Non-canonical Functions Research Articles

Non-canonical functions of EZH2 in cancer.

Mutations in chromatin modifying genes frequently occur in many kinds of cancer. Most mechanistic studies focus on their canonical functions, while therapeutic approaches target their enzymatic activity. Recent studies, however, demonstrate that non-canonical functions of chromatin modifiers may be equally important and therapeutically actionable in different types of cancer. One epigenetic regulator that demonstrates such a dual role in cancer is the histone methyltransferase EZH2. EZH2 is a core component of the polycomb repressive complex 2 (PRC2), which plays a crucial role in cell identity, differentiation, proliferation, stemness and plasticity. While much of the regulatory functions and oncogenic activity of EZH2 have been attributed to its canonical, enzymatic activity of methylating lysine 27 on histone 3 (H3K27me3), a repressive chromatin mark, recent studies suggest that non-canonical functions that are independent of H3K27me3 also contribute towards the oncogenic activity of EZH2. Contrary to PRC2's canonical repressive activity, mediated by H3K27me3, outside of the complex EZH2 can directly interact with transcription factors and oncogenes to activate gene expression. A more focused investigation into these non-canonical interactions of EZH2 and other epigenetic/chromatin regulators may uncover new and more effective therapeutic strategies. Here, we summarize major findings on the non-canonical functions of EZH2 and how they are related to different aspects of carcinogenesis.

Non-canonical functions of autophagy proteins in cardiovascular diseases

Non-canonical functions of autophagy proteins in cardiovascular diseases

Apoptotic cell clearance components in inflammatory arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints that affects ~1% of the human population. Joint swelling and bone erosion, hallmarks of RA, contribute to disability and, sometimes, loss of life. Mechanistically, disease is driven by immune dysregulation characterized by circulating autoantibodies, inflammatory mediators, tissue degradative enzymes, and metabolic dysfunction of resident stromal and recruited immune cells. Cell death by apoptosis has been therapeutically explored in animal models of RA due to the comparisons drawn between synovial hyperplasia and paucity of apoptosis in RA with the malignant transformation of cancer cells. Several efforts to induce cell death have shown benefits in reducing the development and/or severity of the disease. Apoptotic cells are cleared by phagocytes in a process known as efferocytosis, which differs from microbial phagocytosis in its "immuno-silent," or anti-inflammatory, nature. Failures in efferocytosis have been linked to autoimmune disease, whereas administration of apoptotic cells in RA models effectively inhibits inflammatory indices, likely though efferocytosis-mediated resolution-promoting mechanisms. However, the nature of signaling pathways elicited and the molecular identity of clearance mediators in RA are understudied. Furthermore, canonical efferocytosis machinery elements also play important non-canonical functions in homeostasis and pathology. Here, we discuss the roles of efferocytosis machinery components in models of RA and discuss their potential involvement in disease pathophysiology.

Noncanonical functions of Ku may underlie essentiality in human cells

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but is involved in other cellular functions like telomere regulation and maintenance, in which Ku’s role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells using CRISPR/Cas9 editing to study the essentiality of Ku70 function. While we observed loss of cell viability upon Ku depletion, we did not detect significant changes in telomere length, nor did we record lethal levels of DNA damage upon loss of Ku. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study suggests that the driving cause of loss of cell viability in Ku70 knockouts is not linked to the functions of Ku in DNA repair or at telomeres. Moreover, our data shows that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.

Implications of Translesion DNA Synthesis Polymerases on Genomic Stability and Human Health.

