Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer

The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodelling1 and is altered in over 20% of cancers2,3. Here we developed a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supra-physiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumour growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers.

Tumor relevant protein functional interactions identified using bipartite graph analyses

An increased surge of -omics data for the diseases such as cancer allows for deriving insights into the affiliated protein interactions. We used bipartite network principles to build protein functional associations of the differentially regulated genes in 18 cancer types. This approach allowed us to combine expression data to functional associations in many cancers simultaneously. Further, graph centrality measures suggested the importance of upregulated genes such as BIRC5, UBE2C, BUB1B, KIF20A and PTH1R in cancer. Pathway analysis of the high centrality network nodes suggested the importance of the upregulation of cell cycle and replication associated proteins in cancer. Some of the downregulated high centrality proteins include actins, myosins and ATPase subunits. Among the transcription factors, mini-chromosome maintenance proteins (MCMs) and E2F family proteins appeared prominently in regulating many differentially regulated genes. The projected unipartite networks of the up and downregulated genes were comprised of 37,411 and 41,756 interactions, respectively. The conclusions obtained by collating these interactions revealed pan-cancer as well as subtype specific protein complexes and clusters. Therefore, we demonstrate that incorporating expression data from multiple cancers into bipartite graphs validates existing cancer associated mechanisms as well as directs to novel interactions and pathways.

Genomic identification, annotation, and comparative analysis of Vacuolar-type ATP synthase subunits in Diaphorina citri

Detailed annotation and comparative analysis were performed on the Asian citrus psyllid (ACP), Diaphorina citri, vacuolar-type ATP synthase (V-ATPase) to support the biological understanding and development of novel therapeutics to manage psyllid vectors. D. citri is a hemipteran insect that vectors the causative agent, the bacteria Candidatus Liberibacter asiaticus (CLas), of the citrus greening disease, Huanglongbing (HLB). Millions of citrus trees have been destroyed by citrus greening and every grove in Florida has been directly impacted. In eukaryotic organisms, V-ATPase is an abundant heterodimeric enzyme that serves the cell with essential compartment acidification through the active processes that transport protons across the membrane. Manual curation was completed on 15 putative V-ATPase genes in the D. citri genome. Comparative genomic analysis reveals that the D. citri V-ATPase subunits share domains and motifs with other insects, including the V-ATPase-A superfamily domain from the V-ATPase catalytic subunit A, which shares a 92% identity with Acyrthosiphon pisum. Phylogenetic analysis separates D. citri V-ATPase subunits into expected clades with orthologous sequences. Based on the results of annotation and comparative genomic analysis, RNAi therapies targeting D. citri V-ATPase genes, which have been successfully utilized in related hemipterans, are being pursued. Annotation of the D. citri genome is a critical step towards the development of directed-pest management that will lead to the reduced spread of the pathogens causing HLB throughout the citrus industry.

SWI/SNF chromatin remodeler complex within the reward pathway is required for behavioral adaptations to stress

Stress exposure is a cardinal risk factor for most psychiatric diseases. Preclinical and clinical studies point to changes in gene expression involving epigenetic modifications within mesocorticolimbic brain circuits. Brahma (BRM) and Brahma-Related-Gene-1 (BRG1) are ATPase subunits of the SWI/SNF complexes involved in chromatin remodeling, a process essential to enduring plastic changes in gene expression. Here, we show that repeated social defeat induces changes in BRG1 nuclear distribution. The inactivation of the Brg1/Smarca4 gene within dopamine-innervated regions or the constitutive inactivation of the Brm/Smarca2 gene leads to resilience to repeated social defeat and decreases the behavioral responses to cocaine without impacting midbrain dopamine neurons activity. Within striatal medium spiny neurons Brg1 gene inactivation reduces the expression of stress- and cocaine-induced immediate early genes, increases levels of heterochromatin and at a global scale decreases chromatin accessibility. Altogether these data demonstrate the pivotal function of SWI/SNF complexes in behavioral and transcriptional adaptations to salient environmental challenges.

