The
            <i>Mycobacterium tuberculosis</i>
            Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway

Becker, Samuel H.; Jastrab, Jordan B.; Dhabaria, Avantika; Chaton, Catherine T.; Rush, Jeffrey S.; Korotkov, Konstantin V.; Ueberheide, Beatrix; Darwin, K. Heran

doi:10.1073/pnas.1819468116

Cited by 26 publications

(30 citation statements)

References 72 publications

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“…150 µg of each protein lysate were proteolytically digested and subjected to quantitative mass spectrometry on an Orbitrap Fusion Lumos mass spectrometer using isobaric tandem mass tags (TMT) similar to previous studies 70, 71 .…”

Section: Methodsmentioning

confidence: 99%

“…A 500 μg aliquot of the pooled sample was fractionated using basic pH reverse-phase HPLC (as described) 71 . Briefly, the sample was loaded onto a 4.6 mm × 250 mm Xbridge C18 column (Waters, 3.5 μm bead size) using an Agilent 1260 Infinity Bio-inert HPLC and separated over a 90 min linear gradient from 10 to 50% solvent B at a flow rate of 0.5 ml/min (Buffer A = 10 mM ammonium formate, pH 10.0; Buffer B = 90% ACN, 10 mM ammonium formate, pH 10.0).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Melanoma-secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Kleffman

Levinson

Wong

et al. 2019

Preprint

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26Brain metastasis is a significant cause of morbidity and mortality in multiple cancer 27 types and represents an unmet clinical need. The mechanisms that mediate metastatic 28 cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the 29 highest rate of brain metastasis among common cancer types, is an ideal model to 30 study how cancer cells adapt to the brain parenchyma. We performed unbiased 31 proteomics analysis of melanoma short-term cultures, a novel model for the study of 32 brain metastasis. Intriguingly, we found that proteins implicated in neurodegenerative 33 pathologies are differentially expressed in melanoma cells explanted from brain 34 metastases compared to those derived from extracranial metastases. This raised the 35 exciting hypothesis that molecular pathways implicated in neurodegenerative disorders 36 are critical for metastatic adaptation to the brain. 37 38Here, we show that melanoma cells require amyloid beta (Ab), a polypeptide heavily 39 implicated in Alzheimer's disease, for growth and survival in the brain parenchyma. 40Melanoma cells produce and secrete Ab, which activates surrounding astrocytes to a 41 pro-metastatic, anti-inflammatory phenotype. Furthermore, we show that 42 pharmacological inhibition of Ab decreases brain metastatic burden. 43 44Our results reveal a mechanistic connection between brain metastasis and Alzheimer's 45 disease -two previously unrelated pathologies, establish Ab as a promising therapeutic 46 target for brain metastasis, and demonstrate suppression of neuroinflammation as a 47 critical feature of metastatic adaptation to the brain parenchyma. 48 49 3 Main 50 51Brain metastasis is the most common form of adult intracranial malignancy 1 and results 52 in severe morbidity and mortality. 40-75% of Stage IV melanoma patients develop brain 53 metastasis 2,3 , reflecting melanoma's striking ability to colonize the brain. Brain 54 metastases are less responsive than extracranial metastases to current cancer 55 therapies 4-6 , and the majority of patients succumb to disease in less than one year 7 . 56 Furthermore, patients with brain metastasis are often excluded from clinical trials and 57 urgently need new clinical options. In recent years, research has started to elucidate the 58 molecular mechanisms contributing to the multi-step process of brain metastasis. Most 59 findings have focused on cancer extravasation across the blood-brain barrier (BBB), 60 which cannot be leveraged therapeutically given that the vast majority of brain 61 metastasis patients will present with extravasated cancer cells at the time of cancer 62 diagnosis. The main bottleneck in the brain metastatic process has been shown to be 63 the successful expansion of a single cell in the brain parenchyma to form a macro-64 metastasis 8 . Recent studies have begun to demonstrate the role of the brain 65 microenvironment in this process. In particular, reactive astrocytes have been shown to 66 interact with cancer cells in the brain 9,10 and exhibit both pro-and anti-...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Melanoma-secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Kleffman

Levinson

Wong

et al. 2019

Preprint

Self Cite

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“…We previously reported the abundance of HrcA-regulated gene products between a wild type (WT) M. tuberculosis strain and an mpa mutant, which cannot degrade pupylated proteins, and found that GroES, GroEL1, and GroEL2 levels are significantly lower in an mpa strain; however, Ruc abundance is unaffected by disruption of mpa under these conditions. This result suggested that proteasomal degradation of HrcA is not sufficient for Ruc production under the conditions tested (23); however, this experiment was performed with cultures incubated at 37°C in minimal medium. We therefore compared Ruc abundance from the same strains incubated at 45°C and observed a striking defect in Ruc production in the mpa mutant ( Fig.…”

Section: Resultsmentioning

confidence: 94%

“…We became interested in Ruc after identifying the transcriptional repressor HrcA as a putative target of the M. tuberculosis Pup-proteasome system (23). In M. tuberculosis , HrcA directly represses four genes; three of them encode highly conserved chaperone proteins of the Hsp60/Hsp10 family, while the fourth gene, ruc , encodes a protein of unknown function (24).…”

