Protein-protein Interaction Domains Research Articles

Mutations and Protein Interaction Landscape Reveal Key Cellular Events Perturbed in Upper Motor Neurons with HSP and PLS.

Hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS) are rare motor neuron diseases, which affect mostly the upper motor neurons (UMNs) in patients. The UMNs display early vulnerability and progressive degeneration, while other cortical neurons mostly remain functional. Identification of numerous mutations either directly linked or associated with HSP and PLS begins to reveal the genetic component of UMN diseases. Since each of these mutations are identified on genes that code for a protein, and because cellular functions mostly depend on protein-protein interactions, we hypothesized that the mutations detected in patients and the alterations in protein interaction domains would hold the key to unravel the underlying causes of their vulnerability. In an effort to bring a mechanistic insight, we utilized computational analyses to identify interaction partners of proteins and developed the protein-protein interaction landscape with respect to HSP and PLS. Protein-protein interaction domains, upstream regulators and canonical pathways begin to highlight key cellular events. Here we report that proteins involved in maintaining lipid homeostasis and cytoarchitectural dynamics and their interactions are of great importance for UMN health and stability. Their perturbation may result in neuronal vulnerability, and thus maintaining their balance could offer therapeutic interventions.

Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein

SummaryThe kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Taken together, these results provide structural insights into the unconventional kinetoplastid kinetochore protein KKT4.

Functional domain studies uncover novel roles for the ZTL Kelch repeat domain in clock function.

The small LOV/F-box/Kelch family of E3 ubiquitin ligases plays an essential role in the regulation of plant circadian clocks and flowering time by sensing dusk. The family consists of three members, ZEITLUPE (ZTL), LOV KELCH PROTEIN 2 (LKP2), and FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1 (FKF1), which share a unique protein domain architecture allowing them to act as photoreceptors that transduce light signals via altering stability of target proteins. Despite intensive study of this protein family we still lack important knowledge about the biochemical and functional roles of the protein domains that comprise these unique photoreceptors. Here, we perform comparative analyses of transgenic lines constitutively expressing the photoreceptor LOV domain or the Kelch repeat protein-protein interaction domains of ZTL, FKF1, and LKP2. Expression of each domain alone is sufficient to disrupt circadian rhythms and flowering time, but each domain differs in the magnitude of effect. Immunoprecipitation followed by mass spectrometry with the ZTL Kelch repeat domain identified a suite of potential interacting partners. Furthermore, the ZTL Kelch repeat domain can interact with the ZTL homologs, LKP2 and FKF1, and the LOV domain of ZTL itself. This suggests a hypothesis that the Kelch repeat domain of ZTL may mediate inter- and intra-molecular interactions of the three LOV/F-box/Kelch proteins and provides added insight into the composition of the protein complexes and an additional role for the Kelch repeat domain.

MicroProteins: Expanding functions and novel modes of regulation

MicroProteins: Expanding functions and novel modes of regulation

Targeted degradation of the enhancer lysine acetyltransferases CBP and p300

The enhancer factors CREB-binding protein (CBP) and p300 (also known as KAT3A and KAT3B) maintain gene expression programs through lysine acetylation of chromatin and transcriptional regulators and by scaffolding functions mediated by several protein-protein interaction domains. Small molecule inhibitors that target some of these domains have been developed; however, they cannot completely ablate p300/CBP function in cells. Here we describe a chemical degrader of p300/CBP, dCBP-1. Leveraging structures of ligand-bound p300/CBP domains, we use in silico modeling of ternary complex formation with the E3 ubiquitin ligase cereblon to enable degrader design. dCBP-1 is exceptionally potent at killing multiple myeloma cells and can abolish the enhancer that drives MYC oncogene expression. As an efficient degrader of this unique class of acetyltransferases, dCBP-1 is a useful tool alongside domain inhibitors for dissecting the mechanism by which these factors coordinate enhancer activity in normal and diseased cells.

Linarin Protects the Kidney against Ischemia/Reperfusion Injury via the Inhibition of Bioactive ETS2/IL-12.

