Protein Transport Research Articles

Plastid HSP90C C-terminal extension region plays a regulatory role in chaperone activity and client binding.

HSP90Cs are essential molecular chaperones localized in the plastid stroma that maintain protein homeostasis and assist the import and thylakoid transport of chloroplast proteins. While HSP90C contains all conserved domains as an HSP90 family protein, it also possesses a unique feature in its variable C-terminal extension (CTE) region. This study elucidated the specific function of this HSP90C CTE region. Our phylogenetic analyses revealed that this intrinsically disordered region contains a highly conserved DPW motif in the green lineages. With biochemical assays, we showed that the CTE is required for the chaperone to effectively interact with client proteins PsbO1 and LHCB2 to regulate ATP-independent chaperone activity and to effectuate its ATP hydrolysis. The CTE truncation mutants could support plant growth and development reminiscing the wild type under normal conditions except for a minor phenotype in cotyledon when expressed at a level comparable to wild type. However, higher HSP90C expression was observed to correlate with a stronger response to specific photosystem II inhibitor DCMU, and CTE truncations dampened the response. Additionally, when treated with lincomycin to inhibit chloroplast protein translation, CTE truncation mutants showed a delayed response to PsbO1 expression repression, suggesting its role in chloroplast retrograde signaling. Our study therefore provides insights into the mechanism of HSP90C in client protein binding and the regulation of green chloroplast maturation and function, especially under stress conditions.

Haploinsufficiency of intraflagellar transport protein 172 causes autism-like behavioral phenotypes in mice through BDNF

IntroductionPrimary cilia are hair-like solitary organelles growing on most mammalian cells that play fundamental roles in embryonic patterning and organogenesis. Defective cilia often cause a suite of inherited diseases called ciliopathies with multifaceted manifestations. Intraflagellar transport (IFT), a bidirectional protein trafficking along the cilium, actively facilitates the formation and absorption of primary cilia. IFT172 is the largest component of the IFT-B complex, and its roles in Bardet-Biedl Syndrome (BBS) have been appreciated with unclear mechanisms. ObjectivesWe performed a battery of behavioral tests with Ift172 haploinsufficiency (Ift172+/-) and WT littermates. We use RNA sequencing to identify the genes and signaling pathways that are differentially expressed and enriched in the hippocampus of Ift172+/- mice. Using AAV-mediated sparse labeling, electron microscopic examination, patch clamp and local field potential recording, western blot, luciferase reporter assay, chromatin immunoprecipitation, and neuropharmacological approach, we investigated the underlying mechanisms for the aberrant phenotypes presented by Ift172+/- mice. ResultsIft172+/- mice displayed excessive self-grooming, elevated anxiety, and impaired cognition. RNA sequencing revealed enrichment of differentially expressed genes in pathways relevant to axonogenesis and synaptic plasticity, which were further confirmed by less spine density and synaptic number. Ift172+/- mice demonstrated fewer parvalbumin-expressing neurons, decreased inhibitory synaptic transmission, augmented theta oscillation, and sharp-wave ripples in the CA1 region. Moreover, Ift172 haploinsufficiency caused less BDNF production and less activated BDNF-TrkB signaling pathway through transcription factor Gli3. Application of 7,8-Dihydroxyflavone, a potent small molecular TrkB agonist, fully restored BDNF-TrkB signaling activity and abnormal behavioral phenotypes presented by Ift172+/- mice. With luciferase and chip assays, we provided further evidence that Gli3 may physically interact with BDNF promoter I and regulate BDNF expression. ConclusionsOur data suggest that Ift172 per se drives neurotrophic effects and, when defective, could cause neurodevelopmental disorders reminiscent of autism-like disorders.

Post-Transcriptional Regulation of Rab7a in Lysosomal Positioning and Drug Resistance in Nutrient-Limited Cancer Cells.

