New progress in roles of TGF-ß signaling crosstalks in cellular functions, immunity and diseases.

The family of secreted dimeric proteins known as the Transforming Growth Factor-ß (TGF-ß) family plays a critical role in facilitating intercellular communication within multicellular animals. A recent symposium on TGF-ß Biology - Signaling, Development, and Diseases, held on December 19-21, 2023, in Hangzhou, China, showcased some latest advances in our understanding TGF-ß biology and also served as an important forum for scientific collaboration and exchange of ideas. More than twenty presentations and discussions at the symposium delved into the intricate mechanisms of TGF-ß superfamily signaling pathways, their roles in normal development and immunity, and the pathological conditions associated with pathway dysregulation.

Nuclear KRT19 is a transcriptional corepressor promoting histone deacetylation and liver tumorigenesis.

BACKGROUND AND AIMS: Epigenetic reprogramming and escape from terminal differentiation are poorly understood enabling characteristics of liver cancer. Keratin 19 (KRT19), classically known to form the intermediate filament cytoskeleton, is a marker of stemness and worse prognosis in liver cancer. This study aimed to address the functional roles of KRT19 in liver tumorigenesis and to elucidate the underlying mechanisms. APPROACH AND RESULTS: Using multiplexed genome editing of hepatocytes in vivo, we demonstrated that KRT19 promoted liver tumorigenesis in mice. Cell fractionation revealed a previously unrecognized nuclear fraction of KRT19. Tandem affinity purification identified histone deacetylase 1 and REST corepressor 1, components of the corepressor of RE-1 silencing transcription factor (CoREST) complex as KRT19-interacting proteins. KRT19 knockout markedly enhanced histone acetylation levels. Mechanistically, KRT19 promotes CoREST complex formation by enhancing histone deacetylase 1 and REST corepressor 1 interaction, thus increasing the deacetylase activity. ChIP-seq revealed hepatocyte-specific genes, such as hepatocyte nuclear factor 4 alpha ( HNF4A ), as direct targets of KRT19-CoREST. In addition, we identified forkhead box P4 as a direct activator of aberrant KRT19 expression in liver cancer. Furthermore, treatment of primary liver tumors and patient-derived xenografts in mice suggest that KRT19 expression has the potential to predict response to histone deacetylase 1 inhibitors especially in combination with lenvatinib. CONCLUSIONS: Our data show that nuclear KRT19 acts as a transcriptional corepressor through promoting the deacetylase activity of the CoREST complex, resulting in dedifferentiation of liver cancer. These findings reveal a previously unrecognized function of KRT19 in directly shaping the epigenetic landscape in cancer.

SNX8 enables lysosome reformation and reverses lysosomal storage disorder.

Lysosomal Storage Disorders (LSDs), which share common phenotypes, including enlarged lysosomes and defective lysosomal storage, are caused by mutations in lysosome-related genes. Although gene therapies and enzyme replacement therapies have been explored, there are currently no effective routine therapies against LSDs. During lysosome reformation, which occurs when the functional lysosome pool is reduced, lysosomal lipids and proteins are recycled to restore lysosome functions. Here we report that the sorting nexin protein SNX8 promotes lysosome tubulation, a process that is required for lysosome reformation, and that loss of SNX8 leads to phenotypes characteristic of LSDs in human cells. SNX8 overexpression rescued features of LSDs in cells, and AAV-based delivery of SNX8 to the brain rescued LSD phenotypes in mice. Importantly, by screening a natural compound library, we identified three small molecules that enhanced SNX8-lysosome binding and reversed LSD phenotypes in human cells and in mice. Altogether, our results provide a potential solution for the treatment of LSDs.

IRF3 activates RB to authorize cGAS-STING-induced senescence and mitigate liver fibrosis.

