Lars Plate | Scripps Research Institute (original) (raw)

Papers by Lars Plate

Cellular proteostasis is maintained by stress-responsive signaling pathways such as the heat shoc... more Cellular proteostasis is maintained by stress-responsive signaling pathways such as the heat shock response (HSR), the oxidative stress response (OSR), and the unfolded protein response (UPR). Activation of these pathways results in the transcriptional upregulation of select subsets of stress-responsive genes that restore proteostasis and adapt cellular physiology to promote recovery following various types of acute insult. The capacity for these pathways to regulate cellular proteostasis makes them attractive therapeutic targets to correct proteostasis defects associated with diverse diseases. High-throughput screening (HTS) using cell-based reporter assays is highly effective for identifying putative activators of stress-responsive signaling pathways. However, the development of these compounds is hampered by the lack of medium-throughput assays to define compound potency and selectivity for a given pathway. Here, we describe a targeted RNA sequencing (RNAseq) assay that allows co...

Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggrega... more Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼ 70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation.

Analytical Chemistry, 2013

Although the objective of any 'omic science is broad measurement of its constituents, such covera... more Although the objective of any 'omic science is broad measurement of its constituents, such coverage has been challenging in metabolomics because the metabolome is comprised of a chemically diverse set of small molecules with variable physical properties. While extensive studies have been performed to identify metabolite isolation and separation methods, these strategies introduce bias toward lipophilic or water-soluble metabolites depending on whether reversedphase (RP) or hydrophilic interaction liquid chromatography (HILIC) is used, respectively. Here we extend our consideration of metabolome isolation and separation procedures to integrate RPLC/MS and HILIC/MS profiling. An aminopropyl-based HILIC/MS method was optimized on the basis of mobile-phase additives and pH, followed by evaluation of reproducibility. When applied to the untargeted study of perturbed bacterial metabolomes, the HILIC method enabled the accurate assessment of key, dysregulated metabolites in central carbon pathways (e.g., amino acids, organic acids, phosphorylated sugars, energy currency metabolites), which could not be retained by RPLC. To demonstrate the value of the integrative approach, bacterial cells, human plasma, and cancer cells were analyzed by combined RPLC/HILIC separation coupled to ESI positive/negative MS detection. The combined approach resulted in the observation of metabolites associated with lipid and central carbon metabolism from a single biological extract, using 80% organic solvent (ACN:MeOH:H 2 O 2:2:1). It enabled the detection of more than 30,000 features from each sample type, with the highest number of uniquely detected features by RPLC in ESI positive mode and by HILIC in ESI negative mode. Therefore, we conclude that when time and sample are limited, the maximum amount of biological information related to lipid and central carbon metabolism can be acquired by combining RPLC ESI positive and HILIC ESI negative mode analysis. M etabolites are a direct readout of biochemical activity and, as the end products of genomic, transcriptomic, and proteomic expression, provide a representation of cellular state. 1−3 In contrast to genes, transcripts, and proteins, which are composed of a defined set of building blocks, metabolites contain a high level of chemical diversity. 3−5 Given the wide range of physical properties characteristic of the metabolome, measuring the complete set of metabolites present in complex biological matrices represents a major challenge for untargeted, mass spectrometry-based metabolomics. 1,3,4,6 Examining a broad range of chemically diverse metabolites, however, is important when studying biological systems. In diabetes, for example, in addition to perturbations in carbohydrate metabolism there are alterations in the lipid profile that also contribute to disease pathogenesis. To study the chemically complex metabolome, liquid chromatography−mass spectrometry (LC/MS) equipped with an electrospray ionization (ESI) source has become a key analytical tool given the number of analytes that can be simultaneously measured. 4,9−11 The number of metabolite features detected by LC/MS analyses, defined by unique m/z and LC retention times, has previously been used as a relevant

Trends in Biochemical Sciences, 2013

Heme-nitric oxide/oxygen binding (H-NOX) domains function as sensors for the gaseous signaling ag... more Heme-nitric oxide/oxygen binding (H-NOX) domains function as sensors for the gaseous signaling agent nitric oxide (NO) in eukaryotes and bacteria. Mammalian NO signaling is well characterized and involves the H-NOX domain of soluble guanylate cyclase. In bacteria, H-NOX proteins interact with bacterial signaling proteins in two-component signaling systems or in cyclic-di-GMP metabolism. Characterization of several downstream signaling processes has shown that bacterial H-NOX proteins share a common role in controlling important bacterial communal behaviors in response to NO. The H-NOX pathways regulate motility, biofilm formation, quorum sensing, and symbiosis. Here, we review the latest structural and mechanistic studies that have elucidated how H-NOX domains selectively bind NO and transduce ligand binding into conformational changes that modulate activity of signaling partners. Furthermore, we summarize the recent advances in understanding the physiological function and biochemical details of the H-NOX signaling pathways.

Proceedings of the National Academy of Sciences, 2013

Author contributions: L.P. and M.A.M. designed research; L.P. performed research; L.P. and M.A.M.... more Author contributions: L.P. and M.A.M. designed research; L.P. performed research; L.P. and M.A.M. analyzed data; and L.P. and M.A.M. wrote the paper.