Ischemia/reperfusion injury (IRI), a participant in acute kidney injury (AKI), can occur as a series of pathological processes such as inflammation. Linarin (LIN) has been widely used for different diseases. To confirm the anti-inflammatory value and relevant mechanism of LIN during IRI, in vivo and vitro models were established. LIN or dissolvent was given, and histologic analysis, quantitative (q)RT-PCR, serum creatinine and blood urea nitrogen testing were used to evaluate kidney injury. Microarray analysis, protein-protein interaction (PPI) analysis and molecular docking were used to identify the target protein of LIN, and small interfering RNA (siRNA) transfection was applied to explore the crucial role of identified protein. First, we found that LIN inhibited kidney injury in an in vivo IRI model and decreased the expression of interleukin-12 (IL-12) p40 in vivo and in vitro IRI models. To explore the mechanism of LIN, we collected raw data from a public microarray database and identified E26 oncogene homolog 2 (ETS2) as a crucial protein of LIN according to microarray analysis and PPI. Meanwhile, qRT-PCR indicated that IL-12 p40 showed no significant difference between ETS2 knock down group and LIN treated ETS2 knock down group after hypoxia reoxygenation treatment. In addition, according to molecular docking the contact area is highly conserved and located on a PPI domain of ETS2 which indicates that LIN may alter the interaction with synergistic proteins in the regulation of IL-12 p40 expression. Our study demonstrated the anti-inflammatory effect of LIN during IRI-AKI, broadening the medicinal value of LIN and the therapeutic options for IRI-AKI.

Global Analysis of Cereal microProteins Suggests Diverse Roles in Crop Development and Environmental Adaptation.

MicroProteins are a class of small single-domain proteins that post-translationally regulate larger multidomain proteins from which they evolved or which they relate to. They disrupt the normal function of their targets by forming microProtein-target heterodimers through compatible protein-protein interaction (PPI) domains. Recent studies confirm the significance of microProteins in the fine-tuning of plant developmental processes such as shoot apical meristem maintenance and flowering time regulation. While there are a number of well-characterized microProteins in Arabidopsis thaliana, studies from more complex plant genomes are still missing. We have previously developed miPFinder, a software for identifying microProteins from annotated genomes. Here we present an improved version where we have updated the algorithm to increase its accuracy and speed, and used it to analyze five cereal crop genomes – wheat, rice, barley, maize and sorghum. We found 20,064 potential microProteins from a total of 258,029 proteins in these five organisms, of which approximately 2000 are high-confidence, i.e., likely to function as actual microProteins. Gene ontology analysis of these 2000 microProtein candidates revealed their roles in stress, light and growth responses, hormone signaling and transcriptional regulation. Using a recently developed rice gene co-expression database, we analyzed 347 potential rice microProteins that are also conserved in other cereal crops and found over 50 of these rice microProteins to be co-regulated with their identified interaction partners. Overall, our study reveals a rich source of biotechnologically interesting small proteins that regulate fundamental plant processes such a growth and stress response that could be utilized in crop bioengineering.

WD40-Repeat Proteins in Ciliopathies and Congenital Disorders of Endocrine System.

WD40-repeat (WDR)-containing proteins constitute an evolutionarily conserved large protein family with a broad range of biological functions. In human proteome, WDR makes up one of the most abundant protein-protein interaction domains. Members of the WDR protein family play important roles in nearly all major cellular signalling pathways. Mutations of WDR proteins have been associated with various human pathologies including neurological disorders, cancer, obesity, ciliopathies and endocrine disorders. This review provides an updated overview of the biological functions of WDR proteins and their mutations found in congenital disorders. We also highlight the significant role of WDR proteins in ciliopathies and endocrine disorders. The new insights may help develop therapeutic approaches targeting WDR motifs.

