Biology subjects

Chiang, A.

Publications and source records attributed to Chiang, A..

10 recordsLinked to original sources

bioRxiv2025

Autism Mutations Preferentially Target Distributed Brain Circuits and Cell Types Associated with Sensory Integration and Decision Making

Autism spectrum disorder (ASD) is a highly heritable psychiatric condition characterized by difficulties in social communication and stereotypic, repetitive behaviors. Genetics studies have discovered many dozens of genes with causal roles in autism, and functional analyses have demonstrated that ASD-associated mutations affect a diverse range of brain regions and cell types. However, the precise mechanisms by which these genetic alterations lead to autism-related phenotypes remain unclear. Psychiatric cognitive and behavioral traits are believed to arise from dysfunction of specific brain circuits formed by anatomically and functionally connected brain areas. To identify the circuits and cell types primarily affected by ASD mutations, we developed an unbiased approach, GENCIC, which computationally integrates genome-wide genetic data with a brain-wide spatial mouse transcriptome and an anatomical mesoscale connectome. Applying this approach to ASD reveals a convergence of mutations on cohesive brain circuits that are enriched in long-distance connections and span both cortical and subcortical brain structures. Furthermore, our analysis of brain-wide single-cell spatial transcriptomics shows that the heterogeneity of circuit structures affected in ASD is matched by the substantial diversity of strongly impacted circuit cell types. Notably, the implicated circuits and cell types play a central role in the integration of multimodal sensory and emotional information and in decision-making based on this information. We also find that different circuit structures exhibit distinct vulnerability patterns that correlate with cognitive phenotypes in ASD. Overall, our study demonstrates how ASD-related genetic mutations impact multiple levels of brain organization, ultimately disrupting functional circuits that drive core autism-related behaviors.

systems biology↗

bioRxiv2024

Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination

Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC50 to cisplatin and whole-cell proteomics reveal an increased abundance of [~]20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2Ds central role in proteome management.

biochemistry↗

bioRxiv2024

Cellular processing of beneficial de novo emerging proteins

Recent evidence demonstrates that eukaryotic genomes encode thousands of evolutionarily novel proteins that originate de novo from non-coding DNA and can contribute to species-specific adaptations. Yet, it remains unclear how these incipient proteins--whose sequences are entirely new to nature--navigate the cellular environment to bring about phenotypic change. Here, we conduct a systematic in vivo investigation of yeast de novo proteins with enhanced growth phenotypes, revealing the early stages of cellular integration. We find that these proteins are strongly enriched at the endoplasmic reticulum (ER) relative to conserved proteins, and that they integrate into cellular systems through conserved membrane targeting, trafficking, and degradation pathways. Despite having unrelated sequences, ER-localized de novo proteins share a common molecular signature: a C-terminal transmembrane domain that likely enables recognition by conserved post-translational ER insertion pathways. After insertion, ER-localized de novo proteins traffic from the ER and their homeostasis is regulated by conserved proteasomal and vacuolar degradation pathways. Our findings demonstrate that ancient targeting and degradation pathways can accommodate young de novo proteins sharing a convergent molecular signature. These pathways may act as selective filters, biasing which young de novo proteins persist. Significance StatementDeeply conserved genes shape the core structure and function of cells, but novel genes are key drivers of biodiversity. Novel genes can arise by divergence from ancient genes, or de novo from non-genic DNA. For de novo genes to develop novel functions, it is necessary for them to be processed by the cell so that their protein levels and localization are regulated. However, little is known about how novel de novo genes obtain the capacity to engage the cellular machinery needed for this regulation. Here, we experimentally assess how a set of endoplasmic reticulum (ER)-localized proteins encoded by recently-evolved de novo genes are localized and degraded in yeast cells. We discover that, despite having entirely unique amino acid sequences, these proteins share biochemical signatures allowing them to engage the same ancient cellular machinery and localize to the ER membrane. Interestingly though, this machinery is not the one that targets most ancient proteins to the ER. These results indicate that even recently emerged proteins without an extensive period of evolutionary adaptation can be recognized by specific ancient cellular pathways, facilitating their localization and homeostasis.