New insights into the human 26S proteasome function and regulation

SummaryThe 26S proteasome is a protease complex essential for proteostasis and strict regulation of diverse critical physiological processes, the mechanisms of which are still not fully described. The human 26S proteasome purification was optimized without exogenous nucleotides, to preserve the endogenous nucleotide occupancy and conformation of its AAA-ATPase subunits. This unveiled important effects on the proteasome function and structure resulting from exposure to Ca2+ or Mg2+, with important physiological implications. This sample, with an added model degron designed to mimic the minimum canonical ubiquitin signal for proteasomal recognition, was analysed by high-resolution cryo-EM. Two proteasome conformations were resolved, with only one capable of degron binding. The structural data show that this occurs without major conformation rearrangements and allows to infer into the allosteric communication between ubiquitin degron binding and the peptidase activities. These results revise existing concepts on the 26S proteasome function and regulation, opening important opportunities for further research.

miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact proteotoxicity and muscle function during aging

Muscle function relies on the precise architecture of dynamic contractile elements, which must be fine-tuned to maintain motility throughout life. Muscle is also plastic, and remodeled in response to stress, growth, neural and metabolic inputs. The conserved muscle-enriched microRNA, miR-1, regulates distinct aspects of muscle development, but whether it plays a role during aging is unknown. Here we investigated Caenorhabditis elegans miR-1 in muscle function in response to proteostatic stress. mir-1 deletion improved mid-life muscle motility, pharyngeal pumping, and organismal longevity upon polyQ35 proteotoxic challenge. We identified multiple vacuolar ATPase subunits as subject to miR-1 control, and the regulatory subunit vha-13/ATP6V1A as a direct target downregulated via its 3′UTR to mediate miR-1 physiology. miR-1 further regulates nuclear localization of lysosomal biogenesis factor HLH-30/TFEB and lysosomal acidification. Our studies reveal that miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact muscle function and health during aging.

An Empirical Energy Landscape Reveals Mechanism of Proteasome in Polypeptide Translocation

The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits. How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is not known. In this study, we developed a novel approach for delineating the nucleotide-dependent free-energy landscape (FEL) of the proteasome's heterohexameric ATPase complex based on complementary structural and kinetic measurements. We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties. The FEL model predictions were widely consistent with experimental observations in this and previous studies and suggested novel features of the mechanism of proteasomal ATPase. We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding state. ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex.

Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease

The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions. Graphic Abstract

Streptococcus salivarius Isolates of Varying Acid Tolerance Exhibit F1F0-ATPase Conservation

Genes encoding the subunits of the membrane-bound F₁F₀-ATPase (responsible for exporting protons from the cytoplasm and contributing to acid tolerance) were sequenced for 24 non-mutans streptococci isolated from carious lesions. Isolates, mostly <i>Streptococcus salivarius</i>, displayed a continuum of acid tolerance thresholds ranging from pH 4.55 to 3.39, but amino acid alignments of F₁F₀-ATPase subunits revealed few non-synonymous substitutions and these were unrelated to acid tolerance. Thus, the F₁F₀-ATPase is highly-conserved among <i>S. salivarius</i> isolates despite varying acid tolerance thresholds, supporting the contention that acid tolerance is determined by the level of gene/protein expression rather than variation in molecular structure.

Monitoring iron-sulfur cluster occupancy across the E. coli proteome using chemoproteomics

Iron-sulfur (Fe-S) clusters are ubiquitous metallocofactors found across diverse protein families, where they perform myriad functions including redox chemistry, radical generation, and gene regulation. Monitoring Fe-S cluster occupancy in protein targets directly within native biological systems has been challenging. Commonly utilized spectroscopic methods to detect Fe-S clusters require purification of proteins prior to analysis. Global iron incorporation into the proteome can be monitored using radiolabeled iron, but limitations include the low resolution afforded by gel-based autoradiography. Here, we report the development of a mass spectrometry-based strategy to assess Fe-S cluster binding in a native proteome. This chemoproteomic strategy relies on monitoring changes in the reactivity of Fe-S cluster cysteine ligands upon disruption of Fe-S cluster incorporation. Application to E. coli cells cultured under iron-depleted conditions enabled monitoring of disruptions to Fe-S cluster incorporation broadly across the E. coli Fe-S proteome. Evaluation of E. coli deletion strains of three scaffold proteins within the Isc Fe-S biogenesis pathway enabled the identification of Fe-S clients that are reliant on each individual scaffold protein for proper cluster installation. Lastly, cysteine-reactivity changes characteristic of Fe-S ligands were used to identify previously unannotated Fe-S proteins, including the tRNA hydroxylase, TrhP, and a member of a family of membrane transporter ATPase subunits, DppD. In summary, the chemoproteomic strategy described herein provides a powerful tool to report on Fe-S cluster incorporation directly within a native proteome, to interrogate the role of scaffold and accessory proteins within Fe-S biogenesis pathways, and to identify previously uncharacterized Fe-S proteins.