CDH1 binds MAD2L2 in a Rev1-like pattern

MAD2L2 (i.e. Rev7) is a central regulatory protein important in several processes, such as translesion synthesis (TLS), DNA damage response and mitosis. In TLS, MAD2L2 binds Rev3 to form Pol zeta (ζ) and promotes formation of the Pol ζ- REV1 complex allowing extension beyond distorted DNA structures. MAD2L2 is also part of the heterotetrameric shieldin complex that regulates DNA repair at sites of damage, where similarly to TLS, it bridges the interaction between SHLD2 and SHLD3. Lastly, during mitosis, MAD2L2 prevents premature activation of the anaphase promoting complex/cyclosome (APC/C), by sequestering its activator, CDH1. MAD2L2 exits in a ‘closed’ active conformation binding Rev3 and Rev1, or SHLD2 and SHLD3, and an ‘open’ inactive conformation, with no binding partners. Moreover, Pol ζ- REV1 forms a homodimer using a protein-protein interaction (PPI) domain comprised of a central αC helix, promoting Rev3-MAD2L2 interaction and C-terminus β-sheets, enabling Rev1-MAD2L2 interaction. While the role of MAD2L2 in TLS is well established, molecular details regarding the CDH1-MAD2L2 interaction and MAD2L2 homodimerization are still missing. Here we demonstrate, in a human cell line, using a series of MAD2L2 mutants, that MAD2L2’s C-terminus interface is essential for the CDH1-MAD2L2 binding as well as for homodimerization. In addition, we show that CDH1 interacts with MAD2L2 in a Rev1-like pattern, using the same C-terminus residues on MAD2L2 which Rev1 binds. Thus, identification of CDH1 as an additional Rev1-like binding protein strengthens the versatility of MAD2L2 as a regulatory protein and emphasizes the complexity involved in MAD2L2’s preferential complex formation.

Abstract B44: The TAZ-CAMTA1 and YAP-TFE3 fusion proteins transform cells by binding to subunits of the histone acetyltransferase Ada2a-containing complex (ATAC)

Abstract Background: Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma characterized by WWTR1(TAZ)-CAMTA1 (85%) and YAP1-TFE3 gene fusions (15%). The fusion proteins contain the N-terminus of TAZ and YAP, including their TEAD binding domains, fused in frame to the C-termini of CAMTA1 and TFE3, respectively. The C-termini of CAMTA1 and TFE3 contain transactivating domains as well as other protein-protein interaction domains. The structure of the fusion proteins led us to hypothesize that the C termini of CAMTA1 and TFE3 interact with novel proteins that potentiate the oncogenic activity of TAZ and YAP. Methods: NIH 3T3 and SW872 cell lines were transduced with either TAZ-CAMTA1(TC) or YAP-TFE3(YT) and anchorage independent growth was assayed via growth in soft agar and on poly-HEMA. 1x107 NIH 3T3 cells or SW872 cells expressing TC, YT, or containing EV were subcutaneously injected in NOD Scid Gamma mice. TC and YT as well as the controls including TAZ, CAMTA1, YAP, and TFE3 were tagged with the activated BirA biotin ligase. Protein-protein interactions were identified using BioID mass spectrometry and a subsequent shRNA screen was conducted to identify prey proteins crucial to driving anchorage-independent growth. Results: We showed that TC and YT promote anchorage-independent growth as well as tumorigenesis and metastasis in vivo. Similar to TC, we showed YT requires interaction with TEAD4 to drive its oncogenic transcriptional program. BioID mass spectrometry identified 41 novel interactions with prey proteins (as compared to full-length TAZ and YAP) mediated by the fusion proteins, 26 of which are epigenetic modifying enzymes. Of these 26 proteins, YEATS2 and ZZZ3, both members of the histone acetyltransferase Ada2a-containing complex (ATAC), were identified by the shRNA screen as critical to TC driven anchorage-independent growth. Mass spectrometry showed that an additional subunit of the ATAC complex, KAT14, also interacted with both the TC and YT fusion proteins. ZZZ3 was demonstrated to coimmunoprecipitate with TC, validating the mass spectrometry results. Kaplan-Meier analysis of the TCGA Illumina HiSeq RNA-Seq data set for sarcomas demonstrates that increased expression of YEATS2 or ZZZ3 predicts worse overall survival (p=0.0049 and p=0.0037 respectively) across all sarcomas, independent of histologic type. Conclusions: Utilizing two serial unbiased assays, we showed that oncogenic properties of TC and YT can be explained in part by their interaction with the ATAC complex, a histone acetyltransferase complex. Future studies will elucidate the mechanisms by which the ATAC complex augments the TAZ and YAP oncogenic transcriptional programs. TAZ and YAP fusion proteins have been identified in other cancers, suggesting that similar epigenetic mechanisms may be at work in these cancers. Furthermore, since the ATAC complex is a histone acetyltransferase complex, and contains a catalytic GCN5 subunit, it represents a potential therapeutic target in EHE and possibly other sarcomas. Citation Format: Nicole Merritt, Keith Garcia, Dushyandi Rajendran, Zhen-Yuan Lin, Xiaomeng Zhang, Katrina Mitchell, Colleen Fullenkamp, Anne-Claude Gingras, Kieran Harvey, Munir Tanas. The TAZ-CAMTA1 and YAP-TFE3 fusion proteins transform cells by binding to subunits of the histone acetyltransferase Ada2a-containing complex (ATAC) [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B44.