Limited nutrient availability in the tumor microenvironment can cause the rewiring of signaling and metabolic networks to confer cancer cells with survival advantages. We show here that the limitation of glucose, glutamine and serum from the culture medium resulted in the survival of a population of cancer cells with high viability and capacity to form tumors in vivo. These cells also displayed a remarkable increase in the abundance and size of lysosomes. Moreover, lysosomes were located mainly in the perinuclear region in nutrient-limited cells; this translocation was mediated by a rapid post-transcriptional increase in the key endolysosomal trafficking protein Rab7a. The acidic lysosomes in nutrient-limited cells could trap weakly basic drugs such as doxorubicin, mediating resistance of the cells to the drug, which could be partially reversed with the lysosomal inhibitor bafilomycin A1. An in vivo chorioallantoic membrane (CAM) assay indicated a remarkable decrease in microtumor volume when nutrient-limited cells were treated with 5-Fluorouracil (5-FU) and bafilomycin A1 compared to cells treated with either agent alone. Overall, our data indicate the activation of complementary pathways with nutrient limitation that can enable cancer cells to survive, proliferate and acquire drug resistance.

Microfluidic measurement of the size and shape of lipid-anchored proteins

The surface of a cell is crowded with membrane proteins. The size, shape, density, and mobility of extracellular surface proteins mediate cell surface accessibility to external molecules, viral particles, and other cells. However, predicting these qualities is not always straightforward, even when protein structures are known. We previously developed an experimental method for measuring flow-driven lateral transport of neutravidin bound to biotinylated lipids in supported lipid bilayers. Here, we use this method to detect hydrodynamic force applied to a series of lipid-anchored proteins with increasing size. We find that the measured force reflects both protein size and shape, making it possible to distinguish these features of intact, folded proteins in their undisturbed orientation and proximity to the lipid membrane. In addition, our results demonstrate that individual proteins are transported large distances by flow forces on the order of femtoNewtons, similar in magnitude to the shear forces resulting from blood circulation or from the swimming motion of microorganisms. Similar protein transport across living cells by hydrodynamic force may contribute to biological flow sensing.

Expression patterns of HDAC6 in correlation to ARID1A status in different subtypes of endometriosis: A retrospective tissue microarray analysis

Endometriosis is a disease affecting approximately 10% of reproductive age women. Loss of the tumor suppressor gene AT-rich interactive domain-containing protein 1A (ARID1A) occurs in some endometriosis cases. Histone deacetylase 6 (HDAC-6) is an enzyme with implication in several diseases including different cancer types and immunological disorders, where it is involved in protein trafficking and degradation, cell shape, and migration. In ARID1A-deficient ovarian cancer increased HDAC-6 expression lead to apoptosis-inhibiting post-translational modification of p53. It is not known if HDAC-6 expression is also altered in ARID1A-deficient endometriosis. The aim of this study was to assess HDAC-6 expression in endometriotic lesions in correlation to ARID1A-status.Two tissue-microarrays with 168 endometriotic lesions, including ovarian (64/168, 38 %), peritoneal (66/168, 39 %) and deep-infiltrating (38/168, 23 %) subtypes, and 73 endometrium of women without endometriosis were assessed. Mean ARID1A immunoreactivity score (IRS) in endometriosis group was 10.83 (±2.36) and 10.78 (±1.94) in the epithelium and stroma, respectively, while the respective mean HDAC6 IRS were 9.16 (±2.76) and 5.94 (±2.88). The comparison of the HDAC6 expression between endometriosis subtypes showed higher expression in deep-infiltrating endometriosis, in both, epithelium (p = 0.032) and stroma (p = 0.007). In ARID1A negative cases, epithelial expression of HDAC6 was higher in endometriosis compared to women without endometriosis (p = 0.031), and this was also specifically observed in the subset of ovarian endometriosis (p = 0.037). There were no significant differences in the stromal expression of HDAC6.In conclusion, our results demonstrate a complex expression pattern of HDAC6 depending on ARID1A status in different endometriosis subtypes. Further studies on HDAC6 and ARID1A are important to elucidate mechanisms involved in malignant transformation of endometriosis.

