Exploring PROTAC Cooperativity with Coarse-Grained Alchemical Methods.

Proteolysis targeting chimera (PROTAC) is a novel drug modality that facilitates the degradation of a target protein by inducing proximity with an E3 ligase. In this work, we present a new computational framework to model the cooperativity between PROTAC-E3 binding and PROTAC-target binding principally through protein-protein interactions (PPIs) induced by the PROTAC. Due to the scarcity and low resolution of experimental measurements, the physical and chemical drivers of these non-native PPIs remain to be elucidated. We develop a coarse-grained (CG) approach to model interactions in the target-PROTAC-E3 complexes, which enables converged thermodynamic estimations using alchemical free energy calculation methods despite an unconventional scale of perturbations. With minimal parametrization, we successfully capture fundamental principles of cooperativity, including the optimality of intermediate PROTAC linker lengths that originates from configurational entropy. We qualitatively characterize the dependency of cooperativity on PROTAC linker lengths and protein charges and shapes. Minimal inclusion of sequence- and conformation-specific features in our current force field, however, limits quantitative modeling to reproduce experimental measurements, but further development of the CG model may allow for efficient computational screening to optimize PROTAC cooperativity.

Human induced mesenchymal stem cells display increased sensitivity to matrix stiffness.

The clinical translation of mesenchymal stem cells (MSCs) is limited by population heterogeneity and inconsistent responses to engineered signals. Specifically, the extent in which MSCs respond to mechanical cues varies significantly across MSC lines. Although induced pluripotent stem cells (iPSCs) have recently emerged as a novel cell source for creating highly homogeneous MSC (iMSC) lines, cellular mechanosensing of iMSCs on engineered materials with defined mechanics is not well understood. Here, we tested the mechanosensing properties of three human iMSC lines derived from iPSCs generated using a fully automated platform. Stiffness-driven changes in morphology were comparable between MSCs and iMSCs cultured atop hydrogels of different stiffness. However, contrary to tissue derived MSCs, no significant changes in iMSC morphology were observed between iMSC lines atop different stiffness hydrogels, demonstrating a consistent response to mechanical signals. Further, stiffness-driven changes in mechanosensitive biomarkers were more pronounced in iMSCs than MSCs, which shows that iMSCs are more adaptive and responsive to mechanical cues than MSCs. This study reports that iMSCs are a promising stem cell source for basic and applied research due to their homogeneity and high sensitivity to engineered mechanical signals.

Proteomic Alterations and Novel Markers of Neurotoxic Reactive Astrocytes in Human Induced Pluripotent Stem Cell Models.

Astrocytes respond to injury, infection, and inflammation in the central nervous system by acquiring reactive states in which they may become dysfunctional and contribute to disease pathology. A sub-state of reactive astrocytes induced by proinflammatory factors TNF, IL-1α, and C1q ("TIC") has been implicated in many neurodegenerative diseases as a source of neurotoxicity. Here, we used an established human induced pluripotent stem cell (hiPSC) model to investigate the surface marker profile and proteome of TIC-induced reactive astrocytes. We propose VCAM1, BST2, ICOSL, HLA-E, PD-L1, and PDPN as putative, novel markers of this reactive sub-state. We found that several of these markers colocalize with GFAP+ cells in post-mortem samples from people with Alzheimer's disease. Moreover, our whole-cells proteomic analysis of TIC-induced reactive astrocytes identified proteins and related pathways primarily linked to potential engagement with peripheral immune cells. Taken together, our findings will serve as new tools to purify reactive astrocyte subtypes and to further explore their involvement in immune responses associated with injury and disease.

Acceleration of opioid-related EMS runs in the spring of 2020: The National Emergency Medical Services Information System data for 2018-2020.