P0537LINARIN PROTECTS KIDNEY AGAINST ISCHEMIA/REPERFUSION INJURY VIA INHIBITION OF ETS2

Abstract Background and Aims Ischemia/reperfusion injury (IRI), a participant in acute kidney injury (AKI), can occur as a series of pathological processes such as inflammation. Linarin (LIN) has been widely used for different diseases. To confirm the anti-inflammatory value and relevant mechanism of LIN during IRI, in vivo and vitro model were established. Method LIN or isovolumetric DMSO was given, and histologic analysis, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), serum creatinine (SCr) testing and blood urea nitrogen (BUN) were used to evaluate kidney injury. Microarray analysis, protein-protein interaction (PPI) analysis and molecular docking were used to identify target protein of LIN. Results At first, we find that LIN could inhabit the kidney injury in vivo IRI model and decrease the expression of IL-12 p40 in vitro IRI model. To explore the mechanism of LIN, we collected raw data from public microarray database and identified E26 oncogene homolog 2 (ETS2) as a target protein of LIN according to microarray analysis, PPI and molecular docking. The contact area is highly conserved and located on a protein-protein interaction domain of ETS2 which indicated that LIN may alter the interaction with synergistical protein in the regulation of IL-12 p40 expression. Conclusion Our study demonstrated the anti-inflammatory effect of LIN during IRI-triggered AKI, broadening the medicinal value of LIN and the therapeutic options for IRI-triggered AKI.

The Selectivity of Fosfosal for STAT5b over STAT5a is Mediated by Arg566 in the Linker Domain

Fosfosal is the O‐phosphorylated derivative of salicylic acid, with documented clinical use as a prodrug for the treatment of inflammatory diseases. We recently discovered that fosfosal itself inhibits the protein‐protein interaction domain, the SH2 domain, of the tumor‐related transcription factor STAT5b. Here, we demonstrate that fosfosal is selective for STAT5b over its close homologue STAT5a. This selectivity is mediated by the STAT5b residue Arg566, located in the SH2 domain‐adjacent linker domain. Our data provide further evidence for the role of the STAT linker domain in determining the activity of small molecules against the SH2 domain. We present a refined binding model for fosfosal and STAT5b, which can serve as the basis for the development of fosfosal‐based STAT5b inhibitors.

Six1 proteins with human branchio-oto-renal mutations differentially affect cranial gene expression and otic development.