The role of endoplasmic reticulum stress in the pathogenesis of oral diseases.

The endoplasmic reticulum (ER) is crucial for protein synthesis, transport, and folding, as well as calcium storage, lipid and steroid synthesis, and carbohydrate metabolism. Endoplasmic reticulum stress (ERS) occurs when misfolded or unfolded proteins accumulate in the ER lumen due to increased protein secretion or impaired folding. While the role of ERS in disease pathogenesis has been widely studied, most research has focused on extraoral diseases, leaving the role of ERS in intraoral diseases unclear. This review examines the role of ERS in oral diseases and oral fibrosis pathogenesis. A systematic search of literature through July 2023 was conducted in the MEDLINE database (via PubMed) using specific terms related to ERS, oral diseases, and fibrosis. The findings were summarized in both table and narrative form. Emerging evidence indicates that ERS significantly contributes to the pathogenesis of oral diseases and fibrosis. ERS-induced dysregulation of protein folding and the unfolded protein response can lead to cellular dysfunction and inflammation in oral tissues. Understanding the relationship between ERS and oral disease pathogenesis could offer new therapeutic targets for managing oral health and fibrosis-related complications.

Developmental and physiological impacts of pathogenic human huntingtin protein in the nervous system.

Huntington's Disease (HD) is a neurodegenerative disorder, part of the nine identified inherited polyglutamine (polyQ) diseases. Most commonly, HD pathophysiology manifests in middle-aged adults with symptoms including progressive loss of motor control, cognitive decline, and psychiatric disturbances. Associated with the pathophysiology of HD is the formation of insoluble fragments of the huntingtin protein (htt) that tend to aggregate in the nucleus and cytoplasm of neurons. To track both the intracellular progression of the aggregation phenotype as well as the physiological deficits associated with mutant htt, two constructs of human HTT were expressed with varying polyQ lengths, non-pathogenic-htt (Q15, NP-htt) and pathogenic-htt (Q138, P-htt), with an N-terminal RFP tag for in vivo visualization. P-htt aggregates accumulate in the ventral nerve cord cell bodies as early as 24 hours post hatching and significant aggregates form in the segmental nerve branches at 48 hours post hatching. Organelle trafficking up-and downstream of aggregates formed in motor neurons showed severe deficits in trafficking dynamics. To explore putative downstream deficits of htt aggregation, ultrastructural changes of presynaptic motor neurons and muscles were assessed, but no significant effects were observed. However, the force and kinetics of muscle contractions were severely affected in P-htt animals, reminiscent of human chorea. Reduced muscle force production translated to altered locomotory behavior. A novel HD aggregation model was established to track htt aggregation throughout adulthood in the wing, showing similar aggregation patterns with larvae. Expressing P-htt in the adult nervous system resulted in significantly reduced lifespan, which could be partially rescued by feeding flies the mTOR inhibitor rapamycin. These findings advance our understanding of htt aggregate progression as well the downstream physiological impacts on the nervous system and peripheral tissues.

Pharmacological chaperones restore proteostasis of epilepsy-associated GABAA receptor variants

Recent advances in genetic diagnosis identified variants in genes encoding GABAA receptors as causative for genetic epilepsy. Here, we selected eight disease-associated variants in the α1 subunit of GABAA receptors causing mild to severe clinical phenotypes and showed that they are loss of function, mainly by reducing the folding and surface trafficking of the α1 protein. Furthermore, we sought client protein-specific pharmacological chaperones to restore the function of pathogenic receptors. Applications of positive allosteric modulators, including Hispidulin and TP003, increase the functional surface expression of the α1 variants. Mechanism of action study demonstrated that they enhance the folding, assembly, and trafficking and reduce the degradation of GABAA variants without activating the unfolded protein response in HEK293T cells and human iPSC-derived neurons. Since these compounds cross the blood-brain barrier, such a pharmacological chaperoning strategy holds great promise to treat genetic epilepsy in a GABAA receptor-specific manner.