Replication fork arrest-induced DNA double strand breaks (DSBs) caused by lesions are effectively suppressed in cells due to the presence of a specialized mechanism, commonly referred to as DNA damage tolerance (DDT). In eukaryotic cells, DDT is facilitated through translesion DNA synthesis (TLS) carried out by a set of DNA polymerases known as TLS polymerases. Another parallel mechanism, referred to as homology-directed DDT, is error-free and involves either template switching or fork reversal. The significance of the DDT pathway is well established. Several diseases have been attributed to defects in the TLS pathway, caused either by mutations in the TLS polymerase genes or dysregulation. In the event of a replication fork encountering a DNA lesion, cells switch from high-fidelity replicative polymerases to low-fidelity TLS polymerases, which are associated with genomic instability linked with several human diseases including, cancer. The role of TLS polymerases in chemoresistance has been recognized in recent years. In addition to their roles in the DDT pathway, understanding noncanonical functions of TLS polymerases is also a key to unraveling their importance in maintaining genomic stability. Here we summarize the current understanding of TLS pathway in DDT and its implication for human health.

RNA-Polymer Hybrids via Direct and Site-Selective Acylation with the ATRP Initiator and Photoinduced Polymerization.

Combining synthetic polymers with RNA paves the way for creating RNA-based materials with non-canonical functions. We have developed an acylation reagent that allows for direct incorporation of the atom transfer radical polymerization (ATRP) initiator into both short synthetic oligoribonucleotides and natural biomass RNA extracted from torula yeast. The acylation was performed in a quantitative yield. The resulting initiator-functionalized RNAs were used for grafting polymer chains from the RNA by photoinduced ATRP, resulting in RNA-polymer hybrids with narrow molecular weight distributions. The RNA initiator was used for the polymerization of oligo(ethylene oxide) methyl ether methacrylate, poly(ethylene glycol) dimethacrylate, and N-isopropylacrylamide monomers, resulting in RNA bottlebrushes, hydrogels, and stimuli-responsive materials. This approach, readily applicable to both post-synthetic and nature-derived RNA, can be used to engineer the properties of a variety of RNA-based macromolecular hybrids and assemblies providing access to a wide variety of RNA-polymer hybrids.

Tau protein binds to the P53 E3 ubiquitin ligase MDM2

Tau gene mutations cause a progressive dementia and neurotoxic Tau forms deposited in neurofibrillary tangles are hallmarks of neurodegenerative tauopathies. Loss of non-canonical Tau functions may contribute to disease. In fact, Tau depletion affects the cellular response to DNA damage and tauopathies exhibit the accumulation of DNA lesions. Moreover, Tau modulates P53 activity and cell fate. Considering that MDM2 is the main antagonist of P53, we investigated, using orthogonal assays, if Tau interacts with MDM2. We report the existence in cells and brain of a Tau-MDM2 complex that, in vitro, exhibits reduced P53 ubiquitination activity in a manner sensitive to a Tau mutation. The Tau-MDM2 interaction involves the microtubule-binding domain of Tau and the acidic domain of MDM2, reminiscent of the binding of Tau to negatively charged microtubules. Notably, MDM2 accumulates aberrantly in neurofibrillary tangles. Aging-associated insults may expose a novel loss-of-function of Tau in neurodegeneration and cancer.

Targeting Epidermal Growth Factor Receptor for Cancer Treatment: Abolishing Both Kinase-Dependent and Kinase-Independent Functions of the Receptor.

