationally-modified tau derived from brain lysates of human AD brain tissue, we found that LRP1-expressing cells, but not LRP1-deficient cells, promote cytosolic tau seeding in a process enhanced by apoE. These studies identify LRP1 as an endocytic receptor that binds and processes monomeric forms of tau leading to its degradation and promotes seeding by pathological forms of tau. The balance of these processes may be fundamental to the spread of neuropathology across the brain in AD.

Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells., Graphical abstract Unlabelled Image, Highlights • Adenovirus entry into cells is guided by specific glycan and protein interactions. • Glycan arrays and shotgun glycomics methods are valuable technologies to identify virus–glycan interactions. • Shotgun proteomics and ligand-based receptor capture are powerful methods for proteinaceous receptor discovery. • A combination of shotgun glycomics and proteomics with CRISPR/Cas9 and RNAi validation holds the promise of generating a systems biology view of virus entry processes.

Because cartilage lacks nerves, blood vessels, and lymphatic vessels, it is thought to contain factors that inhibit the growth and development of those tissues. Chondroitin sulfate proteoglycans (CSPGs) are a major extracellular component in cartilage. CSPGs contribute to joint flexibility and regulate extracellular signaling via their attached glycosaminoglycan, chondroitin sulfate (CS). CS and CSPG inhibit axonal regeneration; however, their role in blood vessel formation is largely unknown. To clarify the function of CSPG in blood vessel formation, we tested salmon nasal cartilage proteoglycan (PG), a member of the aggrecan family of CSPG, for endothelial capillary-like tube formation. Treatment with salmon PG inhibited endothelial cell adhesion and in vitro tube formation. The anti-angiogenic activity was derived from CS in the salmon PG but not the core protein. Salmon PG also reduced matrix metalloproteinase expression and inhibited angiogenesis in the chick chorioallantoic membrane. All of these data support an anti-angiogenic role for CSPG in cartilage., Highlights • The role of CSPGs in blood vessel formation in cartilage is largely unknown. • Treatment of salmon PG inhibited in vitro and in vivo angiogenesis. • The CS portion of salmon PG was responsible for the anti-angiogenic activity. • Salmon PG also reduced MMP expression and inhibited cell adhesion. • Our results support an anti-angiogenic role for CSPG in cartilage.

The heparan sulfate (HS) proteoglycan perlecan is a major component of basement membranes, plays a key role in extracellular matrix (ECM) structure, interacts with growth factors and adhesion molecules, and regulates the adhesion, differentiation and proliferation of vascular cells. Atherosclerosis is characterized by chronic inflammation and the presence of oxidized materials within lesions, with the majority of protein damage present on ECM, rather than cell, proteins. Weakening of ECM structure plays a key role in lesion rupture, the major cause of heart attacks and strokes. In this study peroxynitrite, a putative lesion oxidant, is shown to damage perlecan structurally and functionally. Exposure of human perlecan to peroxynitrite decreases recognition by antibodies raised against both the core protein and heparan sulfate chains; dose-dependent formation of 3-nitrotyrosine was also detected. These effects were modulated by bicarbonate and reaction pH. Oxidant exposure resulted in aggregate formation, consistent with oxidative protein crosslinking. Peroxynitrite treatment modified functional properties of perlecan that are dependent on both the protein core (decreased binding of human coronary artery endothelial cells), and the HS chains (diminished fibroblast growth factor-2 (FGF-2) receptor-mediated proliferation of Baf-32 cells). The latter is consistent with a decrease in FGF-2 binding to the HS chains of modified perlecan. Immunofluorescence of advanced human atherosclerotic lesions provided evidence for the presence of perlecan and extensive formation of 3-nitrotyrosine epitopes within the intimal region; these materials showing marked co-localization. These data indicate that peroxynitrite induces major structural and functional changes to perlecan and that damage to this material occurs within human atherosclerotic lesions.