Degradation Of Extracellular Proteins Research Articles

Directed evolution of genetically encoded LYTACs for cell-mediated delivery

Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here, we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin-like growth factor 2 (IGF2). After showing initial efficacy with wild-type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially selective targeted protein degradation.

Targeted Extracellular Protein Degradation by Dendronized DNA Chimeras.

Extracellular soluble proteins are key agents in the development of various diseases. However, strategies to remove therapeutically relevant extracellular targets are still scarce. Here, we establish dendronized DNA chimera (DENTAC) as an efficient approach for targeted degradation of the extracellular protein of interest (ePOI). DENTAC consists of a DNA dendron against cell-surface scavenger receptors (SRs), a protein ligand, and a connecting linker, which harnesses SRs as a lysosome-trafficking receptor to mediate the lysosomal degradation of the ePOI. We interrogate and optimize structure-activity relationships of DENTAC. Using neutravidin as a model ePOI, we show that both branch number and DNA length in the DNA dendron are important determinants for efficient lysosomal delivery and degradation of the protein. We demonstrate three branches and 10 nucleotide-length polythymidine as the optimal DNA dendron components to construct DENTAC. We further exemplify the anticancer application of DENTAC by targeting matrix metalloproteinase-9 (MMP-9), where we find linker property as another factor important for DENTAC performance. We reveal that MMP-9-targeting DENTAC effectively restrain cancer cell proliferation, migration, and invasion. This study thus provides a potent strategy to delete extracellular proteins that are commonly difficult to target.

Anti-tumor immunotherapy using engineered bacterial outer membrane vesicles fused to lysosome-targeting chimeras mediated by transferrin receptor.

The lysosome-targeting chimera (LYTAC) approach has shown promise for the targeted degradation of secreted and membrane proteins via lysosomes. However, there have been challenges in design, development, and targeting. Here, we have designed a genetically engineered transferrin receptor (TfR)-mediated lysosome-targeting chimera (TfR-LYTAC) that is efficiently internalized via TfR-mediate endocytosis and targets PD-L1 for lysosomal degradation in cultured cells but not invivo due to short half-life and poor tumor targeting. A delivery platform was developed by fusing TfR-LYTAC to the surface of bacterial outer membrane vesicles (OMVs). The engineered OMV-LYTAC combines PD-1/PD-L1 pathway inhibition with LYTAC and immune activation by bacterial OMVs. OMV-LYTAC significantly reduced tumor growth invivo. We have provided a modular and simple genetic strategy for lysosomal degradation as well as a delivery platform for invivo tumor targeting. The study paves the way for the targeting and degradation of extracellular proteins using the TfR-LYTAC system.

Discovery of Repurposable Drugs in the Combination Therapy of Breast Cancer: A Virtual Drug Screening Study

Cathepsin D (Cat D) is a lysosomal aspartic acid protease encoded by CTSD gene and has significant biological roles such as degradation of extracellular and intracellular proteins, regulation of apoptosis, hormone processing, antigen processing etc. Furthermore, it is overexpressed by breast cancer cells and it acts a role in many processes affecting the cancer prognosis such as metastasis, angiogenesis, invasion, and drug resistance through regulation of the metabolic pathways and digesting the extracellular matrix (ECM) proteins. Due to that there is no drug targeting Cat D in clinical trial phases, a virtual drug screening in order to reveal possible drugs with high Cat D inhibitory activity from a library composed of 12,111 ligands is carried out with this study. Results have demonstrated that ZINC000003922429 (Adozelesin), ZINC000012358610 (Phthalocyanine), ZINC000051951669 (Bemcentinib), ZINC000003786250 (YM022), and ZINC000150338819 (Ledipasvir) have high binding affinity to Cat D. Among these chemical ligands, YM022 from Drugs in Clinical Trials dataset has been evaluated as most promising one that might be repurposed in the treatment of breast cancer due to its high affinity, convenient ADME and Toxicity properties, and highest bioactivity profiles. However, the possible activity of YM022 should be analyzed with further molecular dynamics (MD) simulations, in vitro and in vivo studies.

Click chemistry in the development of PROTACs.

Proteolysis-targeting chimeras or PROTACs are hetero-bifunctional molecules designed to mediate the disposal of a target protein via recruitment of the ubiquitination-proteasome degradation machinery. Because of the chimeric nature of such molecules, their synthesis requires a key step of "assembling" whether in the lab or in situ. Furthermore, targeted PROTACs often are hetero-trifunctional and require a second "assembling" step. Click chemistry has the unique advantages of tethering two or more molecular entities of choice under near physiological conditions and therefore has been applied to the development of PROTACs in various ways. This review provides a succinct summary of this field with a critical analysis of various factors that need to be considered for optimal results. Specifically, we examine issues including applications of click chemistry in in situ assembly for improved delivery, conjugation with a targeting group for selectivity, rapid synthesis for linker optimization, and lysosomal degradation of extracellular and membrane-associated proteins. We also examine reaction kinetics issues whenever possible or warranted.