Section: Resultsmentioning

confidence: 99%

Mycobacterium tuberculosisRv0991c is a redox-regulated molecular chaperone

Becker

Ulrich

Dhabaria

et al. 2020

Preprint

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21The bacterial pathogen Mycobacterium (M.) tuberculosis is the leading cause of death by 22 an infectious disease among humans. Here, we describe a previously uncharacterized 23 M. tuberculosis protein, Rv0991c, as a molecular chaperone that is activated by oxidation. 24Rv0991c has homologues in most bacterial lineages and appears to function analogously 25 to the well-characterized Escherichia coli redox-regulated chaperone Hsp33, despite a 26 dissimilar protein sequence. Rv0991c is transcriptionally co-regulated with hsp60 and 27 hsp70 chaperone genes in M. tuberculosis, suggesting that Rv0991c functions with these 28 chaperones in maintaining protein quality control. Supporting this hypothesis, we found 29 that, like oxidized Hsp33, oxidized Rv0991c prevents the aggregation of a model unfolded 30 protein in vitro, and promotes its refolding by the M. tuberculosis Hsp70 chaperone 31 system. Furthermore, Rv0991c interacts with DnaK and associates with many other M. 32 tuberculosis proteins. Importantly, we found Rv0991c is required for the full virulence of 33 M. tuberculosis in mice. We therefore propose that Rv0991c, which we named "Ruc" 34 (redox-regulated protein with unstructured C-terminus), represents a founding member of 35 a new chaperone family that protects M. tuberculosis and other species from 36 proteotoxicity during oxidative stress. 37 38 IMPORTANCE 39 M. tuberculosis infections are responsible for more than one million human deaths per 40year. Developing effective strategies to combat this disease requires a greater 41 understanding of M. tuberculosis biology. As in all cells, protein quality control is essential 42 for the viability of M. tuberculosis, which likely faces proteome stress within a host. Here, 43we identify an M. tuberculosis protein, Ruc, that gains chaperone activity upon oxidation. 44Ruc represents a previously unrecognized family of redox-regulated chaperones found 45 throughout the bacterial super-kingdom. In addition to elucidating the activity of this 46 chaperone, we found that Ruc was required for full M. tuberculosis virulence in mice. This 47 6 discovery that the uncharacterized M. tuberculosis gene Rv0991c encodes a chaperone, 97 which we named Ruc (redox-regulated chaperone with unstructured C-terminus). Ruc 98 belongs to a previously unacknowledged, but evolutionarily widespread family of bacterial 99 proteins with little predicted structural similarity to other chaperones. Upon oxidation, Ruc 100 is capable of inhibiting protein aggregation and delivering unfolded proteins to DnaKJE 101 for refolding. Ruc was also found to interact with DnaK in M. tuberculosis and, while 102 dispensable for in vitro growth, was required for full M. tuberculosis virulence in mice. 103These observations ultimately suggest that Ruc is important for the ability of M. 104 tuberculosis, and potentially many other bacterial species, to withstand oxidation-105 associated proteotoxicity. 106 107 RESULTS 108

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“…Other studies in yeast and metazoan species showed mRNA-protein correlations at the gene-to-gene basis of above 0.5 (Alli Shaik et al, 2014;Beyer et al, 2004;Brockmann et al, 2007;Ghaemmaghami et al, 2003). A possible explanation for the apparent increased post-transcriptional regulation in Mtb may be the presence of the proteasome system responsible for protein degradation and homeostasis that is rarely found in other bacteria (Becker et al, 2019). Due to the substantial post-transcriptional regulation in Mtb, RNA data in isolation can lead to incomplete or misleading mechanistic understanding.…”

Section: Post-transcriptional Regulation Is Prevalent In Mtb Clinicalmentioning

confidence: 97%

Network analysis identifies regulators of lineage-specific phenotypes inMycobacterium tuberculosis

Banaei‐Esfahani

Trauner

Borrell

et al. 2020

Preprint

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Highlights Proteomic and transcriptomic characterization of fully sequenced diverse L1 and L2 clinical isolates of Mtb. Post-transcriptional control mechanisms for regulatory and virulence genes are mitigated in Mtb L2. By applying a genome-scale transcriptional framework, DosR, Rv1985c, Lsr2 and Rv0691c are identified as master transcription factors responsible for differential target gene expression in L2 strains compared to L1. L1 and L2 DosR proteins respond differently to nitric oxide stress, thus determining a relevant phenotype. SummaryThe Mycobacterium tuberculosis (Mtb) complex comprises seven phylogenetically distinct human-adapted lineages exhibiting different geographical distribution and degrees of pathogenicity. Among these, Lineage 1 (L1) has been associated with low virulence whereas Lineage 2 (L2) has been linked to hyper-virulence, enhanced transmission and drug resistance.Here, we conducted multi-layer comparative analyses using whole genome sequencing data combined with quantitative transcriptomic and proteomic profiling of a set of L1 and L2 clinical strains, each grown under two different conditions in vitro. Our data revealed different degrees of correlation between transcript and protein abundances across clinical strains and functional gene categories, indicating variable levels of post-transcriptional regulation in the tested lineages. Contrasting genomic and gene expression data showed that the magnitude of the transcriptional and translational changes was proportional to the phylogenetic distance between strains, with one out of three single nucleotide polymorphisms leading to a transcriptional and/or translational change on average. We devised a new genome-scale transcriptional regulatory model and identified several master transcription factors, strongly linked to the sigma factor network, whose targets were differentially regulated between the two lineages. These differences resulted in a higher basal expression of DosR proteins and a stronger response to nitric oxide (NO) exposure in L2 compared to L1. These patterns are most likely responsible for the shorter NO-induced growth arrest in L2 observed. Given the limited genetic variation between strains, it appears that phenotypic differences in Mtb are substantially driven by differences in the regulation of biochemical networks through master transcriptional regulators.

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The Mycobacterium tuberculosis Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway

Cited by 26 publications

References 72 publications

Melanoma-secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Melanoma-secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

Mycobacterium tuberculosisRv0991c is a redox-regulated molecular chaperone

Network analysis identifies regulators of lineage-specific phenotypes inMycobacterium tuberculosis

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