SIRT5 induces autophagy and alleviates myocardial infarction via desuccinylation of TOM1

Myocardial infarction (MI) is a prevalent form of ischemic heart disease, significantly contributing to heart disease-related deaths worldwide. This condition is primarily caused by myocardial ischemic-reperfusion injury (MIRI). Sirtuin 5 (SIRT5) is a desuccinylase known for its ability to reduce protein succinylation. Recent studies have highlighted the potential role of SIRT5 in various human diseases, including MIRI. This study aims to investigate the specific role of SIRT5 in modulating autophagy and cardiomyocyte death in a MIRI model, as well as to identify the downstream protein targets of SIRT5. Initially, we established a hypoxia/reoxygenation (H/R)-induced MIRI cell model to measure SIRT5 expression and assess its functions. Our results indicated that H/R induction led to a downregulation of SIRT5 expression, decreased autophagy, and increased cell death. Notably, overexpression of SIRT5 effectively promoted autophagy and inhibited cell death in the MIRI cell model. Mechanistically, SIRT5 was found to directly interact with the target of myb1 membrane trafficking protein (TOM1) at the K48 site, inducing its desuccinylation and stabilization. Further rescue assays revealed that TOM1 knockdown reversed the changes in autophagy and apoptosis caused by SIRT5 overexpression in the MIRI cell model. In vivo experiments demonstrated that SIRT5 alleviated myocardial injury in MI models. In conclusion, this study uncovers the role of SIRT5-mediated desuccinylation of TOM1 in regulating autophagy-related cell death in MIRI, providing new insights into potential therapeutic strategies for MI.

A Humanized Yeast Model for Studying TRAPP Complex Mutations; Proof-of-Concept Using Variants from an Individual with a TRAPPC1-Associated Neurodevelopmental Syndrome.

Variants in membrane trafficking proteins are known to cause rare disorders with severe symptoms. The highly conserved transport protein particle (TRAPP) complexes are key membrane trafficking regulators that are also involved in autophagy. Pathogenic genetic variants in specific TRAPP subunits are linked to neurological disorders, muscular dystrophies, and skeletal dysplasias. Characterizing these variants and their phenotypes is important for understanding the general and specialized roles of TRAPP subunits as well as for patient diagnosis. Patient-derived cells are not always available, which poses a limitation for the study of these diseases. Therefore, other systems, like the yeast Saccharomyces cerevisiae, can be used to dissect the mechanisms at the intracellular level underlying these disorders. The development of CRISPR/Cas9 technology in yeast has enabled a scar-less editing method that creates an efficient humanized yeast model. In this study, core yeast subunits were humanized by replacing them with their human orthologs, and TRAPPC1, TRAPPC2, TRAPPC2L, TRAPPC6A, and TRAPPC6B were found to successfully replace their yeast counterparts. This system was used for studying the first reported individual with an autosomal recessive disorder caused by biallelic TRAPPC1 variants, a girl with a severe neurodevelopmental disorder and myopathy. We show that the maternal variant (TRAPPC1 p.(Val121Alafs*3)) is non-functional while the paternal variant (TRAPPC1 p.(His22_Lys24del)) is conditional-lethal and affects secretion and non-selective autophagy in yeast. This parallels defects seen in fibroblasts derived from this individual which also showed membrane trafficking defects and altered Golgi morphology, all of which were rescued in the human system by wild-type TRAPPC1. This study suggests that humanized yeast can be an efficient means to study TRAPP subunit variants in the absence of human cells and can assign significance to variants of unknown significance (VUS). This study lays the foundation for characterizing further TRAPP variants through this system, rapidly contributing to disease diagnosis.