Cytosolic double-stranded DNA surveillance by cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) signaling triggers cellular senescence, autophagy, biased mRNA translation, and interferon-mediated immune responses. However, detailed mechanisms and physiological relevance of STING-induced senescence are not fully understood. Here, we unexpectedly found that interferon regulatory factor 3 (IRF3), activated during innate DNA sensing, forms substantial endogenous complexes in the nucleus with retinoblastoma (RB), a key cell cycle regulator. The IRF3-RB interaction attenuates cyclin-dependent kinase 4/6 (CDK4/6)-mediated RB hyperphosphorylation that mobilizes RB to deactivate E2 family (E2F) transcription factors, thereby driving cells into senescence. STING-IRF3-RB signaling plays a notable role in hepatic stellate cells (HSCs) within various murine models, pushing activated HSCs toward senescence. Accordingly, IRF3 global knockout or conditional deletion in HSCs aggravated liver fibrosis, a process mitigated by the CDK4/6 inhibitor. These findings underscore a straightforward yet vital mechanism of cGAS-STING signaling in inducing cellular senescence and unveil its unexpected biology in limiting liver fibrosis.

Loss of ZNRF3/RNF43 Unleashes EGFR in Cancer.

ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the most negatively correlated protein with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptor, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.

A chaperone-like function of FUS ensures TAZ condensate dynamics and transcriptional activation.

The Hippo pathway has important roles in organ development, tissue homeostasis and tumour growth. Its downstream effector TAZ is a transcriptional coactivator that promotes target gene expression through the formation of biomolecular condensates. However, the mechanisms that regulate the biophysical properties of TAZ condensates to enable Hippo signalling are not well understood. Here using chemical crosslinking combined with an unbiased proteomics approach, we show that FUS associates with TAZ condensates and exerts a chaperone-like effect to maintain their proper liquidity and robust transcriptional activity. Mechanistically, the low complexity sequence domain of FUS targets the coiled-coil domain of TAZ in a phosphorylation-regulated manner, which ensures the liquidity and dynamicity of TAZ condensates. In cells lacking FUS, TAZ condensates transition into gel-like or solid-like assembles with immobilized TAZ, which leads to reduced expression of target genes and inhibition of pro-tumorigenic activity. Thus, our findings identify a chaperone-like function of FUS in Hippo regulation and demonstrate that appropriate biophysical properties of transcriptional condensates are essential for gene activation.

Innate immune sensing of lysosomal dysfunction drives multiple lysosomal storage disorders.

Lysosomal storage disorders (LSDs), which are characterized by genetic and metabolic lysosomal dysfunctions, constitute over 60 degenerative diseases with considerable health and economic burdens. However, the mechanisms driving the progressive death of functional cells due to lysosomal defects remain incompletely understood, and broad-spectrum therapeutics against LSDs are lacking. Here, we found that various gene abnormalities that cause LSDs, including Hexb, Gla, Npc1, Ctsd and Gba, all shared mutual properties to robustly autoactivate neuron-intrinsic cGAS-STING signalling, driving neuronal death and disease progression. This signalling was triggered by excessive cytoplasmic congregation of the dsDNA and DNA sensor cGAS in neurons. Genetic ablation of cGAS or STING, digestion of neuronal cytosolic dsDNA by DNase, and repair of neuronal lysosomal dysfunction alleviated symptoms of Sandhoff disease, Fabry disease and Niemann-Pick disease, with substantially reduced neuronal loss. We therefore identify a ubiquitous mechanism mediating the pathogenesis of a variety of LSDs, unveil an inherent connection between lysosomal defects and innate immunity, and suggest a uniform strategy for curing LSDs.

Computational design and genetic incorporation of lipidation mimics in living cells.

Protein lipidation, which regulates numerous biological pathways and plays crucial roles in the pharmaceutical industry, is not encoded by the genetic code but synthesized post-translationally. In the present study, we report a computational approach for designing lipidation mimics that fully recapitulate the biochemical properties of natural lipidation in membrane association and albumin binding. Furthermore, we establish an engineered system for co-translational incorporation of these lipidation mimics into virtually any desired position of proteins in Escherichia coli and mammalian cells. We demonstrate the utility of these length-tunable lipidation mimics in diverse applications, including improving the half-life and activity of therapeutic proteins in living mice, anchoring functional proteins to membrane by substituting natural lipidation, functionally characterizing proteins carrying different lengths of lipidation and determining the plasma membrane-binding capacity of a given compound. Our strategy enables gain-of-function studies of lipidation in hundreds of proteins and facilitates the creation of superior therapeutic candidates.

