Over a century ago, colloidal phase separation of matter into non-membranous bodies was recognized as a fundamental organizing principal of cell “protoplasm.” Recent insights into the molecular ...properties of such phase-separated bodies present challenges to our understanding of cellular protein interaction networks, as well as opportunities for interpreting and understanding of native and pathological genetic and molecular interactions. Here, we briefly review examples of and discuss physical principles of phase-separated cellular bodies and then reflect on how knowledge of these principles may direct future research on their functions.
This Perspective explores the nature of protein-protein interactions that enable organization of cellular matter through phase separation and the functional implications for the resulting membrane-free compartments.
Heterobifunctional small-molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we ...currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize a comprehensive characterization of the ligand-dependent CRBN-BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low-energy binding conformations that are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying interprotein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic interprotein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands.
Poly(A)-binding protein (PABPC1) is involved in multiple aspects of mRNA processing and translation. It is a component of RNA stress granules and binds the RNA-induced silencing complex to promote ...degradation of silenced mRNAs. Here, we report the crystal structures of the C-terminal Mlle (or PABC) domain in complex with peptides from GW182 (TNRC6C) and Ataxin-2. The structures reveal overlapping binding sites but with unexpected diversity in the peptide conformation and residues involved in binding. The mutagenesis and binding studies show low to submicromolar binding affinity with overlapping but distinct specificity determinants. These results rationalize the role of the Mlle domain of PABPC1 in microRNA-mediated mRNA deadenylation and suggest a more general function in the assembly of cytoplasmic RNA granules.
The other double helix: The molecular structure of double‐stranded poly(rA) was predicted in 1961, but it has only now been confirmed. The crystal structure of the parallel double helix of (rA)11 ...obtained at physiological pH was solved using ab initio direct methods and refined to 1.0 Å resolution. The crystals have bound ammonium ions that are complexed by the RNA phosphates and adenine N1 atoms (see section of the structure).
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LPG3 is the Leishmania homologue of the mammalian endoplasmic reticulum (ER) chaperone GRP94. This molecule has an essential role in synthesis of the lipophosphoglycan (LPG), the most ...abundant surface molecule identified on promastigotes of all Leishmania species. This paper describes the utilization of two vaccination strategies including DNA and prim/boost vaccination of LPG3 in susceptible model of Leishmania major infection. In addition different truncated forms of LPG3 were produce and sera reactivities of human CL and VL were tested as biomarker.
One of the main issues in vaccine development is implementation of new adjuvants to improve the antigen presentation and eliciting the protective immune response. Heat shock protein (HSP) molecules are known as natural adjuvants. They can stimulate the innate and adaptive immune response against infectious diseases and cancer. Lipophosphoglycan 3 (LPG3), the Leishmania homologous with GRP94 (glucose regulated protein 94), a member of HSP90 family, is involved in assembly of LPG as the most abundant macromolecule on the surface of Leishmania promastigotes. In the present study as a primary step, we tested LPG3 as a vaccine candidate in two regimens, DNA/DNA and prime-boost (DNA/Protein), against Leishmania major infection in BALB/c mice model. Our results showed that LPG3 and its fragment (rNT-LPG3) are highly immunogenic in BALB/c mice and can stimulate the production of both IgG1 and IgG2a. In prime-boost immunization strategy, the level of antibody response was higher compared with DNA/DNA immunization. The levels of IFN-γ in the supernatant of splenocytes from mice immunized with DNA/DNA and prime-boost regimens were significantly higher when compared to control groups. In fact, immunization with prime-boost vaccination has higher ratio of IFN-γ/IL-5, suggesting a shift towards a Th1 response.
In addition, sera reactivity against LPG3 in visceral leishmaniasis (VL) patients was significantly higher in comparison with cutaneous leishmaniasis (CL) patients. Therefore, we recommend further investigations on the usage of LPG3 co-delivery with candidate antigens for vaccine development against leishmaniasis.
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Translation is regulated in cells. Key among the different stages of translation, translation initiation is controlled by regulatory interactions of eIFs and also translation regulatory ...proteins interacting with eIFs and PABP. PABP-interacting protein (Paip) 1 and 2 have been identified as regulatory proteins affecting the rate of translation initiation through their interactions with PABP. These interactions are mediated by the PABP interacting Motifs (PAM) 1 and 2 of Paips. Paip1 enhances the rate of translation while Paip2 suppresses it. To further understand their mechanisms of actions, here, we have studied the interactions of PABP RRMs with the PAM1 regions of Paip1 and 2. Compared to Paip1, affinity measurements using ITC show that Paip2 binds to PABP with higher affinity. While Paip2 dissociates poly(A) from PABP RRMs, Paip1 can not compete with poly(A) binding to PABP. Our binding studies show that Paip1 increases eIF4G-binding to PABP RRM1-2, supporting its stimulatory role in translation. Upon binding, Paip1 and Paip2 affect the conformation of the PABP RRMs differently. Compared to RRMs–Paip1, the complex of RRMs–Paip2 has a compact conformation. Our data suggest that Paip1 and Paip2 regulate PABP function through modulating its conformation, stimulating its incorporation in the pre-initiation complex, or sequestering it from translational machinery.