Nuclear proteasomes buffer cytoplasmic proteins during autophagy compromise

Autophagy is a conserved pathway where cytoplasmic contents are engulfed by autophagosomes, which then fuse with lysosomes enabling their degradation. Mutations in core autophagy genes cause neurological conditions, and autophagy defects are seen in neurodegenerative diseases such as Parkinson’s disease and Huntington’s disease. Thus, we have sought to understand the cellular pathway perturbations that autophagy-perturbed cells are vulnerable to by seeking negative genetic interactions such as synthetic lethality in autophagy-null human cells using available data from yeast screens. These revealed that loss of proteasome and nuclear pore complex components cause synergistic viability changes akin to synthetic fitness loss in autophagy-null cells. This can be attributed to the cytoplasm-to-nuclear transport of proteins during autophagy deficiency and subsequent degradation of these erstwhile cytoplasmic proteins by nuclear proteasomes. As both autophagy and cytoplasm-to-nuclear transport are defective in Huntington’s disease, such cells are more vulnerable to perturbations of proteostasis due to these synthetic interactions.

Hemifusomes and Interacting Proteolipid Nanodroplets: Formation of a Novel Cellular Organelle Complex.

Within cells, vesicle fusion, scission, and the formation of intraluminal vesicles are critical processes that facilitate traffic, degradation, and recycling of cellular components, and maintenance of cellular homeostasis. Despite significant advancements in elucidating the molecular mechanisms that drive these dynamic processes, the direct in situ visualization of membrane remodeling intermediates remains challenging. Here, through the application of cryo-electron tomography in mammalian cells, we have identified a previously undescribed vesicular organelle complex with unique membrane topology: heterotypic hemifused vesicles that share extended hemifusion diaphragms (HDs) with a 42 nm proteolipid nanodroplet (PND) at their rim. We have termed these organelle complexes "hemifusomes". The HDs of hemifusomes exhibit a range of sizes and curvatures, including the formation of lens-shaped compartments encapsulated within the membrane bilayer. The morphological diversity of the lens-shaped vesicle aligns with a step-wise process of their intraluminal budding, ultimately leading to their scission and the generation of intraluminal vesicles. We propose that hemifusomes function as versatile platforms for protein and lipid sorting and as central hubs for the biogenesis of intraluminal vesicles and the formation of multivesicular bodies.

Mutant induced neurons and humanized mice enable identification of Niemann-Pick type C1 proteostatic therapies.

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step toward the development of disease-modifying therapies.

NEMF-mediated CAT-tailing defines distinct branches of translocation-associated quality control.

Ribosome stalling during co-translational translocation at the endoplasmic reticulum (ER) causes translocon clogging and impairs ER protein biogenesis. Mammalian cells resolve translocon clogging vial a poorly characterized translocation-associated quality control (TAQC) process. Here, we combine genome-wide CRISPR screen with live cell imaging to dissect the molecular linchpin of TAQC. We show that substrates translated from mRNAs bearing a ribosome stalling poly(A) sequence are degraded by lysosomes and the proteasome, while substrates encoded by non-stop mRNAs are degraded by an unconventional ER-associated degradation (ERAD) mechanism involving ER-to-Golgi trafficking and KDEL-dependent substrate retrieval. The triaging diversity appears to result from the heterogeneity of NEMF-mediated CATylation, because a systematic characterization of representative CAT-tail mimetics establishes an AT-rich tail as a "degron" for ERAD, whereas an AG-rich tail can direct a secretory protein to the lysosome. Our study reveals an unexpected protein sorting function for CAT-tailing that safeguards ER protein biogenesis.

Ultrafast and Chemoselective Biotinylation of Living Cell Surfaces for Time-Resolved Surfaceome Analysis.