Smad4 sequestered in SFPQ condensates prevents TGF-ß tumor-suppressive signaling.

Loss of TGF-ß growth-inhibitory responses is a hallmark of human cancer. However, the molecular mechanisms underlying the TGF-ß resistance of cancer cells remain to be fully elucidated. Splicing factor proline- and glutamine-rich (SFPQ) is a prion-like RNA-binding protein that is frequently upregulated in human cancers. In this study, we identified SFPQ as a potent suppressor of TGF-ß signaling. The ability of SFPQ to suppress TGF-ß responses depends on its prion-like domain (PrLD) that drives liquid-liquid phase separation (LLPS). Mechanistically, SFPQ physically restrained Smad4 in its condensates, which excluded Smad4 from the Smad complex and chromatin occupancy and thus functionally dampened Smad-dependent transcriptional responses. Accordingly, SFPQ deficiency or loss of phase separation activities rendered human cells hypersensitive to TGF-ß responses. Together, our data identify an important function of SFPQ through LLPS that suppresses Smad transcriptional activation and TGF-ß tumor-suppressive activity.

High-sensitivity profiling of SARS-CoV-2 noncoding region-host protein interactome reveals the potential regulatory role of negative-sense viral RNA.

A deep understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-host interactions is crucial to developing effective therapeutics and addressing the threat of emerging coronaviruses. The role of noncoding regions of viral RNA (ncrRNAs) has yet to be systematically scrutinized. We developed a method using MS2 affinity purification coupled with liquid chromatography-mass spectrometry and designed a diverse set of bait ncrRNAs to systematically map the interactome of SARS-CoV-2 ncrRNA in Calu-3, Huh7, and HEK293T cells. Integration of the results defined the core ncrRNA-host protein interactomes among cell lines. The 5' UTR interactome is enriched with proteins in the small nuclear ribonucleoproteins family and is a target for the regulation of viral replication and transcription. The 3' UTR interactome is enriched with proteins involved in the stress granules and heterogeneous nuclear ribonucleoproteins family. Intriguingly, compared with the positive-sense ncrRNAs, the negative-sense ncrRNAs, especially the negative-sense of 3' UTR, interacted with a large array of host proteins across all cell lines. These proteins are involved in the regulation of the viral production process, host cell apoptosis, and immune response. Taken together, our study depicts the comprehensive landscape of the SARS-CoV-2 ncrRNA-host protein interactome and unveils the potential regulatory role of the negative-sense ncrRNAs, providing a new perspective on virus-host interactions and the design of future therapeutics. Given the highly conserved nature of UTRs in positive-strand viruses, the regulatory role of negative-sense ncrRNAs should not be exclusive to SARS-CoV-2. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic affecting millions of lives. During replication and transcription, noncoding regions of the viral RNA (ncrRNAs) may play an important role in the virus-host interactions. Understanding which and how these ncrRNAs interact with host proteins is crucial for understanding the mechanism of SARS-CoV-2 pathogenesis. We developed the MS2 affinity purification coupled with liquid chromatography-mass spectrometry method and designed a diverse set of ncrRNAs to identify the SARS-CoV-2 ncrRNA interactome comprehensively in different cell lines and found that the 5' UTR binds to proteins involved in U1 small nuclear ribonucleoprotein, while the 3' UTR interacts with proteins involved in stress granules and the heterogeneous nuclear ribonucleoprotein family. Interestingly, negative-sense ncrRNAs showed interactions with a large number of diverse host proteins, indicating a crucial role in infection. The results demonstrate that ncrRNAs could serve diverse regulatory functions.

Imatinib blocks tyrosine phosphorylation of Smad4 and restores TGF-ß growth-suppressive signaling in BCR-ABL1-positive leukemia.