IGF2 Peptide-Based LYTACs for Targeted Degradation of Extracellular and Transmembrane Proteins.

Lysosome-targeting chimeras (LYTACs) have recently been developed to facilitate the lysosomal degradation of specific extracellular and transmembrane molecular targets. However, the LYTAC particles described to date are based on glycopeptide conjugates, which are difficult to prepare and produce on a large scale. Here, we report on the development of pure protein LYTACs based on the non-glycosylated IGF2 peptides, which can be readily produced in virtually any facility capable of monoclonal antibody production. These chimeras utilize the IGF2R/CI-M6PR pathway for lysosomal shuttling and, in our illustrative example, target programmed death ligand 1 (PD-L1), eliciting physiological effects analogous to immune checkpoint blockade. Results from in vitro assays significantly exceed the effects of anti-PD-L1 antibodies alone.

Insulin-like Growth Factor 2 (IGF2)-Fused Lysosomal Targeting Chimeras for Degradation of Extracellular and Membrane Proteins.

Targeted degradation of the cell-surface and extracellular proteins via the endogenous lysosomal degradation pathways, such as lysosome-targeting chimeras (LYTACs), has recently emerged as an attractive tool to expand the scope of extracellular chemical biology. Herein, we report a series of recombinant proteins genetically fused to insulin-like growth factor 2 (IGF2), which we termed iLYTACs, that can be conveniently obtained in high yield by standard cloning and bacterial expression in a matter of days. We showed that both type-I iLYTACs, in which IGF2 was fused to a suitable affibody or nanobody capable of binding to a specific protein target, and type-II iLYTAC (or IGF2-Z), in which IGF2 was fused to the IgG-binding Z domain that served as a universal antibody-binding adaptor, could be used for effective lysosomal targeting and degradation of various extracellular and membrane-bound proteins-of-interest. These heterobifunctional iLYTACs are fully genetically encoded and can be produced on a large scale from conventional E. coli expression systems without any form of chemical modification. In the current study, we showed that iLYTACs successfully facilitated the cell uptake, lysosomal localization, and efficient lysosomal degradation of various disease-relevant protein targets from different mammalian cell lines, including EGFR, PD-L1, CD20, and α-synuclein. The antitumor properties of iLYTACs were further validated in a mouse xenograft model. Overall, iLYTACs represent a general and modular strategy for convenient and selective targeted protein degradation, thus expanding the potential applications of current LYTACs and related techniques.

Conjugation with glucagon like peptide-1 enables targeted protein degradation

Lysosome-targeting chimeras (LYTACs) have emerged as a promising technique to extend the scope of targeted protein degradation to extracellular proteins, e.g., secreted proteins and membrane-anchored proteins. However, up to now, only a small number of lysosomal targeting receptors (LTRs), such as cation-independent mannose 6-phosphate receptor (CI-M6PR) and asialoglycoprotein receptor (ASGPR), were reported to build LYTACs for degradation of extracellular proteins. Therefore, it is important to explore more functionalized ligands for the relevant LTRs to expand the LYTAC framework. Herein, we demonstrate a new LTR ligand-glucagon like peptide 1 (GLP-1) based targeted degradation platform, termed GLP-1 receptor-targeting chimeras (GLP-1-LYTAC). GLP-1-LYTACs are formed by conjugating GLP-1 with targeted binder (such as antibody) through Click Chemistry, showing efficiently lysosomal degradation of both extracellular proteins (GFP and Neutravidin) as well as cell membrane proteins (EGFR and PD-L1). We believe that this novel GLP-1-LYTAC will open up a new dimension for targeted protein breakdown.

The Phylogeny, Metabolic Potentials, and Environmental Adaptation of an Anaerobe, Abyssisolibacter sp. M8S5, Isolated from Cold Seep Sediments of the South China Sea.

