Phosphorylation of a Human Microprotein Promotes Dissociation of Biomolecular Condensates.

Proteogenomic identification of translated small open reading frames in humans has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. However, the vast majority of microproteins remain functionally uncharacterized, and many lack secondary structure and exhibit limited evolutionary conservation. One such intrinsically disordered microprotein is NBDY, a 68-amino acid component of membraneless organelles known as P-bodies. In this work, we show that NBDY can undergo liquid-liquid phase separation, a biophysical process thought to underlie the formation of membraneless organelles, in the presence of RNA in vitro. Phosphorylation of NBDY drives liquid phase remixing in vitro and macroscopic P-body dissociation in cells undergoing growth factor signaling and cell division. These results suggest that NBDY phosphorylation enables regulation of P-body dynamics during cell proliferation and, more broadly, that intrinsically disordered microproteins may contribute to liquid-liquid phase separation and remixing behavior to affect cellular processes.

Uncovering the Molecular Interactions in the Catalytic Loop That Modulate the Conformational Dynamics in Protein Tyrosine Phosphatase 1B.

Active-site loops are integral to the function of numerous enzymes. They enable substrate and product binding and release, sequester reaction intermediates, and recruit catalytic groups. Here, we examine the catalytic loop in the enzyme protein tyrosine phosphatase 1B (PTP1B). PTP1B has a mobile so-called WPD loop (named for its three N-terminal residues) that initiates the dephosphorylation of phosphor-tyrosine substrates upon loop closure. We have combined X-ray crystallography, solution NMR, and pre-steady-state kinetics experiments on wild-type and five WPD loop mutants to identify the relationships between the loop structure, dynamics, and function. The motions of the WPD loop are modulated by the formation of weak molecular interactions, where perturbations of these interactions modulate the conformational equilibrium landscape. The point mutants in the WPD loop alter the loop equilibrium position from a predominantly open state (P185A) to 50:50 (F182A), 35:65 (P188A), and predominantly closed states (T177A and P188A). Surprisingly, there is no correlation between the observed catalytic rates in the loop mutants and changes to the WPD loop equilibrium position. Rather, we observe a strong correlation between the rate of dephosphorylation of the phosphocysteine enzyme intermediate and uniform millisecond motions, not only within the loop but also in the adjacent α-helical domain of PTP1B. Thus, the control of loop motion and thereby catalytic activity is dispersed and resides within not only the loop sequence but also the surrounding protein architecture. This has broad implications for the general mechanistic understanding of enzyme reactions and the role that flexible loops play in the catalytic cycle.

Leveraging Reciprocity to Identify and Characterize Unknown Allosteric Sites in Protein Tyrosine Phosphatases.

Drug-like molecules targeting allosteric sites in proteins are of great therapeutic interest; however, identification of potential sites is not trivial. A straightforward approach to identify hidden allosteric sites is demonstrated in protein tyrosine phosphatases (PTP) by creation of single alanine mutations in the catalytic acid loop of PTP1B and VHR. This approach relies on the reciprocal interactions between an allosteric site and its coupled orthosteric site. The resulting NMR chemical shift perturbations (CSPs) of each mutant reveal clusters of distal residues affected by acid loop mutation. In PTP1B and VHR, two new allosteric clusters were identified in each enzyme. Mutations in these allosteric clusters altered phosphatase activity with changes in kcat/KM ranging from 30% to nearly 100-fold. This work outlines a simple method for identification of new allosteric sites in PTP, and given the basis of this method in thermodynamics, it is expected to be generally useful in other systems.

Asymmetric Anchoring Is Required for Efficient Ω-Loop Opening and Closing in Cytosolic Phosphoenolpyruvate Carboxykinase.

Mobile Ω-loops play essential roles in the function of many enzymes. Here we investigated the importance of a residue lying outside of the mobile Ω-loop element in the catalytic function of an H477R variant of cytosolic phosphoenolpyruvate carboxykinase using crystallographic, kinetic, and computational analysis. The crystallographic data suggest that the efficient transition of the Ω-loop to the closed conformation requires stabilization of the N-terminus of the loop through contacts between R461 and E588. In contrast, the C-terminal end of the Ω-loop undergoes changing interactions with the enzyme body through contacts between H477 at the C-terminus of the loop and E591 located on the enzyme body. Potential of mean force calculations demonstrated that altering the anchoring of the C-terminus of the Ω-loop via the H477R substitution results in the destabilization of the closed state of the Ω-loop by 3.4 kcal mol-1. The kinetic parameters for the enzyme were altered in an asymmetric fashion with the predominant effect being observed in the direction of oxaloacetate synthesis. This is exemplified by a reduction in kcat for the H477R mutant by an order of magnitude in the direction of OAA synthesis, while in the direction of PEP synthesis, it decreased by a factor of only 2. The data are consistent with a mechanism for loop conformational exchange between open and closed states in which a balance between fixed anchoring of the N-terminus of the Ω-loop and a flexible, unattached C-terminus drives the transition between a disordered (open) state and an ordered (closed) state.

Enhancing tumor cell response to chemotherapy through nanoparticle-mediated codelivery of siRNA and cisplatin prodrug.

Cisplatin and other DNA-damaging chemotherapeutics are widely used to treat a broad spectrum of malignancies. However, their application is limited by both intrinsic and acquired chemoresistance. Most mutations that result from DNA damage are the consequence of error-prone translesion DNA synthesis, which could be responsible for the acquired resistance against DNA-damaging agents. Recent studies have shown that the suppression of crucial gene products (e.g., REV1, REV3L) involved in the error-prone translesion DNA synthesis pathway can sensitize intrinsically resistant tumors to chemotherapy and reduce the frequency of acquired drug resistance of relapsed tumors. In this context, combining conventional DNA-damaging chemotherapy with siRNA-based therapeutics represents a promising strategy for treating patients with malignancies. To this end, we developed a versatile nanoparticle (NP) platform to deliver a cisplatin prodrug and REV1/REV3L-specific siRNAs simultaneously to the same tumor cells. NPs are formulated through self-assembly of a biodegradable poly(lactide-coglycolide)-b-poly(ethylene glycol) diblock copolymer and a self-synthesized cationic lipid. We demonstrated the potency of the siRNA-containing NPs to knock down target genes efficiently both in vitro and in vivo. The therapeutic efficacy of NPs containing both cisplatin prodrug and REV1/REV3L-specific siRNAs was further investigated in vitro and in vivo. Quantitative real-time PCR results showed that the NPs exhibited a significant and sustained suppression of both genes in tumors for up to 3 d after a single dose. Administering these NPs revealed a synergistic effect on tumor inhibition in a human Lymph Node Carcinoma of the Prostate xenograft mouse model that was strikingly more effective than platinum monotherapy.