evolutionary biology↗

bioRxiv2024

Protein structure, a genetic encoding for glycosylation

Unlike DNA, RNA, and protein biosynthesis, dogma describes glycosylation as primarily determined by intrinsic cellular limitations, such as glycosyltransferase expression and precursor availability. However, this cannot explain the commonly-observed differences between glycans on the same protein. By examining site-specific glycosylation on diverse human proteins, we detected associations between protein structure and glycan structure, broadly generalizable to human-expressed glycoproteins. Through structural analysis of site-specific glycosylation data, we found protein-sequence and structural features consistently correlated with specific glycan features. To quantify these relationships, we present a new amino acid substitution matrix describing "glycoimpact", i.e., the association of primary protein structure and glycosylation. High-glycoimpact amino acids co-evolve with glycosites, and glycoimpact is high when estimates of amino acid conservation and variant pathogenicity diverge. We report thousands of disease variants near glycosites with high-glycoimpact, including several with known links to aberrant glycosylation (e.g., Oculocutaneous Albinism, Jakob-Creutzfeldt disease, Gerstmann-Straussler-Scheinker, and Gauchers Disease). Finally, glycoimpact quantification is validated by studying oligomannose-complex glycan ratios on HIV ENV, differential sialylation on IgG3 Fc, differential glycosylation on SARS-CoV-2 Spike, and fucose-modulated function of a tuberculosis monoclonal antibody. Finally, to test the causality of protein-glycan associations, we created 5 glycoimpact-designed novel Rituximab variants, 4 of which substantially changed glycoprofiles as predicted. In all, we report that site-specific glycan biosynthesis is influenced by underlying protein structure, enabling glycan structure prediction and genetic sequence-guided glycoengineering.

genetics↗

bioRxiv2023

ProteoMixture: A Cell Type Deconvolution Tool for Bulk Tissue Proteomics Data

Numerous multi-omic investigations of cancer tissue have documented varying and poor pairwise transcript:protein quantitative correlations and most deconvolution tools aiming to predict cell type proportions (cell admixture) have been developed and credentialed using transcript-level data alone. To estimate cell admixture using protein abundance data, we analyzed proteome and transcriptome data generated from contrived admixtures of tumor, stroma, and immune cell models or those selectively harvested from the tissue microenvironment by laser microdissection from high grade serous ovarian cancer (HGSOC) tumors. Co-quantified transcripts and proteins performed similarly to estimate stroma and immune cell admixture in two commonly used deconvolution algorithms, ESTIMATE and ConsensusTME (r [≥] 0.63). Here we have developed and optimized protein-based signatures to estimate cell admixture proportions and benchmarked these using bulk tumor proteomics data from over 150 HGSOC patients. The optimized protein signatures supporting cell type proportion estimates from bulk tissue proteomics data are available at (https://lmdomics.org/ProteoMixture/.

bioinformatics↗

bioRxiv2022

Intramuscular injection of nerve growth factor as a model of temporomandibular disorder: nature, time-course, and sex differences characterising the pain experience

BackgroundTemporomandibular disorder (TMD) is a common condition that frequently transitions to chronic symptoms. Experimental pain models that mimic the symptoms of clinical TMD may be useful in understanding the mechanisms, and sex differences, present in this disorder. Here we aimed to comprehensively characterise the nature and time-course of pain, functional impairment and hyperalgesia induced by repeated intramuscular injection of nerve growth factor (NGF) into the masseter muscle, and to investigate sex differences in the NGF-induced pain experience. Methods94 healthy individuals participated in a longitudinal observational study with 30-day follow-up. NGF was injected into the right masseter muscle on Day 0 and Day 2. Participants attended laboratory sessions to assess pain (Numerical Rating Scale; NRS), functional limitation (mouth opening distance, Jaw Functional Limitation Scale; JFLS) and mechanical sensitization (pressure pain thresholds; PPTs) on Days 0, 2 and 5 and completed twice daily electronic pain dairies from Day 0 to day 30. ResultsPeak pain averaged 2.0/10 (95 % CI: 1.6-2.4) at rest and 4.3/10 (95 % CI: 3.9-4.8) on chewing. Pain-free mouth opening distance reduced from 5.0 cm (95 % CI: 4.8-5.1 cm) on Day 0 to 3.7 cm (95 % CI: 3.5-3.9 cm) on Day 5. The greatest reduction in PPTs was observed over the masseter muscle. Females experienced higher pain, greater functional impairment, and greater sensitivity to mechanical stimuli than males. ConclusionIntramuscular injection of NGF is a useful model with which to explore the mechanisms, and sex differences, present in clinical TMD.

