Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states-a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last β-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pK a value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pHsensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals., a 76-residue signaling protein, is found ubiquitously in cells. Two or more Ub molecules can be covalently linked to form a diubiquitin (diUb) and then a polyubiquitin (polyUb), as an isopeptide bond is formed between the carboxylate group of one Ub (called the distal Ub) and the amine group of another Ub (called the proximal Ub). Owing to the characteristic quaternary structures of polyUb and specific interactions between polyUb and its target proteins (1, 2), a polyUb with a specific linkage can be involved in a distinctive set of cellular functions (3).The heterogeneity of Ub also arises from other types of covalent modifications. Proteomics studies have indicated that Ub is phosphorylated at multiple sites (4, 5). However, PINK1 is the only Ub kinase known to date, which specifically phosphorylates Ub at S65 (6, 7). Under normal conditions, only a fraction of Ub is S65-phosphorylated. However, upon oxidative stress, neurodegeneration, or aging, the level of S65 phosphorylation increases significantly (4, 8). S65-phosphorylated Ub (pUb) in turn can activate PARKIN, a ubiquitin ligase, and induce mitophagy (9-12). However, no other pUb-specific targets have been clearly identified, and how phosphorylation affects Ub signaling in general remains unclear.Previously, Komander and coworkers showed that pUb gives two distinct sets of NMR peaks, which correspond to the two conformational states of pUb exchanging at a slow timescale (13). Based on NMR long-r...

Plants can sense both intracellular and extracellular mechanical forces and can respond through morphological changes. The signaling components responsible for mechanotransduction of the touch response are largely unknown. Here, we performed a high-throughput SILIA (stable isotope labeling in Arabidopsis)-based quantitative phosphoproteomics analysis to profile changes in protein phosphorylation resulting from 40 seconds of force stimulation in Arabidopsis thaliana. Of the 24 touch-responsive phosphopeptides identified, many were derived from kinases, phosphatases, cytoskeleton proteins, membrane proteins and ion transporters. TOUCH-REGULATED PHOSPHOPROTEIN1 (TREPH1) and MAP KINASE KINASE 2 (MKK2) and/or MKK1 became rapidly phosphorylated in touch-stimulated plants. Both TREPH1 and MKK2 are required for touch-induced delayed flowering, a major component of thigmomorphogenesis. The treph1-1 and mkk2 mutants also exhibited defects in touch-inducible gene expression. A nonphosphorylatable site-specific isoform of TREPH1 (S625A) failed to restore touch-induced flowering delay of treph1-1, indicating the necessity of S625 for TREPH1 function and providing evidence consistent with the possible functional relevance of the touch-regulated TREPH1 phosphorylation. Bioinformatic analysis and biochemical subcellular fractionation of TREPH1 protein indicate that it is a soluble protein. Altogether, these findings identify new protein players in Arabidopsis thigmomorphogenesis regulation, suggesting that protein phosphorylation may play a critical role in plant force responses.Like neural systems in animals, plants respond to a delicate force signal, such as a light touch, with extreme sensitivity, being it thigmotropism, thigmonastic movement, and thigmomorphogenesis. To understand the complex force signaling networks in plants, we applied SILIA-based quantitative PTM proteomics to measure 40 seconds of protein phosphorylation changes in Arabidopsis in response to cotton and human hair touches, and identified 4895 repeatable and non-redundant phosphopeptides, 579 of which are novel phosphosites derived from the 509 phosphoprotein groups, and finally identified 24 TOUCH-REGUALTED PHOSPHOPROTEIN (TREPHs) groups. Consequent molecular biological and bioinformatic studies revealed that both TREPH1 and MKK2 proteins are required for Arabidopsis touch response. These studies suggest that protein phosphorylation is involved in mechanotransduction of plant thigmomorphogenesis.

No abstract

We used an antibody array to compare the protein expression of matrix metalloproteinases (MMPs)-1, -2, -3, -8, -9, -10, and -13, as well as the tissue inhibitors of metalloproteinases (TIMPs)-1, -2, and -4 during blastema formation in amputated hindlimbs of regeneration-competent wild-type axolotls and stage-54 Xenopus, and regeneration-deficient short-toes axolotls and Xenopus froglets. Expression of MMP-9 and -2 was also compared by zymography. Both short-toes and froglet failed to up-regulate MMPs in a pattern comparable to the wild-type axolotl, suggesting that subnormal histolysis is at least in part responsible for the poor blastema formation characteristic of both short-toes and froglet. MMP levels were much lower in amputated stage-54 Xenopus limb buds than in the other animals, suggesting that blastema formation in these limb buds requires much less extracellular matrix degradation than in fully differentiated limbs. TIMP expression patterns followed the same trends as the MMP's in each group of animals. Developmental

The interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13 NTD ) and Ub monomer has been characterized, while it remains unclear how Rpn13 specifically recognizes K48-linked Ub chain. Using single-molecule FRET, here we show that K48-linked diubiquitin (K48-diUb) fluctuates among distinct conformational states, and a preexisting compact state is selectively enriched by Rpn13 NTD . The same binding mode is observed for full-length Rpn13 and longer K48-linked Ub chain. Using solution NMR spectroscopy, we have determined the complex structure between Rpn13 NTD and K48-diUb. In this structure, Rpn13 NTD simultaneously interacts with proximal and distal Ub subunits of K48-diUb that remain associated in the complex, thus corroborating smFRET findings. The proximal Ub interacts with Rpn13 NTD similarly as the Ub monomer in the known Rpn13 NTD :Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13 NTD . Thus, a charge-reversal mutation in Rpn13 NTD weakens the interaction between Rpn13 and K48-linked Ub chain, causing accumulation of ubiquitinated proteins. Moreover, physical blockage of the access of the distal Ub to Rpn13 NTD with a proximity-attached Ub monomer can disrupt the interaction between Rpn13 and K48-diUb. Taken together, the bivalent interaction of K48-linked Ub chain with Rpn13 provides the structural basis for Rpn13 linkage selectivity, which opens a new window for modulating proteasomal function.