Loss of TGF-ß-mediated growth suppression is a major contributor to the development of cancers, best exemplified by loss-of-function mutations in genes encoding components of the TGF-ß signaling pathway in colorectal and pancreatic cancers. Alternatively, gain-of-function oncogene mutations can also disrupt antiproliferative TGF-ß signaling. However, the molecular mechanisms underlying oncogene-induced modulation of TGF-ß signaling have not been extensively investigated. Here, we show that the oncogenic BCR-ABL1 of chronic myelogenous leukemia (CML) and the cellular ABL1 tyrosine kinases phosphorylate and inactivate Smad4 to block antiproliferative TGF-ß signaling. Mechanistically, phosphorylation of Smad4 at Tyr195, Tyr301, and Tyr322 in the linker region interferes with its binding to the transcription co-activator p300/CBP, thereby blocking the ability of Smad4 to activate the expression of cyclin-dependent kinase (CDK) inhibitors and induce cell cycle arrest. In contrast, the inhibition of BCR-ABL1 kinase with Imatinib prevented Smad4 tyrosine phosphorylation and re-sensitized CML cells to TGF-ß-induced antiproliferative and pro-apoptotic responses. Furthermore, expression of phosphorylation-site-mutated Y195F/Y301F/Y322F mutant of Smad4 in Smad4-null CML cells enhanced antiproliferative responses to TGF-ß, whereas the phosphorylation-mimicking Y195E/Y301E/Y322E mutant interfered with TGF-ß signaling and enhanced the in vivo growth of CML cells. These findings demonstrate the direct role of BCR-ABL1 tyrosine kinase in suppressing TGF-ß signaling in CML and explain how Imatinib-targeted therapy restored beneficial TGF-ß anti-growth responses.

Protein phosphatase PPM1A inhibition attenuates osteoarthritis via regulating TGF-ß/Smad2 signaling in chondrocytes.

TGF-ß signaling is crucial for modulating osteoarthritis (OA), and protein phosphatase magnesium-dependent 1A (PPM1A) has been reported as a phosphatase of SMAD2 and regulates TGF-ß signaling, while the role of PPM1A in cartilage homeostasis and OA development remains largely unexplored. In this study, we found increased PPM1A expression in OA chondrocytes and confirmed the interaction between PPM1A and phospho-SMAD2 (p-SMAD2). Importantly, our data show that PPM1A KO substantially protected mice treated with destabilization of medial meniscus (DMM) surgery against cartilage degeneration and subchondral sclerosis. Additionally, PPM1A ablation reduced the cartilage catabolism and cell apoptosis after the DMM operation. Moreover, p-SMAD2 expression in chondrocytes from KO mice was higher than that in WT controls with DMM induction. However, intraarticular injection with SD-208, repressing TGF-ß/SMAD2 signaling, dramatically abolished protective phenotypes in PPM1A-KO mice. Finally, a specific pharmacologic PPM1A inhibitor, Sanguinarine chloride (SC) or BC-21, was able to ameliorate OA severity in C57BL/6J mice. In summary, our study identified PPM1A as a pivotal regulator of cartilage homeostasis and demonstrated that PPM1A inhibition attenuates OA progression via regulating TGF-ß/SMAD2 signaling in chondrocytes and provided PPM1A as a potential target for OA treatment.

HERC3 promotes YAP/TAZ stability and tumorigenesis independently of its ubiquitin ligase activity.