Bacillota are widely distributed in various environments, owing to their versatile metabolic capabilities and remarkable adaptation strategies. Recent studies reported that Bacillota species were highly enriched in cold seep sediments, but their metabolic capabilities, ecological functions, and adaption mechanisms in the cold seep habitats remained obscure. In this study, we conducted a systematic analysis of the complete genome of a novel Bacillota bacterium strain M8S5, which we isolated from cold seep sediments of the South China Sea at a depth of 1151 m. Phylogenetically, strain M8S5 was affiliated with the genus Abyssisolibacter within the phylum Bacillota. Metabolically, M8S5 is predicted to utilize various carbon and nitrogen sources, including chitin, cellulose, peptide/oligopeptide, amino acids, ethanolamine, and spermidine/putrescine. The pathways of histidine and proline biosynthesis were largely incomplete in strain M8S5, implying that its survival strictly depends on histidine- and proline-related organic matter enriched in the cold seep ecosystems. On the other hand, strain M8S5 contained the genes encoding a variety of extracellular peptidases, e.g., the S8, S11, and C25 families, suggesting its capabilities for extracellular protein degradation. Moreover, we identified a series of anaerobic respiratory genes, such as glycine reductase genes, in strain M8S5, which may allow it to survive in the anaerobic sediments of cold seep environments. Many genes associated with osmoprotectants (e.g., glycine betaine, proline, and trehalose), transporters, molecular chaperones, and reactive oxygen species-scavenging proteins as well as spore formation may contribute to its high-pressure and low-temperature adaptations. These findings regarding the versatile metabolic potentials and multiple adaptation strategies of strain M8S5 will expand our understanding of the Bacillota species in cold seep sediments and their potential roles in the biogeochemical cycling of deep marine ecosystems.

Clinicopathological correlation of Cathepsin K expression in salivary gland carcinomas; relation to patients` outcome

BackgroundSalivary gland carcinomas (SGCs) represent various groups of tumors that demonstrate marked diversity in their prognosis owing to different histology and clinical characteristics. One of the poor prognostic indicators is distant metastasis which is considered the major reason for death in SGC patients. Discovering new biomarkers is urgently required to aid in the detection of cancer onset and progression. Cathepsin K (CTSK), the lysosomal cysteine protease has a principal role in cancer invasion and progression through interaction with the tumor microenvironment, degradation of extracellular membrane proteins and destruction of the elastic lamina of blood vessels. In the English literature, little information was present about the role of CTSK in SGCs. The current study aimed to assess the immunohistochemical expression of CTSK in SGCs and correlate its expression to different clinicopathologic parameters.MethodsThe retrospective study applied to 45 cases of SGCs categorized as high-grade (33 cases) and low-grade SGCs (12 cases) following the criteria of WHO classification (2017) of head and neck tumors. All patients` clinicopathological and follow-up records were retrieved. The following statistical tests were used to study the variance of CTSK expression in SGCs concerning different clinicopathological parameters; Pearson`s Chi-square test, unpaired two-tailed student t-test, One-way ANOVA, and Post Hoc tests. Disease-free survival (DFS) and Overall survival (OS) were calculated and displayed with the Kaplan–Meier strategy and analyzed with the log-rank test. Univariate and multivariate survival analyses were performed with Cox regression. A P-value lesser than 0.05 was considered statistically significant.ResultsStrong CTSK expression was significantly related to high-grade SGCs (P = 0.000), large infiltrating carcinomas (P = 0.000), presence of nodal (P = 0.041) and distant metastasis (P = 0.009), advanced TNM clinical stage (P = 0.000), the incidence of recurrence (P = 0.009), and reduced DFS (P = 0.006). Distant metastasis was the independent predictor for DFS using Cox regression model.ConclusionsCTSK has a great role in cancer progression by triggering many signaling pathways. Its level in cancerous tissue is considered an effective index for predicting the severity and prognosis of cancer. Therefore, we indicate its utility as a prognostic tool and therapeutic target for cancer treatment.Trial registrationRetrospectively registered.

Concise Review on Modulation Mechanism of Matrix Metalloproteinase via Oncoviruses in Aggressiveness of Malignancies

"Matrix metalloproteinases (MMPs) are a member of the protein family intimately associated with zinc-containing endopeptidases, which are the main proteases implicated in extracellular matrix (ECM) degradation. The high ability of MMPs in the degradation of extracellular proteins is an important event in many normal biological as well as pathologic processes, including tumor progression, tissue repair, wound healing, developmental morphogenesis, and inflammatory diseases. This review is focused on the description of the modulation mechanism of MMPs via oncoviruses. Up to now, a large number of oncogenic viruses have been detected and recognized as etiologic factors of multiple human cancers. Actually, by expression of several viral oncoproteins of tumorigenic viruses with the simple genetic system, we are able to deregulate or modulate the pathways of cell transformation procedure in malignancy. In brief, upregulated expression of MMPs genes by direct (through DNA binding) , as well as indirect interactions (by activating PI-3K/AKT, ERK/MAPK, IFN/JAK/STAT, JNK, NF-κB signaling pathways and/or immune response) with tumorigenic viruses, have a noticeable role in the proliferation of cells, as well as angiogenesis, mobility, EMT, invasiveness, and metastatic features. In fact, the growth of tumor-related researchers offers a future perspective for the efficiency of targeted, molecular-based therapies and its anticancer effects via diverse mechanisms such as the suppression of viral oncoproteins. "

