Biology subjects

Skariah, G.

Publications and source records attributed to Skariah, G..

6 recordsLinked to original sources

bioRxiv2026

ENPP1 buffers extracellular cGAMP in brown adipose tissue to limit insulin resistance

The ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has long been linked with metabolic diseases, with the common ENPP1 K173Q (historically K121Q) variant conferring increased risk for type 2 diabetes (T2D). However, the mechanistic basis of this association has remained unclear. Here, we demonstrate that the K173Q variant has decreased cGAMP hydrolysis activity, suggesting that this loss of enzymatic function could contribute to its pathogenesis. Using a cGAMP-hydrolysis-deficient knock-in mouse (Enpp1H362A), we show that selective loss of this activity leads to a primary defect in energy expenditure and exacerbates high-fat diet (HFD)-induced weight gain and insulin resistance. An unbiased in vivo glucose-uptake screen reveals brown adipose tissue (BAT) as a focal site of metabolic impairment, characterized by profound extracellular cGAMP accumulation and a selective failure of insulin-stimulated glucose uptake. Mechanistically, we demonstrate that nutrient excess drives mitochondrial DNA leakage in brown adipocytes, triggering cGAMP production and export. Excess cGAMP directly propagates STING-dependent suppression of glucose uptake and lipogenesis in brown adipocytes. Additionally, when ENPP1-mediated clearance is compromised, extracellular cGAMP acts as a paracrine immunotransmitter that remodels the BAT microenvironment by recruiting and polarizing macrophages toward an M1-like phenotype. Together, our findings nominate the impaired ENPP1-dependent buffering of extracellular cGAMP as one mechanism by which ENPP1 variants influence metabolic homeostasis.

immunology↗

bioRxiv2023

ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway

ENPP1 expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer STING pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single cell RNA-seq (scRNA-seq), we show that ENPP1 overexpression drives primary breast tumor growth and metastasis by synergistically dampening extracellular cGAMP-STING mediated antitumoral immunity and activating immunosuppressive extracellular adenosine (eADO) signaling. In addition to cancer cells, stromal and immune cells in the tumor microenvironment (TME) also express ENPP1 that restrains their response to tumor-derived cGAMP. Enpp1 loss-of-function in both cancer cells and normal tissues slowed primary tumor initiation and growth and prevented metastasis in an extracellular cGAMP- and STING-dependent manner. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied total ENPP1 knockout, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Strikingly, we find that breast cancer patients with low ENPP1 expression have significantly higher immune infiltration and improved response to therapeutics impacting cancer immunity upstream or downstream of the cGAMP-STING pathway, like PARP inhibitors and anti-PD1. Altogether, selective inhibition of ENPP1s cGAMP hydrolase activity alleviates an innate immune checkpoint to boost cancer immunity and is therefore a promising therapeutic approach against breast cancer that may synergize with other cancer immunotherapies.

cancer biology↗

bioRxiv2021

Probing pathophysiology of extracellular cGAMP with substrate-selective ENPP1

The biology of the immune second messenger cGAMP depends on its cellular localization. cGAMP, which is synthesized in response to cytosolic double-stranded DNA, also exists in the extracellular space as a paracrine immunotransmitter that enhances the anticancer immune response. However, the role of extracellular cGAMP is unexplored outside of cancer due to a lack of tools to systemically manipulate it. The extracellular enzyme ENPP1, the only known hydrolase of cGAMP, is a promising target. However, because ENPP1 also degrades extracellular ATP, using genetic knockouts of ENPP1 to study extracellular cGAMP leads to confounding effects. Here we report the H362A point mutation in ENPP1, the dominant cGAMP hydrolase, which selectively abolishes ENPP1s ability to degrade cGAMP, while retaining activity toward other substrates. H362 is not necessary for binding cGAMP or the catalytically-essential zinc atoms but instead supports the in-line reaction geometry. H362 is evolutionarily conserved down to bacteria, suggesting an ancient origin for extracellular cGAMP biology. Enpp1H362A mice do not display the systemic calcification seen in Enpp1-/- mice, highlighting the substrate-specific phenotype of ENPP1. Remarkably, Enpp1H362A mice were resistant to HSV-1 infection, demonstrating the antiviral role of endogenous extracellular cGAMP. The ENPP1H362A mutation is the first genetic tool to enable exploration of extracellular cGAMP biology in a wide range of tissues and diseases.

immunology↗

bioRxiv2019

Neuropathology of RAN translation proteins in Fragile X-associated Tremor/Ataxia Syndrome

CGG repeat expansions in FMR1 cause the neurodegenerative disorder Fragile X-associated Tremor/Ataxia Syndrome (FXTAS). Ubiquitinated neuronal intranuclear inclusions (NIIs) are the neuropathological hallmark of FXTAS. Both sense strand derived CGG repeats and antisense strand derived CCG repeats support non-AUG initiated (RAN) translation of homopolymeric proteins in potentially 6 different reading frames. However, the relative abundance of these proteins in FXTAS brains and their co-localization with each other and NIIs is lacking. Here we describe rater-blinded assessment of immunohistochemical and immunofluorescence staining with newly generated antibodies to different CGG RAN translation products in FXTAS and control brains as well as co-staining with ubiquitin, p62/SQSTM1, and ubiquilin 2. We find that both FMRpolyG and a second CGG repeat derived RAN translation product, FMRpolyA, accumulate in aggregates in FXTAS brains. FMRpolyG is a near-obligate component of both ubiquitin-positive and p62-positive NIIs in FXTAS, with occurrence of aggregates in 20% of all hippocampal neurons and >90% of all inclusions. A subset of these inclusions also stain positive for the ALS/FTD associated protein ubiquilin 2. Ubiquitinated inclusions and FMRpolyG+ aggregates are rarer in cortex and cerebellum. Intriguingly, FMRpolyG staining is also visible in control neuronal nuclei. In contrast to FMRpolyG, staining for FMRpolyA and CCG antisense derived RAN translation products were less abundant and were infrequent components of FMRpolyG+ inclusions. In conclusion, RAN translated FMRpolyG is a common component of ubiquitin and p62 positive inclusions in FXTAS patient brains.

neuroscience↗

bioRxiv2019

2'3'-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

23-cyclic GMP-AMP (cGAMP) is characterized as an intracellular second messenger that is synthesized in response to cytosolic dsDNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, using mass spectrometry, we detected that cGAMP is continuously exported as a soluble factor by an engineered cell line but then efficiently cleared by ENPP1, explaining why it has escaped detection until now. By developing a potent, specific, and cell impermeable ENPP1 inhibitor, we detected cGAMP export in cancer cell lines commonly used for mouse tumor models. In tumors, depletion of extracellular cGAMP using neutralizing proteins decreased tumor-associated dendritic cells. Boosting extracellular cGAMP by genetic knockout and pharmacological inhibition of ENPP1 increased tumor-associated dendritic cells, shrunk tumors, and synergized with ionizing radiation and anti-CTLA-4 to cure tumors. In conclusion, cGAMP is an anti-cancer immunotransmitter released by tumors and detected by host innate immunity.

biochemistry↗

bioRxiv2018

Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells

Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5 untranslated region of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing, and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRPs pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the CRISPR/dCAS9 system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCAS9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 mRNA expression despite promoter and repeat methylation. These studies demonstrate that FMR1 mRNA expression can be selectively reactivated in human patient cells, creating a pathway forward for therapeutic development in Fragile X Syndrome.

neuroscience↗