YAP/TAZ transcriptional co-activators play pivotal roles in tumorigenesis. In the Hippo pathway, diverse signals activate the MST-LATS kinase cascade that leads to YAP/TAZ phosphorylation, and subsequent ubiquitination and proteasomal degradation by SCFß-TrCP . When the MST-LATS kinase cascade is inactive, unphosphorylated or dephosphorylated YAP/TAZ translocate into the nucleus to mediate TEAD-dependent gene transcription. Hippo signaling-independent YAP/TAZ activation in human malignancies has also been observed, yet the mechanism remains largely elusive. Here, we report that the ubiquitin E3 ligase HERC3 can promote YAP/TAZ activation independently of its enzymatic activity. HERC3 directly binds to ß-TrCP, blocks its interaction with YAP/TAZ, and thus prevents YAP/TAZ ubiquitination and degradation. Expression levels of HERC3 correlate with YAP/TAZ protein levels and expression of YAP/TAZ target genes in breast tumor cells and tissues. Accordingly, knockdown of HERC3 expression ameliorates tumorigenesis of breast cancer cells. Our results establish HERC3 as a critical regulator of the YAP/TAZ stability and a potential therapeutic target for breast cancer.

Targeting proteostasis of the HEV replicase to combat infection in preclinical models.

BACKGROUND & AIMS: Appropriate treatment options are lacking for hepatitis E virus (HEV)-infected pregnant women and immunocompromised individuals. Thus, we aimed to identify efficient anti-HEV drugs through high-throughput screening, validate them in vitro and in vivo (in a preclinical animal study), and elucidate their underlying antiviral mechanism of action. METHODS: Using appropriate cellular and rodent HEV infection models, we studied a critical pathway for host-HEV interactions and performed a preclinical study of the corresponding antivirals, which target proteostasis of the HEV replicase. RESULTS: We found 17 inhibitors that target HEV-HSP90 interactions by unbiased compound library screening on human hepatocytes harboring an HEV replicon. Inhibitors of HSP90 (iHSP90) markedly suppressed HEV replication with efficacy exceeding that of conventional antivirals (IFNα and ribavirin) in vitro. Mechanistically, iHSP90 treatment released the viral replicase ORF1 protein from the ORF1-HSP90 complex and triggered rapid ubiquitin/proteasome-mediated degradation of ORF1, resulting in abrogated HEV replication. Furthermore, a preclinical trial in a Mongolian gerbil HEV infection model showed this novel anti-HEV strategy to be safe, efficient, and able to prevent HEV-induced liver damage. CONCLUSIONS: In this study, we uncover a proteostatic pathway that is critical for host-HEV interactions and we provide a foundation from which to translate this new understanding of the HEV life cycle into clinically promising antivirals. IMPACT AND IMPLICATIONS: Appropriate treatment options for hepatitis E virus (HEV)-infected pregnant women and immunocompromised patients are lacking; hence, there is an urgent need for safe and effective HEV-specific therapies. This study identified new antivirals (inhibitors of HSP90) that significantly limit HEV infection by targeting the viral replicase for degradation. Moreover, these anti-HEV drugs were validated in an HEV rodent model and were found to be safe and efficient for prevention of HEV-induced liver injury in preclinical experiments. Our findings substantially promote the understanding of HEV pathobiology and pave the way for antiviral development.

Energy sensor AMPK gamma regulates translation via phosphatase PPP6C independent of AMPK alpha.

Maintenance of energy level to drive movements and material exchange with the environment is a basic principle of life. AMP-activated protein kinase (AMPK) senses energy level and is a major regulator of cellular energy responses. The gamma subunit of AMPK senses elevated ratio of AMP to ATP and allosterically activates the alpha catalytic subunit to phosphorylate downstream effectors. Here, we report that knockout of AMPKγ, but not AMPKα, suppressed phosphorylation of eukaryotic translation elongation factor 2 (eEF2) induced by energy starvation. We identified PPP6C as an AMPKγ-regulated phosphatase of eEF2. AMP-bound AMPKγ sequesters PPP6C, thereby blocking dephosphorylation of eEF2 and thus inhibiting translation elongation to preserve energy and to promote cell survival. Further phosphoproteomic analysis identified additional targets of PPP6C regulated by energy stress in an AMPKγ-dependent manner. Thus, AMPKγ senses cellular energy availability to regulate not only AMPKα kinase, but also PPP6C phosphatase and possibly other effectors.

AMPK directly phosphorylates TBK1 to integrate glucose sensing into innate immunity.