ABSTRACTSingle-nucleotide mutations in human SIX1 result in amino acid substitutions in either the protein-protein interaction domain or the homeodomain, and cause ∼4% of branchio-otic (BOS) and branchio-oto-renal (BOR) cases. The phenotypic variation between patients with the same mutation, even within affected members of the same family, make it difficult to functionally distinguish between the different SIX1 mutations. We made four of the BOS/BOR substitutions in the Xenopus Six1 protein (V17E, R110W, W122R, Y129C), which is 100% identical to human in both the protein-protein interaction domain and the homeodomain, and expressed them in embryos to determine whether they cause differential changes in early craniofacial gene expression, otic gene expression or otic morphology. We confirmed that, similar to the human mutants, all four mutant Xenopus Six1 proteins access the nucleus but are transcriptionally deficient. Analysis of craniofacial gene expression showed that each mutant causes specific, often different and highly variable disruptions in the size of the domains of neural border zone, neural crest and pre-placodal ectoderm genes. Each mutant also had differential effects on genes that pattern the otic vesicle. Assessment of the tadpole inner ear demonstrated that while the auditory and vestibular structures formed, the volume of the otic cartilaginous capsule, otoliths, lumen and a subset of the hair cell-containing sensory patches were reduced. This detailed description of the effects of BOS/BOR-associated SIX1 mutations in the embryo indicates that each causes subtle changes in gene expression in the embryonic ectoderm and otocyst, leading to inner ear morphological anomalies.

De Novo Designed Protein-Interaction Modules for In-Cell Applications.

Protein-protein interactions control a wide variety of natural biological processes. α-Helical coiled coils frequently mediate such protein-protein interactions. Due to the relative simplicity of their sequences and structures and the ease with which properties such as strength and specificity of interaction can be controlled, coiled coils can be designed de novo to deliver a variety of non-natural protein-protein interaction domains. Herein, several de novo designed coiled coils are tested for their ability to mediate protein-protein interactions in Escherichia coli cells. The set includes a parallel homodimer, a parallel homotetramer, an antiparallel homotetramer, and a newly designed heterotetramer, all of which have been characterized in vitro by biophysical and structural methods. Using a transcription repression assay based on reconstituting the Lac repressor, we find that the modules behave as designed in the cellular environment. Each design imparts a different property to the resulting Lac repressor-coiled coil complexes, resulting in the benefit of being able to reconfigure the system in multiple ways. Modification of the system also allows the interactions to be controlled: assembly can be tuned by controlling the expression of the constituent components, and complexes can be disrupted through helix sequestration. The small and straightforward de novo designed components that we deliver are highly versatile and have considerable potential as protein-protein interaction domains in synthetic biology where proteins must be assembled in highly specific ways. The relative simplicity of the designs makes them amenable to future modifications to introduce finer control over their assembly and to adapt them for different contexts.

A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells.

Protein interactions guide the majority of cellular processes. Orthogonal hetero-specific protein-protein interaction domains may facilitate better control of engineered biological systems. Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (the NICP set) and its application for the regulation of diverse cellular processes, from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR/dCas9-based transcriptional activators and to amplify the response of light- and small-molecule inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biology.

Comprehensive in silico screening and molecular dynamics studies of missense mutations in Sjogren-Larsson syndrome associated with the ALDH3A2 gene.

Sjögren-Larsson syndrome (SLS) is an autoimmune disorder inherited in an autosomal recessive pattern. To date, 80 missense mutations have been identified in association with the Aldehyde Dehydrogenase 3 Family Member A2 (ALDH3A2) gene causing SLS. Disruption of the function of ALDH3A2 leads to excessive accumulation of fat in the cells, which interferes with the normal function of protective membranes or materials that are necessary for the body to function normally. We retrieved 54 missense mutations in the ALDH3A2 from the OMIM, UniProt, dbSNP, and HGMD databases that are known to cause SLS. These mutations were examined with various in silico stability tools, which predicted that the mutations p.S308N and p.R423H that are located at the protein-protein interaction domains are the most destabilizing. Furthermore, to determine the atomistic-level differences within the protein-protein interactions owing to mutations, we performed macromolecular simulation (MMS) using GROMACS to validate the motion patterns and dynamic behavior of the biological system. We found that both mutations (p.S380N and p.R423H) had significant effects on the protein-protein interaction and disrupted the dimeric interactions. The computational pipeline provided in this study helps to elucidate the potential structural and functional differences between the ALDH3A2 native and mutant homodimeric proteins, and will pave the way for drug discovery against specific targets in the SLS patients.