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is activated by ligand binding, overexpression, or mutation. It is well known for its tyrosine kinase-dependent oncogenic activities in a variety of human cancers. A large number of EGFR inhibitors have been developed for cancer treatment, including monoclonal antibodies, tyrosine kinase inhibitors, and a vaccine. The EGFR inhibitors are aimed at inhibiting the activation or the activity of EGFR tyrosine kinase. However, these agents have shown efficacy in only a few types of cancers. Drug resistance, both intrinsic and acquired, is common even in cancers where the inhibitors have shown efficacy. The drug resistance mechanism is complex and not fully known. The key vulnerability of cancer cells that are resistant to EGFR inhibitors has not been identified. Nevertheless, it has been increasingly recognized in recent years that EGFR also possesses kinase-independent oncogenic functions and that these noncanonical functions may play a crucial role in cancer resistance to EGFR inhibitors. In this review, both kinase-dependent and -independent activities of EGFR are discussed. Also discussed are the mechanisms of actions and therapeutic activities of clinically used EGFR inhibitors and sustained EGFR overexpression and EGFR interaction with other receptor tyrosine kinases to counter the EGFR inhibitors. Moreover, this review discusses emerging experimental therapeutics that have shown potential for overcoming the limitation of the current EGFR inhibitors in preclinical studies. The findings underscore the importance and feasibility of targeting both kinase-dependent and -independent functions of EGFR to enhance therapeutic efficacy and minimize drug resistance. SIGNIFICANCE STATEMENT: EGFR is a major oncogenic driver and therapeutic target, but cancer resistance to current EGFR inhibitors remains a significant unmet clinical problem. This article reviews the cancer biology of EGFR as well as the mechanisms of actions and the therapeutic efficacies of current and emerging EGFR inhibitors. The findings could potentially lead to development of more effective treatments for EGFR-positive cancers.

An EZH2-NF-κB regulatory axis drives expression of pro-oncogenic gene signatures in triple negative breast cancer

The histone methyltransferase EZH2 has been studied most extensively in the context of PRC2-dependent gene repression. Accumulating evidence indicates non-canonical functions for EZH2 in cancer contexts including promoting paradoxical gene expression through interactions with transcription factors, including NF-κB in triple negative breast cancer (TNBC). We profile EZH2 and NF-κB factor co-localization and positive gene regulation genome-wide, and define a subset of NF-κB targets and genes associated with oncogenic functions in TNBC that is enriched in patient datasets. We demonstrate interaction between EZH2 and RelA requiring the recently identified transactivation domain (TAD) which mediates EZH2 recruitment to, and activation of certain NF-κB-dependent genes, and supports downstream migration and stemness phenotypes in TNBC cells. Interestingly, EZH2-NF-κB positive regulation of genes and stemness does not require PRC2. This study provides new insight into pro-oncogenic regulatory functions for EZH2 in breast cancer through PRC2-independent, and NF-κB-dependent regulatory mechanisms.

The Structure, Expression, and Non-Canonical Functions of Human rDNA: The Role of Non-Coding Regions

The Structure, Expression, and Non-Canonical Functions of Human rDNA: The Role of Non-Coding Regions

The circadian clock CRY1 regulates pluripotent stem cell identity and somatic cell reprogramming

Distinct metabolic conditions rewire circadian-clock-controlled signaling pathways leading to the de novo construction of signal transduction networks. However, it remains unclear whether metabolic hallmarks unique to pluripotent stem cells (PSCs) are connected to clock functions. Reprogramming somatic cells to a pluripotent state, here we highlighted non-canonical functions of the circadian repressor CRY1 specific to PSCs. Metabolic reprogramming, including AMPK inactivation and SREBP1 activation, was coupled with the accumulation of CRY1 in PSCs. Functional assays verified that CRY1 is required for the maintenance of self-renewal capacity, colony organization, and metabolic signatures. Genome-wide occupancy of CRY1 identified CRY1-regulatory genes enriched in development and differentiation in PSCs, albeit not somatic cells. Last, cells lacking CRY1 exhibit differential gene expression profiles during induced PSC (iPSC) reprogramming, resulting in impaired iPSC reprogramming efficiency. Collectively, these results suggest the functional implication of CRY1 in pluripotent reprogramming and ontogenesis, thereby dictating PSC identity.

Complement: Functions, location and implications.

The complement system, an arm of the innate immune system plays a critical role in both health and disease. The complement system is highly complex with dual possibilities, helping or hurting the host, depending on the location and local microenvironment. The traditionally known functions of complement include surveillance, pathogen recognition, immune complex trafficking, processing and pathogen elimination. The noncanonical functions of the complement system include their roles in development, differentiation, local homeostasis and other cellular functions. Complement proteins are present in both, the plasma and on the membranes. Complement activation occurs both extra- and intracellularly, which leads to considerable pleiotropy in their activity. In order to design more desirable and effective therapies, it is important to understand the different functions of complement, and its location-based and tissue-specific responses. This manuscript will provide a brief overview into the complex nature of the complement cascade, outlining some of their complement-independentfunctions, their effects at different locale, and their implication in disease settings.