Frontispiz: Aptamer‐LYTACs for Targeted Degradation of Extracellular and Membrane Proteins

Aptamers. In their Communication (e202218106), Zhi Zhu et al. report the development of aptamer-LYTACs (lysosome targeting chimeras) for the efficient degradation of the platelet-derived growth factor and the membrane protein PTK7 through the lysosomal degradation pathway.

Frontispiece: Aptamer‐LYTACs for Targeted Degradation of Extracellular and Membrane Proteins

Aptamers. In their Communication (e202218106), Zhi Zhu et al. report the development of aptamer-LYTACs (lysosome targeting chimeras) for the efficient degradation of the platelet-derived growth factor and the membrane protein PTK7 through the lysosomal degradation pathway.

Nano-LYTACs for Degradation of Membrane Proteins and Inhibition of CD24/Siglec-10 Signaling Pathway.

Lysosome-targeting chimeras (LYTACs) are an emerging therapeutic modality that effectively degrade cancer cell membranes and extracellular target proteins. In this study, a nanosphere-based LYTAC degradation system is developed. The amphiphilic peptide-modified N-acetylgalactosamine (GalNAc) can self-assemble into nanospheres with a strong affinity for asialoglycoprotein receptor targets. They can degrade different membranes and extracellular proteins by linking with the relevant antibodies. CD24, a heavily glycosylated glycosylphosphatidylinositol-anchored surface protein, interacts with Siglec-10 to modulate the tumor immune response. The novel Nanosphere-AntiCD24, synthesized by linking nanospheres with CD24 antibody, accurately regulates the degradation of CD24 protein and partially restores the phagocytic function of macrophages toward tumor cells by blocking the CD24/Siglec-10 signaling pathway. When Nanosphere-AntiCD24 is combined with glucose oxidase, an enzyme promoting the oxidative decomposition of glucose, the combination not only effectively restores the function of macrophages in vitro but also suppresses tumor growth in xenograft mouse models without detectable toxicity to normal tissues. The results indicate that GalNAc-modified nanospheres, as a part of LYTACs, can be successfully internalized and are an effective drug-loading platform and a modular degradation strategy for the lysosomal degradation of cell membrane and extracellular proteins, which can be broadly applied in the fields of biochemistry and tumor therapeutics.

Development of Oligomeric Mannose-6-phosphonate Conjugates for Targeted Protein Degradation.

Lysosome targeting chimeras (LYTACs) are a new protein degradation strategy that has recently emerged. LYTACs utilize the native cell internalization process in the body to target and degrade therapeutically relevant extracellular proteins via the lysosomal pathways. The first lysosomal internalization receptor recently used for LYTACs is the mannose-6-phosphate receptor (M6PR). M6PR is expressed across most cell types, making it ideal for internalization and degradation of numerous extracellular proteins. Herein, we report the development of a series of structurally well-defined mannose-6-phosphonate (M6Pn)-peptide conjugates that are capable of linking to a variety of targeting ligands for proteins of interest and successfully internalizing and degrading those proteins through M6PR. This will greatly facilitate the development of M6Pn based LYTACs for therapeutic applications.

Aptamer‐LYTACs for Targeted Degradation of Extracellular and Membrane Proteins

AbstractRecently, lysosome targeting chimeras (LYTACs) have emerged as a promising technology that expands the scope of targeted protein degradation to extracellular targets. However, the preparation of chimeras by conjugation of the antibody and trivalent N‐acetylgalactosamine (tri‐GalNAc) is a complex and time‐consuming process. The large uncertainty in number and position and the large molecular weights of the chimeras result in low internalization efficiency. To circumvent these problems, we developed the first aptamer‐based LYTAC (Apt‐LYTAC) to realize liver‐cell‐specific degradation of extracellular and membrane proteins by conjugating aptamers to tri‐GalNAc. Taking advantage of the facile synthesis and low molecular weight of the aptamer, the Apt‐LYTACs can efficiently and quickly degrade the extracellular protein PDGF and the membrane protein PTK7 through a lysosomal degradation pathway. We anticipate that the novel Apt‐LYTACs will expand the usage of aptamers and provide a new dimension for targeted protein degradation.