Cell surface proteins participate in many important biological processes, such as cell-to-cell interaction, signal transduction, cell adhesion, and protein transportation. In-depth study of the cell surface protein group is of great significance. Nevertheless, detection and analysis of the surfaceome remain a significant challenge due to their low abundance and hydrophobicity. Herein, we reported an ultrafast and chemoselective labeling method using our newly developed trifunctional probe, the OPA-S-S-alkyne, which labeled cell surface lysine residues, and then established a novel cell surfaceome profiling approach. According to our experimental results, the OPA-S-S-alkyne probe can react extremely fast with living cells, labeling cells in only 1 min, while traditional NHS (labeling cell surface lysine with N-hydroxysuccinimide ester probe) and CSC (labeling cell surface glycan with hydrazide biotin probe) methods normally take longer time of more than 30 min and 1 h, respectively. Taking advantage of this ultrafast property of the method, we highlight the utility of this method by exploring the temporal dynamic changes of surfaceome upon EGF stimulation in living Hela cells and reported "early" and "late" EGF-regulated cell surface proteins, which are difficult to be distinguished by the current cell surface profiling approaches.

Clinical Spectrum, Diagnosis, and Management of TANGO2 Deficiency Disorder: A Comprehensive Review

TANGO2 deficiency disorder is an autosomal recessive disease caused by biallelic pathogenic variants in the TANGO2 gene. First described in 2016, the disorder is characterized by acute metabolic crises, neurological dysfunctions, developmental delays, and intellectual disabilities. Laboratory findings indicate a mitochondrial fatty acid oxidation defect, with metabolic crises often triggered by stressors such as illness or fasting. The TANGO2 protein, implicated in lipid metabolism, membrane trafficking, and cellular homeostasis, is critical for maintaining lipid droplets, regulating phospholipid levels, and facilitating protein and lipid transport between cellular compartments. Symptoms of TANGO2 deficiency are diverse, including psychomotor delays, intellectual impairments, seizures, ataxia, and episodic neurological symptoms known as TANGO2 spells. Diagnosis involves genetic testing, with several pathogenic variants identified. During metabolic crises, patients may experience severe cardiac and metabolic disturbances. No curative treatment exists; management includes B-vitamin supplementation, antiepileptic drugs, and interventions for spasticity, dystonia, thyroid dysfunction, and arrhythmias. TANGO2 deficiency disorder can co-occur with DiGeorge syndrome, complicating diagnosis due to overlapping symptoms. Improved awareness and diagnostic strategies are crucial for effective management of this underrecognized condition.

Endoplasmic reticulum stress in diseases.

Theendoplasmic reticulum (ER) is a key organelle in eukaryotic cells, responsible for a wide range of vital functions, including the modification, folding, and trafficking of proteins, as well as the biosynthesis of lipids and the maintenance of intracellular calcium homeostasis. A variety of factors can disrupt the function of the ER, leading to the aggregation of unfolded and misfolded proteins within its confines and the induction of ER stress. A conserved cascade of signaling events known as the unfolded protein response (UPR) has evolved to relieve the burden within the ER and restore ER homeostasis. However, these processes can culminate in cell death while ER stress is sustained over an extended period and at elevated levels. This review summarizes the potential role of ER stress and the UPR in determining cell fate and function in various diseases, including cardiovascular diseases, neurodegenerative diseases, metabolic diseases, autoimmune diseases, fibrotic diseases, viral infections, and cancer. It also puts forward that the manipulation of this intricate signaling pathway may represent a novel target for drug discovery and innovative therapeutic strategies in the context of human diseases.

Mechanism of proton transport in SLC25 family proteins

Mechanism of proton transport in SLC25 family proteins

Acute Neuroinflammation Alters the Transport of a Model Therapeutic Protein from the Brain into Lymph and Blood.