Autophagy and LRRK2 in the Aging Brain.

Autophagy is a highly conserved process by which long-lived macromolecules, protein aggregates and dysfunctional/damaged organelles are delivered to lysosomes for degradation. Autophagy plays a crucial role in regulating protein quality control and cell homeostasis in response to energetic needs and environmental challenges. Indeed, activation of autophagy increases the life-span of living organisms, and impairment of autophagy is associated with several human disorders, among which neurodegenerative disorders of aging, such as Parkinson’s disease. These disorders are characterized by the accumulation of aggregates of aberrant or misfolded proteins that are toxic for neurons. Since aging is associated with impaired autophagy, autophagy inducers have been viewed as a strategy to counteract the age-related physiological decline in brain functions and emergence of neurodegenerative disorders. Parkinson’s disease is a hypokinetic, multisystemic disorder characterized by age-related, progressive degeneration of central and peripheral neuronal populations, associated with intraneuronal accumulation of proteinaceous aggregates mainly composed by the presynaptic protein α-synuclein. α-synuclein is a substrate of macroautophagy and chaperone-mediated autophagy (two major forms of autophagy), thus impairment of its clearance might favor the process of α-synuclein seeding and spreading that trigger and sustain the progression of this disorder. Genetic factors causing Parkinson’s disease have been identified, among which mutations in the LRRK2 gene, which encodes for a multidomain protein encompassing central GTPase and kinase domains, surrounded by protein-protein interaction domains. Six LRRK2 mutations have been pathogenically linked to Parkinson’s disease, the most frequent being the G2019S in the kinase domain. LRRK2-associated Parkinson’s disease is clinically and neuropathologically similar to idiopathic Parkinson’s disease, also showing age-dependency and incomplete penetrance. Several mechanisms have been proposed through which LRRK2 mutations can lead to Parkinson’s disease. The present article will focus on the evidence that LRRK2 and its mutants are associated with autophagy dysregulation. Studies in cell lines and neurons in vitro and in LRRK2 knock-out, knock-in, kinase-dead and transgenic animals in vivo will be reviewed. The role of aging in LRRK2-induced synucleinopathy will be discussed. Possible mechanisms underlying the LRRK2-mediated control over autophagy will be analyzed, and the contribution of autophagy dysregulation to the neurotoxic actions of LRRK2 will be examined.

Abstract C050: A novel, potent, and selective hypoxia-inducible factor (HIF)-2α antagonist

Abstract The microenvironment of solid tumors is known to be hypoxic and requires induction of genes associated with metabolism, growth, proliferation, and angiogenesis for tumor cells to survive and metastasize. The master transcriptional regulator of hypoxia-induced genes is the Hypoxia-Inducible Factor (HIF), consisting of an oxygen-regulated alpha monomer, of which there are three isoforms (HIF-1α, HIF-2α, and HIF-3α), that can heterodimerize with a constitutively-expressed beta monomer (HIF-1β/ARNT) using Per-ARNT-SIM (PAS) protein-protein interaction domains. In normoxia, proline residues present in the oxygen-dependent degradation domain of the HIF-α subunits are hydroxylated, allowing for recognition by the von Hippel-Lindau (pVHL) E3-ubiquitin ligase complex and subsequent proteasomal degradation. Upon exposure to low oxygen conditions or in the case of VHL mutation or silencing, HIF-α subunits accumulate in the cell and mediate transcription of various pro-tumorigenic gene sets. In patients, overexpression of HIF is associated with poor prognosis, and both preclinical and clinical evidence is mounting that suggests inhibiting HIF-2α is a valid approach to destroy tumor cells, particularly in clear cell renal carcinoma, warranting development of next-generation inhibitors. Using a suite of in vitro and in vivo assays designed to evaluate HIF-2α-specific effects, herein we describe pharmacological properties associated with novel, potent, and selective small-molecule antagonists of HIF-2α. These compounds inhibited HIF-dependent reporter gene transcription as well as VEGF protein secretion in a human renal cell adenocarcinoma line. Compounds that were confirmed to bind the HIF-2α PAS-B domain by Microscale thermophoresis (MST) and Thermal shift assay (TSA) also significantly inhibited HIF-2α, but not HIF-1α, target gene expression in a hepatocellular carcinoma cell line (P<0.05). Further, NanoString analyses revealed pathway signatures upregulated in hypoxic conditions, including angiogenesis, metabolism, and metastasis, that were significantly decreased with antagonist treatment. Selective antagonists were evaluated in vivo to determine efficacy and PK-PD relationships. Additionally, syngeneic tumor models were used to characterize the effects of HIF-2α inhibition (alone or in combination with anti-PD-1 and anti-adenosine agents) on tumor growth, immune cell infiltration, and expression of genes associated with adenosine production/signaling. Collectively, these data support the rationale to target HIF-2α for the treatment of cancers, particularly indications with a HIF-2α-driven hypoxia signature, and describe the properties of novel and selective HIF-2α antagonists. Citation Format: Kelsey E Sivick Gauthier, Kenneth V Lawson, Dana Piovesan, Matthew J Walters, Ada Chen, Xiaoning Zhao, Cesar Meleza, Nikki Kimura, Tim Park, Steve Young, Anh Tran, Samuel L Drew, Lixia Jin, Manmohan Leleti, Elaine Ginn, Jay P Powers. A novel, potent, and selective hypoxia-inducible factor (HIF)-2α antagonist [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C050. doi:10.1158/1535-7163.TARG-19-C050