BACKGROUND: State- and county-level reports suggest that the COVID-19 pandemic exacerbated the opioid crisis. We examined US national trends of nonfatal opioid overdose in 2020 in comparison to pre-COVID years 2018-2019. METHODS: We used National Emergency Medical Services Information System (NEMSIS) data to conduct a temporal analysis from 2018 to 2020. Opioid-related EMS run was defined using five scenarios of naloxone administration. To determine annual patterns and slope inflection points, we used the Prophet model of the time series analysis. Linear slopes and their 95% confidence intervals (CIs) were calculated for pre-stay-at-home (pre-SaH) and SaH periods in 2020 and compared to the slopes during the same time in 2018-2019. Three cut-points for SaH start were considered: March 19, 24, and 29. RESULTS: We identified 91,065, 144,802, and 242,904 opioid-related EMS runs in 2018-2020, respectively. In 2020, opioid-related runs increased in January-June, with a pronounced acceleration in March, which coincides with the stay-at-home (SaH) orders. In both 2018 and 2019, opioid-related runs increased in January-August without the spring acceleration. In 2020, weekly increases (95% CI) during SaH for all examined cut-points were significantly greater than in pre-SaH: 18.09 (16.03-20.16) vs. 6.44 (3.42-9.47) for March 19, 17.77 (15.57-19.98) vs. 4.85 (2.07-7.64) for March 24, 18.03 (15.68-20.39) vs. 4.97(2.4-7.54) for March 29. No significant difference was found between these periods in 2018-2019. CONCLUSIONS: The acceleration of opioid-related EMS runs during the SaH period of 2020 suggests that EMS data may serve as an early warning system for local health jurisdictions to deploy harm reduction/prevention resources.

Coordination of -1 programmed ribosomal frameshifting by transcript and nascent chain features revealed by deep mutational scanning.

Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the alphavirus structural polyprotein, the efficiency of -1PRF is coordinated by a 'slippery' sequence in the transcript, an adjacent RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop reduce the efficiency of -1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of -1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that the coupling between cotranslational folding and -1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how -1PRF is coordinated by features within both the transcript and nascent chain.

Force transduction creates long-ranged coupling in ribosomes stalled by arrest peptides.

Force-sensitive arrest peptides regulate protein biosynthesis by stalling the ribosome as they are translated. Synthesis can be resumed when the nascent arrest peptide experiences a pulling force of sufficient magnitude to break the stall. Efficient stalling is dependent on the specific identity of a large number of amino acids, including amino acids that are tens of angstroms away from the peptidyl transferase center (PTC). The mechanism of force-induced restart and the role of these essential amino acids far from the PTC is currently unknown. We use hundreds of independent molecular dynamics trajectories spanning over 120 µs in combination with kinetic analysis to characterize multiple barriers along the force-induced restart pathway for the arrest peptide SecM. We find that the essential amino acids far from the PTC play a major role in controlling the transduction of applied force. In successive states along the stall-breaking pathway, the applied force propagates up the nascent chain until it reaches the C-terminus of SecM and the PTC, inducing conformational changes that allow for restart of translation. A similar mechanism of force propagation through multiple states is observed in the VemP stall-breaking pathway, but secondary structure in VemP allows for heterogeneity in the order of transitions through intermediate states. Results from both arrest peptides explain how residues that are tens of angstroms away from the catalytic center of the ribosome impact stalling efficiency by mediating the response to an applied force and shielding the amino acids responsible for maintaining the stalled state of the PTC.

Residue-by-residue analysis of cotranslational membrane protein integration in vivo.

We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the 'sliding' model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon.

Isolation of Human CD49f+ Astrocytes and In Vitro iPSC-Based Neurotoxicity Assays.

Given the critical roles of astrocytes in neuroinflammation and neurological diseases, models for studying human astrocyte biology are in increasing demand. Here, we present a protocol to isolate human astrocytes from induced pluripotent stem cell (iPSC)-based cultures, neural organoids, and primary tissue, using the surface marker CD49f. Moreover, we provide protocols for in vitro co-cultures of human iPSC-derived neurons and astrocytes, as well as for neurotoxicity assays that expose neurons to conditioned media from reactive astrocytes. For complete details on the use and execution of this protocol, please refer to Barbar et al. (2020).

Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer.