neuroscience↗

bioRxiv2022

α-Arrestins maintain phospholipid balance and Atg18 distribution to permit efficient autophagy

Cells selectively reorganize their membrane proteome in response to stressors via selective protein trafficking. The -arrestins, a family of conserved protein trafficking adaptors, bind to select membrane proteins and interact with the ubiquitin ligase Rsp5. The -arrestins recruit Rsp5 to its membrane protein substrates, permitting their ubiquitination and endocytosis. To identify new -arrestin functions, we performed a genetic screen to isolate mutants that alter -arrestin-mediated resistance to rapamycin, a drug that inhibits TORC1. Interestingly, loss of many of the ATG genes, which encode the machinery needed for the self-degradative process of autophagy, disrupted -arrestins ability to promote growth on rapamycin. Herein we define a genetic network linking -arrestins to autophagy. We show autophagy impairment in the absence of select -arrestins, with increased autophagosome lifetimes and delayed/reduced delivery of autophagosomes to the vacuole. The -arrestin mutants that impeded autophagy had vacuole morphology defects and increased vacuolar retention of Atg18, a member of the PROPPIN family that is needed to maintain vacuole shape and facilitate lipid transfer to expanding autophagosomes. Atg18 binds phosphatidylinositol 3 phosphate (PI3P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), and we observed increased PI3P on the vacuole membrane in -arrestin mutants. The levels of Vps34 and Fab1, the kinases responsible for the generation of PI3P and PI(3,5)P2, respectively, were also elevated at vacuole membranes in cells lacking -arrestins. We posit that altered phospholipids in the vacuolar membrane form the basis for the Atg18-Atg2 mislocalization and autophagy defect. These data demonstrate a previously unappreciated link between the -arrestins and autophagy, expanding the functional impact of these trafficking adaptors in responding to nutrient stress. Author SummaryCells survive nutrient starvation by degrading parts of themselves through the process of autophagy. During autophagy, cells make a double membrane, known as an autophagosome (AP), around bits of cytoplasm or organelles. The AP and its engulfed material are delivered to the vacuole, an organelle that helps break down proteins and lipids. These materials can then be used as building blocks to generate the essential components needed for the cell to survive starvation. For cells to undergo efficient autophagy, they need -arrestins, a group of proteins important for deciding where membrane proteins localize. In cells lacking -arrestins, the AP forms slowly, likely due to a problem in growing the AP membrane. This results in less material being delivered to the vacuole via APs when cells do not have -arrestins. This study defines a new role for -arrestins in promoting AP formation and starvation survival.

cell biology↗

bioRxiv2022

Dynamic changes in the niche and transcription trigger early murine and human pluripotent stem cell-derived liver organogenesis

The shift from collective migration to differentiation is a crucial process in epithelial biology but recreating this intricate transition has thus far proved elusive. We provide experimental, mechanistic, in vivo, and bioinformatic data supporting an undoubtable link between human pluripotent stem cell (hPSC)- derived collectively migrating hepatoblasts (MHB), and transcriptionally mature, functional hPSC- hepatocytes (HEPs), which incorporates two unrecognized steps. The protocol induces FOXA-dependent induction of HBs, leading to TBX3-positive, YAP-TEAD active MHBs which provide a transcriptional match with murine liver E9.5 MHBs. Simple cultivation changes trigger MHBs to rapidly form functional day 18 HEPs, predicted by a deep-learning designed gene circuit, resulting in a [~]236% fold- increase in maturation (PACNet), on par with the highest score, but with enhanced global transcriptional shaping. Overall, incorporating the MHB to HEP transition establishes a new, unrecognized, and highly efficient mechanism for differentiation that can be cumulatively integrated with existing methods to overcome barriers to maturation.