The CHARGE syndrome-associated protein FAM172A controls AGO2 nuclear import.

CHARGE syndrome is a neural crest-related disorder mainly caused by mutation of the chromatin remodeler-coding gene CHD7 Alternative causes include mutation of other chromatin and/or splicing factors. One of these additional players is the poorly characterized FAM172A, which we previously found in a complex with CHD7 and the small RNA-binding protein AGO2 at the chromatin-spliceosome interface. Focusing on the FAM172A-AGO2 interplay, we now report that FAM172A is a direct binding partner of AGO2 and, as such, one of the long sought-after regulators of AGO2 nuclear import. We show that this FAM172A function mainly relies on its classical bipartite nuclear localization signal and associated canonical importin-α/β pathway, being enhanced by CK2-induced phosphorylation and abrogated by a CHARGE syndrome-associated missense mutation. Overall, this study thus strengthens the notion that noncanonical nuclear functions of AGO2 and associated regulatory mechanisms might be clinically relevant.

A non-canonical function of Arabidopsis ERECTA proteins and a role of the SWI3B subunit of the SWI/SNF chromatin remodeling complex in gibberellin signaling.

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs) comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERECTA-LIKE 2 (ERL2) controls epidermal patterning, inflorescence architecture, and stomata development and patterning. These proteins are reported to be plasma membrane associated. Here we show that the er/erl1/erl2 mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. The ERf kinase domains were found to localize to the nucleus where they interact with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. Similar to swi3c and brm plants with inactivated subunits of SWI/SNF CRCs, it also does not accumulate DELLA RGA and GAI proteins. The ER kinase phosphorylates SWI3B in vitro, and the inactivation of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. The identified correlation between DELLA overaccumulation and SWI3B proteasomal degradation, and the physical interaction of SWI3B with DELLA proteins indicate an important role of SWI3B-containing SWI/SNF CRCs in gibberellin signaling. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in the transcriptional control of gene expression, and observed similar features for human HER2 (epidermal growth family receptor member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors.

Claudin Barriers on the Brink: How Conflicting Tissue and Cellular Priorities Drive IBD Pathogenesis.

Inflammatory bowel diseases (IBDs) are characterized by acute or chronic recurring inflammation of the intestinal mucosa, often with increasing severity over time. Life-long morbidities and diminishing quality of life for IBD patients compel a search for a better understanding of the molecular contributors to disease progression. One unifying feature of IBDs is the failure of the gut to form an effective barrier, a core role for intercellular complexes called tight junctions. In this review, the claudin family of tight junction proteins are discussed as they are a fundamental component of intestinal barriers. Importantly, claudin expression and/or protein localization is altered in IBD, leading to the supposition that intestinal barrier dysfunction exacerbates immune hyperactivity and disease. Claudins are a large family of transmembrane structural proteins that constrain the passage of ions, water, or substances between cells. However, growing evidence suggests non-canonical claudin functions during mucosal homeostasis and healing after injury. Therefore, whether claudins participate in adaptive or pathological IBD responses remains an open question. By reviewing current studies, the possibility is assessed that with claudins, a jack-of-all-trades is master of none. Potentially, a robust claudin barrier and wound restitution involve conflicting biophysical phenomena, exposing barrier vulnerabilities and a tissue-wide frailty during healing in IBD.