Trimeric porins in the outer membrane (OM) of Gram-negative bacteria are the conduits by which nutrients and antibiotics diffuse passively into cells. The narrow gateways that porins form in the OM are also exploited by bacteriocins to translocate into cells by a poorly understood process. Here, using single-channel electrical recording in planar lipid bilayers in conjunction with protein engineering, we explicate the mechanism by which the intrinsically unstructured N-terminal translocation domain (IUTD) of the endonuclease bacteriocin ColE9 is imported passively across the Escherichia coli OM through OmpF. We show that the import is dominated by weak interactions of OmpF pores with binding epitopes within the IUTD that are orientationally biased and result in the threading of over 60 amino acids through 2 subunits of OmpF. Single-molecule kinetic analysis demonstrates that the IUTD enters from the extracellular side of OmpF and translocates to the periplasm where the polypeptide chain does an about turn in order to enter a neighboring subunit, only for some of these molecules to pop out of this second subunit before finally re-entering to form a stable complex. These intimately linked transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer.

CD49f Is a Novel Marker of Functional and Reactive Human iPSC-Derived Astrocytes.

New methods for investigating human astrocytes are urgently needed, given their critical role in the central nervous system. Here we show that CD49f is a novel marker for human astrocytes, expressed in fetal and adult brains from healthy and diseased individuals. CD49f can be used to purify fetal astrocytes and human induced pluripotent stem cell (hiPSC)-derived astrocytes. We provide single-cell and bulk transcriptome analyses of CD49f+ hiPSC-astrocytes and demonstrate that they perform key astrocytic functions in vitro, including trophic support of neurons, glutamate uptake, and phagocytosis. Notably, CD49f+ hiPSC-astrocytes respond to inflammatory stimuli, acquiring an A1-like reactive state, in which they display impaired phagocytosis and glutamate uptake and fail to support neuronal maturation. Most importantly, we show that conditioned medium from human reactive A1-like astrocytes is toxic to human and rodent neurons. CD49f+ hiPSC-astrocytes are thus a valuable resource for investigating human astrocyte function and dysfunction in health and disease.

Dynamics of Co-translational Membrane Protein Integration and Translocation via the Sec Translocon.

An important aspect of cellular function is the correct targeting and delivery of newly synthesized proteins. Central to this task is the machinery of the Sec translocon, a transmembrane channel that is involved in both the translocation of nascent proteins across cell membranes and the integration of proteins into the membrane. Considerable experimental and computational effort has focused on the Sec translocon and its role in nascent protein biosynthesis, including the correct folding and expression of integral membrane proteins. However, the use of molecular simulation methods to explore Sec-facilitated protein biosynthesis is hindered by the large system sizes and long (i.e., minute) time scales involved. In this work, we describe the development and application of a coarse-grained simulation approach that addresses these challenges and allows for direct comparison with both in vivo and in vitro experiments. The method reproduces a wide range of experimental observations, providing new insights into the underlying molecular mechanisms, predictions for new experiments, and a strategy for the rational enhancement of membrane protein expression levels.

Cotranslational folding stimulates programmed ribosomal frameshifting in the alphavirus structural polyprotein.

Viruses maximize their genetic coding capacity through a variety of biochemical mechanisms, including programmed ribosomal frameshifting (PRF), which facilitates the production of multiple proteins from a single mRNA transcript. PRF is typically stimulated by structural elements within the mRNA that generate mechanical tension between the transcript and ribosome. However, in this work, we show that the forces generated by the cotranslational folding of the nascent polypeptide chain can also enhance PRF. Using an array of biochemical, cellular, and computational techniques, we first demonstrate that the Sindbis virus structural polyprotein forms two competing topological isomers during its biosynthesis at the ribosome-translocon complex. We then show that the formation of one of these topological isomers is linked to PRF. Coarse-grained molecular dynamics simulations reveal that the translocon-mediated membrane integration of a transmembrane domain upstream from the ribosomal slip site generates a force on the nascent polypeptide chain that scales with observed frameshifting. Together, our results indicate that cotranslational folding of this viral protein generates a tension that stimulates PRF. To our knowledge, this constitutes the first example in which the conformational state of the nascent polypeptide chain has been linked to PRF. These findings raise the possibility that, in addition to RNA-mediated translational recoding, a variety of cotranslational folding or binding events may also stimulate PRF.

GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells.

BACKGROUND: Human mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines. METHODS: In the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium). RESULTS: The results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum. CONCLUSIONS: The findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine.

Derivation and characterization of the NIH registry human stem cell line NYSCF101 under defined feeder-free conditions.

The human embryonic stem cell line NYSCFe002-A was derived from a day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe002-A line expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro. The line presents normal karyotype and is mycoplasma free. This line is registered as NYSCF101 on the NIH Registry.

Derivation and characterization of the NIH registry human stem cell line NYSCF100 line under defined feeder-free conditions.

The human embryonic stem cell line NYSCFe001-A was derived from a day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe001-A line, registered as NYSCF100 on the NIH registry, presents normal karyotype, is mycoplasma free, expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro.

Derivation and characterization of the NYSCFe003-A human embryonic stem cell line.

The human embryonic stem cell line NYSCFe003-A was derived from a day 5 to day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe003-A line expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro. The line presents normal karyotype and is mycoplasma free.

CRISPR/Cas9-Correctable mutation-related molecular and physiological phenotypes in iPSC-derived Alzheimer's PSEN2 N141I neurons.

Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD, and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs, using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected, cell lines harboring the PSEN2 N141I mutation displayed an increase in the Aß42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit, restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased Aß42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in Aß42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology.

Directed Differentiation of Human Pluripotent Stem Cells to Microglia.

Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the CNS, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines.

Deficiency in prohormone convertase PC1 impairs prohormone processing in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is caused by a loss of paternally expressed genes in an imprinted region of chromosome 15q. Among the canonical PWS phenotypes are hyperphagic obesity, central hypogonadism, and low growth hormone (GH). Rare microdeletions in PWS patients define a 91-kb minimum critical deletion region encompassing 3 genes, including the noncoding RNA gene SNORD116. Here, we found that protein and transcript levels of nescient helix loop helix 2 (NHLH2) and the prohormone convertase PC1 (encoded by PCSK1) were reduced in PWS patient induced pluripotent stem cell-derived (iPSC-derived) neurons. Moreover, Nhlh2 and Pcsk1 expression were reduced in hypothalami of fasted Snord116 paternal knockout (Snord116p-/m+) mice. Hypothalamic Agrp and Npy remained elevated following refeeding in association with relative hyperphagia in Snord116p-/m+ mice. Nhlh2-deficient mice display growth deficiencies as adolescents and hypogonadism, hyperphagia, and obesity as adults. Nhlh2 has also been shown to promote Pcsk1 expression. Humans and mice deficient in PC1 display hyperphagic obesity, hypogonadism, decreased GH, and hypoinsulinemic diabetes due to impaired prohormone processing. Here, we found that Snord116p-/m+ mice displayed in vivo functional defects in prohormone processing of proinsulin, pro-GH-releasing hormone, and proghrelin in association with reductions in islet, hypothalamic, and stomach PC1 content. Our findings suggest that the major neuroendocrine features of PWS are due to PC1 deficiency.

Genetic Drift Can Compromise Mitochondrial Replacement by Nuclear Transfer in Human Oocytes.

Replacement of mitochondria through nuclear transfer between oocytes of two different women has emerged recently as a strategy for preventing inheritance of mtDNA diseases. Although experiments in human oocytes have shown effective replacement, the consequences of small amounts of mtDNA carryover have not been studied sufficiently. Using human mitochondrial replacement stem cell lines, we show that, even though the low levels of heteroplasmy introduced into human oocytes by mitochondrial carryover during nuclear transfer often vanish, they can sometimes instead result in mtDNA genotypic drift and reversion to the original genotype. Comparison of cells with identical oocyte-derived nuclear DNA but different mtDNA shows that either mtDNA genotype is compatible with the nucleus and that drift is independent of mitochondrial function. Thus, although functional replacement of the mitochondrial genome is possible, even low levels of heteroplasmy can affect the stability of the mtDNA genotype and compromise the efficacy of mitochondrial replacement.