bioengineering↗

bioRxiv2021

The influence of somatosensory and auditory evoked potentials on concurrent transcranial-magnetic stimulation electroencephalography recordings

BackgroundTranscranial magnetic stimulation (TMS) evoked potentials (TEPs) can be used to index cortical excitability. However, it remains unclear to what extent TEPs reflect somatosensory and auditory-evoked potentials which arise from the scalp sensation and click of the TMS coil, as opposed to transcranial stimulation of cortical circuits. ObjectivesThe present study had two aims; a) to determine the extent to which sensory potentials contaminate TEPs using a spatially matched sham condition, and b) to determine whether sensory potentials reflect auditory or somatosensory potentials alone, or a combination of the two. MethodsTwenty healthy participants received active or sham stimulation, with the latter consisting of the click of a sham coil combined with scalp electrical stimulation. Earplugs/headphones were used to suppress the TMS click noise. Two additional control conditions i) electrical stimulation alone and ii) auditory stimulation alone were included in a subset of 13 participants. ResultsSignals from active and sham stimulation were correlated in spatial and temporal domains, especially >70ms post-stimulation. Relative to auditory or electrical stimulation alone, combined (sham) stimulation resulted in a) larger evoked responses b) stronger correlations with active stimulation and c) a signal that could not be explained by the linear sum of electrical and auditory stimulation alone. ConclusionsSensory potentials can confound data interpretations of TEPs at timepoints >70ms post-TMS, while earlier timepoints appear reflective of cortical excitability. Furthermore, contamination of TEPs cannot be explained by auditory or somatosensory potentials alone, but instead reflects a non-linear interaction between both sources. Future studies may benefit from controlling for sensory contamination using sham conditions that are spatially matched to active TMS, and which consist of combined auditory and somatosensory stimulation.

neuroscience↗

bioRxiv2021

Alpha-arrestins Aly1 and Aly2 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis

Phosphatidylinositol (PI) is an essential phospholipid and critical component of membrane bilayers. The complete deacylation of PI by phospholipases of the B-type leads to the production of intracellular and extracellular glycerophosphoinositol (GPI), a water-soluble glycerophosphodiester. Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters that resides at the plasma membrane in yeast. Once internalized, GPI can be degraded to produce inositol, phosphate and glycerol, thereby contributing to reserves of these building blocks. Not surprisingly, GIT1 gene expression is controlled by nutrient balance, with limitation for phosphate or inositol each increasing GIT1 expression to facilitate GPI uptake. Less is known about how Git1 protein levels or localization are controlled. Here we show that the -arrestins, an important class of protein trafficking adaptor, regulate the localization of Git1 in a manner dependent upon their association with the ubiquitin ligase Rsp5. Specifically, -arrestin Aly2 is needed for effective Git1 internalization from the plasma membrane under basal conditions. However, in response to GPI-treatment of cells, either Aly1 or Aly2 can promote Git1 trafficking to the vacuole. Retention of Git1 at the cell surface, as occurs in aly1{Delta} aly2{Delta} cells, results in impaired growth in the presences of excess exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of this metabolite or its downstream products leads to cellular toxicity. We further show that regulation of -arrestin Aly1 by the protein phosphatase calcineurin improves both steady-state and ligand-induced trafficking of Git1 when a mutant allele of Aly1 that mimics the dephosphorylated state at calcineurin-regulated residues is employed. Thus, calcineurin regulation of Aly1 is important for the GPI-ligand induced trafficking of Git1 by this -arrestin, however, the role of calcineurin in regulating Git1 trafficking is much broader than can simply be explained by regulation of the -arrestins. Finally, we find that loss of Aly1 and Aly2 leads to an increase in phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole and this alteration is further exacerbated by addition of GPI, suggesting that the effect is at least partially linked to Git1 function. Indeed, loss of Aly1 and Aly2 leads to increased incorporation of inositol label from 3H-inositol-labelled GPI into PI, confirming that internalized GPI influences PI synthesis and indicating a role for the -arrestins in regulating the process.

cell biology↗