Nutrient sensing and damage sensing are two fundamental processes in living organisms. While hyperglycemia is frequently linked to diabetes-related vulnerability to microbial infection, how body glucose levels affect innate immune responses to microbial invasion is not fully understood. Here, we surprisingly found that viral infection led to a rapid and dramatic decrease in blood glucose levels in rodents, leading to robust AMPK activation. AMPK, once activated, directly phosphorylates TBK1 at S511, which triggers IRF3 recruitment and the assembly of MAVS or STING signalosomes. Consistently, ablation or inhibition of AMPK, knockin of TBK1-S511A, or increased glucose levels compromised nucleic acid sensing, while boosting AMPK-TBK1 cascade by AICAR or TBK1-S511E knockin improves antiviral immunity substantially in various animal models. Thus, we identify TBK1 as an AMPK substrate, reveal the molecular mechanism coupling a dual sensing of glucose and nuclei acids, and report its physiological necessity in antiviral defense.

To Ub or not to Ub: a regulatory question in TGF-ß signaling.

The transforming growth factor ß (TGF-ß) superfamily controls a wide spectrum of biological processes in metazoans, including cell proliferation, apoptosis, differentiation, cell-fate determination, and embryonic development. Deregulation of TGF-ß-Smad signaling contributes to developmental anomalies and a variety of disorders and diseases such as tumorigenesis, fibrotic disorders, and immune diseases. In cancer, TGF-ß has dual effects through its antiproliferative and prometastatic actions. At the cellular level, TGF-ß functions mainly through the canonical Smad-dependent pathway in a cell type-specific and context-dependent manner. Accumulating evidence has demonstrated that ubiquitination plays a vital role in regulating TGF-ß-Smad signaling. We summarize current progress on ubiquitination (Ub) and the ubiquitin ligases that regulate TGF-ß-Smad signaling.

FUNDC2 promotes liver tumorigenesis by inhibiting MFN1-mediated mitochondrial fusion.

Mitochondria generate ATP and play regulatory roles in various cellular activities. Cancer cells often exhibit fragmented mitochondria. However, the underlying mechanism remains elusive. Here we report that a mitochondrial protein FUN14 domain containing 2 (FUNDC2) is transcriptionally upregulated in primary mouse liver tumors, and in approximately 40% of human hepatocellular carcinoma (HCC). Importantly, elevated FUNDC2 expression inversely correlates with patient survival, and its knockdown inhibits liver tumorigenesis in mice. Mechanistically, the amino-terminal region of FUNDC2 interacts with the GTPase domain of mitofusin 1 (MFN1), thus inhibits its activity in promoting fusion of outer mitochondrial membrane. As a result, loss of FUNDC2 leads to mitochondrial elongation, decreased mitochondrial respiration, and reprogrammed cellular metabolism. These results identified a mechanism of mitochondrial fragmentation in cancer through MFN1 inhibition by FUNDC2, and suggested FUNDC2 as a potential therapeutic target of HCC.

Liver cancer heterogeneity modeled by in situ genome editing of hepatocytes.

Mechanistic study and precision treatment of primary liver cancer (PLC) are hindered by marked heterogeneity, which is challenging to recapitulate in any given liver cancer mouse model. Here, we report the generation of 25 mouse models of PLC by in situ genome editing of hepatocytes recapitulating 25 single or combinations of human cancer driver genes. These mouse tumors represent major histopathological types of human PLCs and could be divided into three human-matched molecular subtypes based on transcriptomic and proteomic profiles. Phenotypical characterization identified subtype- or genotype-specific alterations in immune microenvironment, metabolic reprogramming, cell proliferation, and expression of drug targets. Furthermore, single-cell analysis and expression tracing revealed spatial and temporal dynamics in expression of pyruvate kinase M2 (Pkm2). Tumor-specific knockdown of Pkm2 by multiplexed genome editing reversed the Warburg effect and suppressed tumorigenesis in a genotype-specific manner. Our study provides mouse PLC models with defined genetic drivers and characterized phenotypical heterogeneity suitable for mechanistic investigation and preclinical testing.