Aptamer-LYTACs for Targeted Degradation of Extracellular and Membrane Proteins.

Recently, lysosome targeting chimeras (LYTACs) have emerged as a promising technology that expands the scope of targeted protein degradation to extracellular targets. However, the preparation of chimeras by conjugation of the antibody and trivalent N-acetylgalactosamine (tri-GalNAc) is a complex and time-consuming process. The large uncertainty in number and position and the large molecular weights of the chimeras result in low internalization efficiency. To circumvent these problems, we developed the first aptamer-based LYTAC (Apt-LYTAC) to realize liver-cell-specific degradation of extracellular and membrane proteins by conjugating aptamers to tri-GalNAc. Taking advantage of the facile synthesis and low molecular weight of the aptamer, the Apt-LYTACs can efficiently and quickly degrade the extracellular protein PDGF and the membrane protein PTK7 through a lysosomal degradation pathway. We anticipate that the novel Apt-LYTACs will expand the usage of aptamers and provide a new dimension for targeted protein degradation.

A cross-sectional study of antibodies toubiquitin proteasome system in different glomerulopathies.

Recently, evidence has emerged that the ubiquitin system, which is involved in extracellular protein degradation, is most susceptible to damage in podocytes in cases of podocytopathies. We studied anti-ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) antibodies in glomerulopathies with proteinuria. 71 patients with glomerulopathy and 11 healthy subjects were included in our study. 44 patients had nephrotic syndrome, and 27 did not. Serum levels of anti-UCHL1 antibodies were measured by ELISA. The levels of anti-UCHL1 antibodies were significantly higher in focal segmental glomerulosclerosis (FSGS) patients than in minimal change disease (MCD), IgA nephropathy, membranous nephropathy, or membranoproliferative glomerulonephritis patients and control group. The levels of UCHL1 antibodies in serum did not correlate with 24-hour proteinuria, blood pressure, glomerulosclerosis percentage, or area of tubulointerstitial fibrosis, but did correlate with serum creatinine and estimated glomerular filtration rate (eGFR). During the development of the ROC curve (AUC = 0.766 (95% CI 0.634-0.897)) for FSGS vs. other forms of glomerulopathies, a readjustment of the sensitivity of 75% and specificity of 61% were established. A former increase in anti-UCHL1 antibody levels above 1.93 ng/mL may be a marker of FSGS OR 3.617 (95% CI 1.051-12.447), p=0.041. An increase in the level of anti-UCHL1 antibodies in the serum was noted in FSGS, which suggests that these antibodies could be a potential biomarker for FSGS patients.

Bifunctional Compounds as Molecular Degraders for Integrin-Facilitated Targeted Protein Degradation.

As effective ways to regulate protein levels, targeted protein degradation technologies have attracted great attention in recent years. Here, we established a novel integrin-facilitated lysosomal degradation (IFLD) strategy to degrade extracellular and cell membrane proteins using bifunctional compounds as molecular degraders. By conjugation of a target protein-binding ligand with an integrin-recognition ligand, the resulting molecular degrader proved to be highly efficient to induce the internalization and subsequent degradation of extracellular or cell membrane proteins in an integrin- and lysosome-dependent manner. As demonstrated in the development of BMS-L1-RGD, which is an efficient programmed death-ligand 1 (PD-L1) degrader validated both in vitro and in vivo, the IFLD strategy expands the toolbox for regulation of secreted and membrane-associated proteins and thus has great potential to be applied in chemical biology and drug discovery.

Modular cytokine receptor-targeting chimeras for targeted degradation of cell surface and extracellular proteins

Targeted degradation of cell surface and extracellular proteins via lysosomal delivery is an important means to modulate extracellular biology. However, these approaches have limitations due to lack of modularity, ease of development, restricted tissue targeting and applicability to both cell surface and extracellular proteins. We describe a lysosomal degradation strategy, termed cytokine receptor-targeting chimeras (KineTACs), that addresses these limitations. KineTACs are fully genetically encoded bispecific antibodies consisting of a cytokine arm, which binds its cognate cytokine receptor, and a target-binding arm for the protein of interest. We show that KineTACs containing the cytokine CXCL12 can use the decoy recycling receptor, CXCR7, to target a variety of target proteins to the lysosome for degradation. Additional KineTACs were designed to harness other CXCR7-targeting cytokines, CXCL11 and vMIPII, and the interleukin-2 (IL-2) receptor-targeting cytokine IL-2. Thus, KineTACs represent a general, modular, selective and simple genetically encoded strategy for inducing lysosomal delivery of extracellular and cell surface targets with broad or tissue-specific distribution.