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Conformational spread and dynamics in allostery of NMDA receptors (2020)

Durham, Ryan J. ; Paudyal, Nabina ; Carrillo, Elisa ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2020; 117(7): 3839-3847. Published 2020 Feb 03. doi: 10.1073/pnas.1910950117.

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Publication Date: 2020-02-03

Description: Allostery can be manifested as a combination of repression and activation in multidomain proteins allowing for fine tuning of regulatory mechanisms. Here we have used single molecule fluorescence resonance energy transfer (smFRET) and molecular dynamics simulations to study the mechanism of allostery underlying negative cooperativity between the two agonists glutamate and glycine in the NMDA receptor. These data show that binding of one agonist leads to conformational flexibility and an increase in conformational spread at the second agonist site. Mutational and cross-linking studies show that the dimer–dimer interface at the agonist-binding domain mediates the allostery underlying the negative cooperativity. smFRET on the transmembrane segments shows that they are tightly coupled in the unliganded and single agonist-bound form and only upon binding both agonists the transmembrane domain explores looser packing which would facilitate activation.

Print ISSN: 0027-8424

Electronic ISSN: 1091-6490

Topics: Biology , Medicine , Natural Sciences in General

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ZNF263 is a transcriptional regulator of heparin and heparan sulfate biosynthesis (2020)

Weiss, Ryan J. ; Spahn, Philipp N. ; Toledo, Alejandro Gómez ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2020; 117(17): 9311-9317. Published 2020 Apr 10. doi: 10.1073/pnas.1920880117.

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Publication Date: 2020-04-10

Description: Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non–heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.

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Electronic ISSN: 1091-6490

Topics: Biology , Medicine , Natural Sciences in General

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Translation of the intrinsically disordered protein α-synuclein is inhibited by a small molecule targeting its structured mRNA (2020)

Zhang, Peiyuan ; Park, Hye-Jin ; Zhang, Jie ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2020; 117(3): 1457-1467. Published 2020 Jan 03. doi: 10.1073/pnas.1905057117.

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Publication Date: 2020-01-03

Description: Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson’s disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5′ untranslated region (5′ UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5′ UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound’s selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.

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Genomic analysis of inherited hearing loss in the Palestinian population (2020)

Abu Rayyan, Amal ; Kamal, Lara ; Casadei, Silvia ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2020; 117(33): 20070-20076. Published 2020 Aug 03. doi: 10.1073/pnas.2009628117.

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Publication Date: 2020-08-03

Description: The genetic characterization of a common phenotype for an entire population reveals both the causes of that phenotype for that place and the power of family-based, population-wide genomic analysis for gene and mutation discovery. We characterized the genetics of hearing loss throughout the Palestinian population, enrolling 2,198 participants from 491 families from all parts of the West Bank and Gaza. In Palestinian families with no prior history of hearing loss, we estimate that 56% of hearing loss is genetic and 44% is not genetic. For the great majority (87%) of families with inherited hearing loss, panel-based genomic DNA sequencing, followed by segregation analysis of large kindreds and transcriptional analysis of participant RNA, enabled identification of the causal genes and mutations, including at distant noncoding sites. Genetic heterogeneity of hearing loss was striking with respect to both genes and alleles: The 337 solved families harbored 143 different mutations in 48 different genes. For one in four solved families, a transcription-altering mutation was the responsible allele. Many of these mutations were cryptic, either exonic alterations of splice enhancers or silencers or deeply intronic events. Experimentally calibrated in silico analysis of transcriptional effects yielded inferences of high confidence for effects on splicing even of mutations in genes not expressed in accessible tissue. Most (58%) of all hearing loss in the population was attributable to consanguinity. Given the ongoing decline in consanguineous marriage, inherited hearing loss will likely be much rarer in the next generation.

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Electronic ISSN: 1091-6490

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Direct lysosome-based autophagy of lipid droplets in hepatocytes (2020)

Schulze, Ryan J. ; Krueger, Eugene W. ; Weller, Shaun G. ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2020; 117(51): 32443-32452. Published 2020 Dec 07. doi: 10.1073/pnas.2011442117.