Targeted protein degradation (TPD) uses small molecules to recruit E3 ubiquitin ligases into the proximity of proteins of interest, inducing ubiquitination-dependent degradation. A major bottleneck ...in the TPD field is the lack of accessible E3 ligase ligands for developing degraders. To expand the E3 ligase toolbox, we sought to convert the Kelch-like ECH-associated protein 1 (KEAP1) inhibitor KI696 into a recruitment handle for several targets. While we were able to generate KEAP1-recruiting degraders of BET family and murine focal adhesion kinase (FAK), we discovered that the target scope of KEAP1 was narrow, as targets easily degraded using a cereblon (CRBN)-recruiting degrader were refractory to KEAP1-mediated degradation. Linking the KEAP1-binding ligand to a CRBN-binding ligand resulted in a molecule that induced degradation of KEAP1 but not CRBN. In sum, we characterize tool compounds to explore KEAP1-mediated ubiquitination and delineate the challenges of exploiting new E3 ligases for generating bivalent degraders.
Many RNA-binding proteins contain multiple single-strand nucleic acid-binding domains and assemble into large multiprotein messenger ribonucleic acid protein (mRNP) complexes. The mechanisms ...underlying the self-assembly of these complexes are largely unknown. In eukaryotes, the association of the translation factors polyadenylate-binding protein-1 (PABP) and eIF4G is essential for high-level expression of polyadenylated mRNAs. Here, we report the crystal structure of the ternary complex poly(A)11·PABP(1–190)·eIF4G(178–203) at 2.0 Å resolution. Our NMR and crystallographic data show that eIF4G interacts with the RRM2 domain of PABP. Analysis of the interaction by small-angle X-ray scattering, isothermal titration calorimetry, and electromobility shift assays reveals that this interaction is allosterically regulated by poly(A) binding to PABP. Furthermore, we have confirmed the importance of poly(A) for the endogenous PABP and eIF4G interaction in immunoprecipitation experiments using HeLa cell extracts. Our findings reveal interdomain allostery as a mechanism for cooperative assembly of RNP complexes.
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► Structure of ternary complex of parts of PABP, eIF4G, and RNA ► eIF4G residues 178–203 bind to PABP RRM2 ► Cooperative binding of eIF4G and RNA to PABP ► Interdomain allostery is the basis of cooperativity
Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this ...structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2'-deoxyribose, 2'-O-methyl-ribose, 2'-deoxy-2'-fluoro-ribose, arabinose and 2'-deoxy-2'-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2' modifications gave a variety of effects. Arabinose and 2'-deoxy-2'-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2'-O-methyl and 2'-deoxy-2'-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability.
KRAS is the most frequently mutated oncogene found in pancreatic, colorectal, and lung cancers. Although it has been challenging to identify targeted therapies for cancers harboring KRAS mutations, ...KRASG12C can be targeted by small-molecule inhibitors that form covalent bonds with cysteine 12 (C12). Here, we designed a library of C12-directed covalent degrader molecules (PROTACs) and subjected them to a rigorous evaluation process to rapidly identify a lead compound. Our lead degrader successfully engaged CRBN in cells, bound KRASG12Cin vitro, induced CRBN/KRASG12C dimerization, and degraded GFP-KRASG12C in reporter cells in a CRBN-dependent manner. However, it failed to degrade endogenous KRASG12C in pancreatic and lung cancer cells. Our data suggest that inability of the lead degrader to effectively poly-ubiquitinate endogenous KRASG12C underlies the lack of activity. We discuss challenges for achieving targeted KRASG12C degradation and proposed several possible solutions which may lead to efficient degradation of endogenous KRASG12C.
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•A comprehensive degrader molecule (PROTAC) library for KRASG12C is described•Lead compound degrades GFP-KRASG12C in a CRBN-dependent manner•Challenges and solutions for achieving endogenous KRASG12C degradation are discussed
KRASG12C is an oncoprotein of high interest for drug development. Zeng et al. investigate targeting KRASG12C for degradation using small-molecule degraders (PROTACs) and document challenges and opportunities in this area.
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