Harnessing the cooperation between DNA-PK and cGAS in cancer therapies: The cooperation between DNA-PK and cGAS shapes tumour immunogenicity: The cooperation between DNA-PK and cGAS shapes tumour immunogenicity.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is central for the initiation of anti-tumoural immune responses. Enormous effort has been made to optimise the design and administration of STING agonists to stimulate tumour immunogenicity. However, in certain contexts the cGAS-STING axis fuels tumourigenesis. Here, we review recent findings on the regulation of cGAS expression and activity. We particularly focus our attention on the DNA-dependent protein kinase (DNA-PK) complex, that recently emerged as an activator of inflammatory responses in tumour cells. We propose that stratification analyses on cGAS and DNA-PK expression/activation status should be carried out to predict treatment efficacy. We herein also provide insights into non-canonical functions borne by cGAS and cGAMP, highlighting how they may influence tumourigenesis. All these parameters should be taken into consideration concertedly to choose strategies aiming to effectively boost tumour immunogenicity.

Human rDNA Structure, Expression, and Non-Canonical Functions: the Role of Non-Coding Regions

The review is dedicated to analyzing and summarizing the data on the part of human genome encoding 45S rRNA. The sequences which seem evolutionary conserved on the first glance astonish one with their variability in structure and a variety of functions on closer examination. The major part of rDNA is non-coding and contains regulatory elements, protein binding sites, pseudogenes, repetitive sequences, and microRNA genes. Ribosomal intergenic spacers are not only in charge with the nucleolus morphology and functioning, namely, the rRNA expression and ribosome biogenesis, but also control nuclear chromatin formation thus mediating cell differentiation. Besides, alterations in the expression of these non-coding regions of rDNA in response to environmental stimuli underlies the keen sense of cell to various types of stressors. Malfunctioning of this process may result in a wide range of pathologies from oncology to neurodegenerative disease and mental illness. Here we observe to-date materials on the structure and transcription of the ribosomal intergenic spacer in humans and its role in rRNA expression, in-born disease development, and cancer.

HMGB1 cleavage by complement C1s and its potent anti-inflammatory product.

Complement C1s association with the pathogenesis of several diseases cannot be simply explained only by considering its main role in activating the classical complement pathway. This suggests that non-canonical functions are to be deciphered for this protease. Here the focus is on C1s cleavage of HMGB1 as an auxiliary target. HMGB1 is a chromatin non-histone nuclear protein, which exerts in fact multiple functions depending on its location and its post-translational modifications. In the extracellular compartment, HMGB1 can amplify immune and inflammatory responses to danger associated molecular patterns, in health and disease. Among possible regulatory mechanisms, proteolytic processing could be highly relevant for HMGB1 functional modulation. The unique properties of HMGB1 cleavage by C1s are analyzed in details. For example, C1s cannot cleave the HMGB1 A-box fragment, which has been described in the literature as an inhibitor/antagonist of HMGB1. By mass spectrometry, C1s cleavage was experimentally identified to occur after lysine on position 65, 128 and 172 in HMGB1. Compared to previously identified C1s cleavage sites, the ones identified here are uncommon, and their analysis suggests that local conformational changes are required before cleavage at certain positions. This is in line with the observation that HMGB1 cleavage by C1s is far slower when compared to human neutrophil elastase. Recombinant expression of cleavage fragments and site-directed mutagenesis were used to confirm these results and to explore how the output of C1s cleavage on HMGB1 is finely modulated by the molecular environment. Furthermore, knowing the antagonist effect of the isolated recombinant A-box subdomain in several pathophysiological contexts, we wondered if C1s cleavage could generate natural antagonist fragments. As a functional readout, IL-6 secretion following moderate LPS activation of RAW264.7 macrophage was investigated, using LPS alone or in complex with HMGB1 or some recombinant fragments. This study revealed that a N-terminal fragment released by C1s cleavage bears stronger antagonist properties as compared to the A-box, which was not expected. We discuss how this fragment could provide a potent brake for the inflammatory process, opening the way to dampen inflammation.