TRIM32, but not its muscular dystrophy-associated mutant, positively regulates and is targeted to autophagic degradation by p62/SQSTM1.

The tripartite motif (TRIM) proteins constitute a family of ubiquitin E3 ligases involved in a multitude of cellular processes, including protein homeostasis and autophagy. TRIM32 is characterized by six protein-protein interaction domains termed NHL, various point mutations in which are associated with limb-girdle-muscular dystrophy 2H (LGMD2H). Here, we show that TRIM32 is an autophagy substrate. Lysosomal degradation of TRIM32 was dependent on ATG7 and blocked by knockout of the five autophagy receptors p62 (also known as SQSTM1), NBR1, NDP52 (also known as CALCOCO2), TAX1BP1 and OPTN, pointing towards degradation by selective autophagy. p62 directed TRIM32 to lysosomal degradation, while TRIM32 mono-ubiquitylated p62 on lysine residues involved in regulation of p62 activity. Loss of TRIM32 impaired p62 sequestration, while reintroduction of TRIM32 facilitated p62 dot formation and its autophagic degradation. A TRIM32LGMD2H disease mutant was unable to undergo autophagic degradation and to mono-ubiquitylate p62, and its reintroduction into the TRIM32-knockout cells did not affect p62 dot formation. In light of the important roles of autophagy and p62 in muscle cell proteostasis, our results point towards impaired TRIM32-mediated regulation of p62 activity as a pathological mechanisms in LGMD2H.

Stafia-1: a STAT5a-Selective Inhibitor Developed via Docking-Based Screening of in Silico O-Phosphorylated Fragments.

We present a new approach for the identification of inhibitors of phosphorylation‐dependent protein–protein interaction domains, in which phenolic fragments are adapted by in silico O‐phosphorylation before docking‐based screening. From a database of 10 369 180 compounds, we identified 85 021 natural product‐derived phenolic fragments, which were virtually O‐phosphorylated and screened for in silico binding to the STAT3 SH2 domain. Nine screening hits were then synthesized, eight of which showed a degree of in vitro inhibition of STAT3. After analysis of its selectivity profile, the most potent inhibitor was then developed to Stafia‐1, the first small molecule shown to preferentially inhibit the STAT family member STAT5a over the close homologue STAT5b. A phosphonate prodrug based on Stafia‐1 inhibited STAT5a with selectivity over STAT5b in human leukemia cells, providing the first demonstration of selective in vitro and intracellular inhibition of STAT5a by a small‐molecule inhibitor.