Molecular Cell, 2012

Nitric oxide (NO) signaling in vertebrates is well characterized and involves the Heme-Nitric oxi... more Nitric oxide (NO) signaling in vertebrates is well characterized and involves the Heme-Nitric oxide/OXygen binding (H-NOX) domain of soluble guanylate cyclase as a selective NO sensor. In contrast, little is known about the biological role or signaling output of bacterial H-NOX proteins. Here, we describe a molecular pathway for H-NOX signaling in Shewanella oneidensis. NO stimulates biofilm formation by controlling the levels of the bacterial secondary messenger cyclic diguanosine monophosphate (c-di-GMP). Phosphotransfer profiling was used to map the connectivity of a multi-component signaling network that involves integration from two histidine kinases and branching to three response regulators. A feed-forward loop between response regulators with phosphodiesterase domains and phosphorylation-mediated activation intricately regulated c-di-GMP levels. Phenotypic characterization established a link between NO signaling and biofilm formation. Cellular adhesion may provide a protection mechanism for bacteria against reactive and damaging NO. These results are broadly applicable to H-NOX-mediated NO signaling in bacteria.

Biochemistry, 2008

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides, prov... more Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides, providing the monomeric precursors required for DNA replication and repair. E. coli RNR is a 1:1 complex of two homodimeric subunits: α2 and β2. The interactions between α2 and β2 are thought to be largely associated with the C-terminal 20 amino acids (residues 356-375) of β2. To study subunit interactions, a single reactive cysteine has been introduced into each of fifteen positions along the C-terminal tail of β2. Each cysteine has been modified with the photo cross-linker benzophenone (BP) and the environmentally sensitive fluorophore, dimethylaminonaphthalene (DAN). Each construct has been purified to homogeneity and characterized by SDS PAGE and ESI-MS. Each BP-β2 has been incubated with 1 equivalent of α2, photolyzed, and the results analyzed quantitatively by SDS-PAGE. Each DAN-β2 was incubated with 50-fold excess of α2 and the emission maximum and intensity measured. A comparison of the results from the two sets of probes reveals that sites with most extensive cross-linking are also associated with the greatest changes in fluorescence. Titration of four different DAN-β2 variants (351, 356, 365 and 367) with α2 gave a K d of ∼0.4 μM for subunit interaction. Disruption of the interaction of α2DAN-β2 complex is accompanied by a decrease in fluorescence intensity and can serve as a high throughput screen for inhibitors of subunit interactions.

Bioconjugate Chemistry, 2008

We proposed the use of opioid drug bound covalently to hyaluronan (HA) via ester linkages as a me... more We proposed the use of opioid drug bound covalently to hyaluronan (HA) via ester linkages as a method to prolong drug delivery and to possibly increase the quality of perioperative pain management. The in vitro release profile of morphine conjugated to HA (1.3 million MW) was studied. The influence of parameters such as conjugation site and steric protection of the labile ester bonds was investigated in phosphate buffered saline (PBS) medium. HA--codeine and HA--naloxone conjugates were used as structural controls. Codeine and morphine conjugated via the allylic hydroxyl group had a release half-life of 14.0 days in PBS. Naloxone conjugated via the phenolic hydroxyl group showed a half-life of 0.3 days, and all drugs admixed in HA showed half-lives of 0.1 days. Methyl, ethyl, or n-propyl introduced in vicinal position to the ester bond prolonged release of naloxone with half-lives of 0.5, 4.0, and 4.0 days in PBS, respectively. Incorporation of a methyl group prolonged codeine release with a half-life of 55.0 days in PBS. Drugs were released chemically unaltered from the conjugates as confirmed by LC-MS/MS. Further, morphine was conjugated to divinylsulfone cross-linked HA (Hylan B) particles and the release profiles in rat plasma were studied in vitro and in vivo. Release in rat plasma was faster than in PBS with a half-life of 2.5 days, but the release was similar (ca. 12 days) when a cocktail of protease inhibitors was added to the plasma. Sustained release of morphine was observed in a rat surgical model over 30 h. Morphine was released chemically unaltered from the conjugate and morphine intermediates were not detected in significant amounts as confirmed by LC-MS/MS. These results suggest that the morphine release profile from the HA conjugates depends on the alkyl groups vicinal to the ester and the nature of the leaving group. In rat plasma, hydrolysis seems to be controlled by esterase activity.

Analytical Biochemistry, 2010

Histidine-aspartic acid phosphotransfer pathways are central components of prokaryotic signal tra... more Histidine-aspartic acid phosphotransfer pathways are central components of prokaryotic signal transduction pathways, and are also found in many eukaryotes. Tools to study histidine kinases, however, are currently quite limited. In this paper, we present a new tool to study histidine-aspartic acid phosphotransfer pathways. We show that many histidine kinases will accept ATPγS as a substrate to form a stable thiophosphohistidine, even when they do not form stable phosphohistidines using the natural substrate ATP. An antibody that has previously been used to detect thiophosphorylated serine, threonine and tyrosine residues is shown to recognize thiophosphohistidine and thiophophoaspartic acid residues. Histidine kinase autothiophosphorylation is regulated by other protein sensor domains in the same way as autophosphorylation, and thiophosphate is transferred to downstream aspartic acid containing response regulators.

Angewandte Chemie, 2009

Investigations into the mechanisms by which heme proteins control ligand affinity and reactivity ... more Investigations into the mechanisms by which heme proteins control ligand affinity and reactivity have been studied for decades, with the globins serving as model systems for histidyl-ligated proteins. The recent discovery of a novel family of heme proteins, Heme Nitric oxide/OXygen (H-NOX) binding domains, has provided an opportunity to investigate if factors previously found to control ligand affinity and reactivity in the globins can be generalized between protein folds or if there are additional determinants.

Angewandte Chemie-international Edition, 2009