Both Maternal and Newborn IgMs Inhibit Transmissible Gastroenteritis Virus Internalization in LLC-PK1 Cells

Immunoglobulins M (IgMs) are the evolutionally oldest class of antibodies in higher eukaryotes. This pool of antibodies is one of the first to appear in humans and begins to be-synthesized at the early stages of the neonatal period. Most of the repertoire of IgMs of the newborns consists of the so-called «natural», or «naive», antibodies synthesized by the body without external antigenic stimulation. In addition to the classical functions of human immunoglobulins M (such as antigen recognition and initiation of innate immune responses), antibodies of this class exhibit a variety of non-canonical functions. The non-canonical functions are the action of antibodies as agonists/antagonists of various receptors, cleavage of antigen due to the catalytic activity of IgM, direct inactivation of pathogens in the absence of effector cells and molecules, etc. The aim of this work was to study and compare the antiviral activity of total preparations of immunoglobulin M of newborns and adults, obtained from umbilical cord and venous blood sera, on the LLC-PK1 cell line model infected with the transmissible gastroenteritis virus (TGEV). In addition, in the course of the studies, a decision was made to investigate the effect of combined preparations of immunoglobulin M on the change in signal transduction in the epidermal growth factor receptor as one of the mechanisms of TGEV internalization during infection of target cells. Methods. Highly purified preparations of total IgM of adults or newborns were obtained using the methodologies of sequential salt fractionation and affinity chromatography. This work used the model of the interaction of the transmissible gastroenteritis virus with cells of the LLC-PK1 line and the monitoring of changes in the phosphorylation state of the epidermal growth factor receptor of these cells during virus infection to study the effect of human IgM on the internalization of the virus and its interaction with the receptor system of the host cell. The degree of cytopathogenic effect of the virus was determined visually by changes in cell morphology. The mean infectious dose for transmissible gastroenteritis virus in the cell culture of the LLC-PK1 line was determined by the Reed-Muench method. Analysis of changes in the phosphorylation of the epidermal growth factor receptor was performed using the Western-blot analysis method. Results. The addition of a total high-purified sample of human IgM reduces the degree of efficiency of TGEV infection of the LLC-PK1 cell line and modulates the phosphorylation levels of these cells. Conclusions. The total preparations of IgM obtained from human venous blood of adults and from umbilical cord blood of newborns can affect the internalization of the transmissible gastroenteritis virus in the LLC-PK1 cell line. The original model of virus (TGEV) — cell line (LLC-PK1) was applied and tested to study the effect of native total preparations of immunoglobulin M on the internalization of the virus into the cell. The obtained data can be useful in further studies for a better understanding of the process of development and functioning of the immune system of newborns.

Working a second job: Cell adhesion proteins that moonlight in the nucleus.

Cells are adept at sensing changes in their environment, transmitting signals internally to coordinate responses to external stimuli, and thereby influencing adaptive changes in cell states and behavior. Often, this response involves modulation of gene expression in the nucleus, which is seen largely as a physically separated process from the rest of the cell. Mechanosensing, whereby a cell senses physical stimuli, and integrates and converts these inputs into downstream responses including signaling cascades and gene regulatory changes, involves the participation of several macromolecular structures. Of note, the extracellular matrix (ECM) and its constituent macromolecules comprise an essential part of the cellular microenvironment, allowing cells to interact with each other, and providing both structural and biochemical stimuli sensed by adhesion transmembrane receptors. This highway of information between the ECM, cell adhesion proteins, and the cytoskeleton regulates cellular behavior, the disruption of which results in disease. Emerging evidence suggests a more direct role for some of these adhesion proteins in chromatin structure and gene regulation, RNA maturation and other non-canonical functions. While many of these discoveries were previously limited to observations of cytoplasmic-nuclear transport, recent advances in microscopy, and biochemical, proteomic and genomic technologies have begun to significantly enhance our understanding of the impact of nuclear localization of these proteins. This review will briefly cover known cell adhesion proteins that migrate to the nucleus, and their downstream functions. We will outline recent advances in this very exciting yet still emerging field, with impact ranging from basic biology to disease states like cancer.