The drainage of fluid and solutes along lymphatic pathways from the brain has been found to be impaired in mouse models of multiple sclerosis, Alzheimer's disease, and Parkinson's disease where neuroinflammation is present. We recently demonstrated that 3H-albumin, a model therapeutic protein (∼65 kDa), undergoes preferential lymphatic transport from the brain using a cervical lymph cannulation model in healthy rats. We thus hypothesized that neuroinflammation would impede the lymphatic transport of 3H-albumin from the brain. Our aim was to quantify the impact of acute neuroinflammation on drainage of the model therapeutic protein (3H-albumin) from the rat brain into blood and deep cervical lymph. To establish the required neuroinflammation model, male Sprague-Dawley rats were administered an intraperitoneal (IP) dose of 0.5-2 mg/kg lipopolysaccharide (LPS, Escherichia coli) or a saline control. After 12 or 24 h, brain samples were collected and analyzed for concentrations of interferon gamma (IFN-γ) using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The impact of neuroinflammation on the drainage of 3H-albumin from the brain was determined via IP administration of 2 mg/kg LPS or saline followed by cannulation of the carotid artery for blood collection 24 h later with/without cannulation or ligation at the efferent deep cervical lymph trunk. Rats were then administered 3H-albumin via direct injection into the brain striatum or via intravenous (IV) injection (lymph-intact group only). Blood ± lymph samples were collected for up to 8 h following dosing. At the end of the study, brain and lymph node samples were harvested for biodistribution analysis, with samples analyzed for radioactivity levels via scintillation counting. Brain concentrations of the pro-inflammatory cytokine IFN-γ were only significantly elevated 24 h after IP administration of 2 mg/kg LPS compared to saline control. Therefore, this induction regimen was utilized for subsequent studies. The plasma concentrations of 3H-albumin over time were elevated in LPS-induced rats compared to saline-injected rats in the lymph-intact and lymph-ligated groups but not in the lymph-cannulated group. In the deep cervical lymph-cannulated animals, the lymph transport of 3H-albumin was not increased and appeared to be slower in the LPS-administered rats. Acute LPS-induced neuroinflammation therefore led to an enhanced overall transport of 3H-albumin from the brain into the systemic circulation. This appeared to be primarily due to increased transport of 3H-albumin from the brain directly into the blood circulation as 3H-albumin transport from the brain via the lymphatics was not increased in the LPS-induced neuroinflammation model. Such changes in the clearance of therapeutic proteins from the brain in the setting of neuroinflammation may impact the therapeutic efficacy and safety.

Proteome asymmetry in mouse and human embryos before fate specification.

Pre-patterning of the embryo, driven by spatially localized factors, is a common feature across several non-mammalian species 1-4 . However, mammals display regulative development and thus it was thought that blastomeres of the embryo do not show such pre-patterning, contributing randomly to the three lineages of the blastocyst: the epiblast, primitive endoderm and trophectoderm that will generate the new organism, the yolk sac and placenta respectively 4-6 . Unexpectedly, early blastomeres of mouse and human embryos have been reported to have distinct developmental fates, potential and heterogeneous abundance of certain transcripts 7-12 . Nevertheless, the extent of the earliest intra-embryo differences remains unclear and controversial. Here, by utilizing multiplexed and label-free single-cell proteomics by mass-spectrometry 13 , we show that 2-cell mouse and human embryos contain an alpha and a beta blastomere as defined by differential abundance of hundreds of proteins exhibiting strong functional enrichment for protein synthesis, transport, and degradation. Such asymmetrically distributed proteins include Gps1 and Nedd8, depletion or overexpression of which in one blastomere of the 2-cell embryo impacts lineage segregation. These protein asymmetries increase at 4-cell stage. Intriguingly, halved mouse zygotes display asymmetric protein abundance that resembles alpha and beta blastomeres, suggesting differential proteome localization already within zygotes. We find that beta blastomeres give rise to a blastocyst with a higher proportion of epiblast cells than alpha blastomeres and that vegetal blastomeres, which are known to have a reduced developmental potential, are more likely to be alpha. Human 2-cell blastomeres also partition into two clusters sharing strong concordance with clusters found in mouse, in terms of differentially abundant proteins and functional enrichment. To our knowledge, this is the first demonstration of intra-zygotic and inter-blastomere proteomic asymmetry in mammals that has a role in lineage segregation.