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Publication Date: 2020-12-07

Description: Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) in the lysosome-directed process of autophagy. An organelle-selective form of this process (macrolipophagy) results in the engulfment of LDs within double-membrane delimited structures (autophagosomes) before lysosomal fusion. Whether this is an exclusive autophagic mechanism used by hepatocytes to catabolize LDs is unclear. It is also unknown whether lysosomes alone might be sufficient to mediate LD turnover in the absence of an autophagosomal intermediate. We performed live-cell microscopy of hepatocytes to monitor the dynamic interactions between lysosomes and LDs in real-time. We additionally used a fluorescent variant of the LD-specific protein (PLIN2) that exhibits altered fluorescence in response to LD interactions with the lysosome. We find that mammalian lysosomes and LDs undergo interactions during which proteins and lipids can be transferred from LDs directly into lysosomes. Electron microscopy (EM) of primary hepatocytes or hepatocyte-derived cell lines supports the existence of these interactions. It reveals a dramatic process whereby the lipid contents of the LD can be “extruded” directly into the lysosomal lumen under nutrient-limited conditions. Significantly, these interactions are not affected by perturbations to crucial components of the canonical macroautophagy machinery and can occur in the absence of double-membrane lipoautophagosomes. These findings implicate the existence of an autophagic mechanism used by mammalian cells for the direct transfer of LD components into the lysosome for breakdown. This process further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying lipid homeostasis in the liver.

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Electronic ISSN: 1091-6490

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Absolute quantification of tumor antigens using embedded MHC-I isotopologue calibrants (2021)

Stopfer, Lauren E. ; Gajadhar, Aaron S. ; Patel, Bhavin ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2021; 118(37): e2111173118. Published 2021 Sep 08. doi: 10.1073/pnas.2111173118.

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Publication Date: 2021-09-08

Description: Absolute quantification measurements (copies per cell) of peptide major histocompatibility complex (pMHC) antigens are necessary to inform targeted immunotherapy drug design; however, existing methods for absolute quantification have critical limitations. Here, we present a platform termed SureQuant-IsoMHC, utilizing a series of pMHC isotopologues and internal standard-triggered targeted mass spectrometry to generate an embedded multipoint calibration curve to determine endogenous pMHC concentrations for a panel of 18 tumor antigens. We apply SureQuant-IsoMHC to measure changes in expression of our target panel in a melanoma cell line treated with a MEK inhibitor and translate this approach to estimate antigen concentrations in melanoma tumor biopsies.

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Stress increases in exopher-mediated neuronal extrusion require lipid biosynthesis, FGF, and EGF RAS/MAPK signaling (2021)

Cooper, Jason F. ; Guasp, Ryan J. ; Arnold, Meghan Lee ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2021; 118(36): e2101410118. Published 2021 Sep 02. doi: 10.1073/pnas.2101410118.

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Publication Date: 2021-09-02

Description: In human neurodegenerative diseases, neurons can transfer toxic protein aggregates to surrounding cells, promoting pathology via poorly understood mechanisms. In Caenorhabditis elegans, proteostressed neurons can expel neurotoxic proteins in large, membrane-bound vesicles called exophers. We investigated how specific stresses impact neuronal trash expulsion to show that neuronal exopher production can be markedly elevated by oxidative and osmotic stress. Unexpectedly, we also found that fasting dramatically increases exophergenesis. Mechanistic dissection focused on identifying nonautonomous factors that sense and activate the fasting-induced exopher response revealed that DAF16/FOXO-dependent and -independent processes are engaged. Fasting-induced exopher elevation requires the intestinal peptide transporter PEPT-1, lipid synthesis transcription factors Mediator complex MDT-15 and SBP-1/SREPB1, and fatty acid synthase FASN-1, implicating remotely initiated lipid signaling in neuronal trash elimination. A conserved fibroblast growth factor (FGF)/RAS/MAPK signaling pathway that acts downstream of, or in parallel to, lipid signaling also promotes fasting-induced neuronal exopher elevation. A germline-based epidermal growth factor (EGF) signal that acts through neurons is also required for exopher production. Our data define a nonautonomous network that links food availability changes to remote, and extreme, neuronal homeostasis responses relevant to aggregate transfer biology.

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Electronic ISSN: 1091-6490

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Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses (2021)

Malonis, Ryan J. ; Earnest, James T. ; Kim, Arthur S. ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2021; 118(37): e2100104118. Published 2021 Sep 10. doi: 10.1073/pnas.2100104118.

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Publication Date: 2021-09-10

Description: Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline–revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

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