AMSH regulates calcium-sensing receptor signaling through direct interactions

Calcium-sensing receptor (CaR) activates intracellular pathways controlling calcium homeostasis. CaR carboxyl-terminal mutants associated with metabolic diseases suggest that unidentified proteins interact with the carboxyl-terminal region of this receptor. To address this possibility, we screened for CaR-interacting proteins using the carboxyl terminus of CaR (CaRDelta895-1075 deletion mutant). We identified AMSH, an ubiquitin isopeptidase, as a CaR-interacting partner. AMSH caused a decrease on the signaling properties of wild-type and mutant CaR. Our results indicate that AMSH, which has been recently characterized as a regulator of the endosomal sorting of epidermal growth factor receptor, represents a novel modulator of CaR signaling.     

STAM-AMSH interaction facilitates the deubiquitination activity in the C-terminal AMSH

Signal transducing adaptor molecule (STAM) complexed with hepatocyte growth factor regulated tyrosine kinase substrate (Hrs) works on sorting of cargo proteins in multivesicular body (MVB) pathway. Associated molecule with SH3 domain of STAM (AMSH), a zinc-containing ubiquitin isopeptidase, is thought to play a role in regulation of ubiquitin-mediated degradation by binding to STAM. We have found that AMSH requires the conformation of Px(V/I)(D/N)RxxKP sequence to bind SH3 domain of STAM with approximately 7 microM affinity, and that the isolated C-terminal domain of AMSH contains the isopeptidase activity. Deubiquitination by AMSH was assisted when ubiquitins were bound to STAM which can bind to AMSH simultaneously. With the specificity toward K63-linked ubiquitins, this facilitated ubiquitin processing activity of AMSH may imply a distinct regulatory mechanism for sorting and degradation through STAM binding.     

The deubiquitinating enzyme AMSH1 is required for rhizobial infection and nodule organogenesis in Lotus japonicus

Legume–rhizobium symbiosis contributes large quantities of fixed nitrogen to both agricultural and natural ecosystems. This global impact and the selective interaction between rhizobia and legumes culminating in development of functional root nodules have prompted detailed studies of the underlying mechanisms. We performed a screen for aberrant nodulation phenotypes using the Lotus japonicus LORE1 insertion mutant collection. Here, we describe the identification of amsh1 mutants that only develop small nodule primordia and display stunted shoot growth, and show that the aberrant nodulation phenotype caused by LORE1 insertions in the Amsh1 gene may be separated from the shoot phenotype. In amsh1 mutants, rhizobia initially became entrapped in infection threads with thickened cells walls. Some rhizobia were released into plant cells much later than observed for the wild‐type; however, no typical symbiosome structures were formed. Furthermore, cytokinin treatment only very weakly induced nodule organogenesis in amsh1 mutants, suggesting that AMSH1 function is required downstream of cytokinin signaling. Biochemical analysis showed that AMSH1 is an active deubiquitinating enzyme, and that AMSH1 specifically cleaves K63‐linked ubiquitin chains. Post‐translational ubiquitination and deubiquitination processes involving the AMSH1 deubiquitinating enzyme are thus involved in both infection and organogenesis in Lotus japonicus.     

Competitive binding of UBPY and ubiquitin to the STAM2 SH3 domain revealed by NMR

To date, the signal transducing adaptor molecule 2 (STAM2) was shown to harbour two ubiquitin binding domains (UBDs) known as the VHS and UIM domains, while the SH3 domain of STAM2 was reported to interact with deubiquitinating enzymes (DUBs) like UBPY and AMSH. In the present study, NMR evidences the interaction of the STAM2 SH3 domain with ubiquitin, demonstrating that SH3 constitutes the third UBD of STAM2. Furthermore, we show that a UBPY-derived peptide can outcompete ubiquitin for SH3 binding and vice versa. These results suggest that the SH3 domain of STAM2 plays versatile roles in the context of ubiquitin mediated receptor sorting.     

Structural basis for autoinhibition of ESCRT-III CHMP3

Endosomal sorting complexes required for transport (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) are selectively recruited to cellular membranes to exert their function in diverse processes, such as multivesicular body biogenesis, enveloped virus budding, and cytokinesis. ESCRT-III is composed of members of the charged multivesicular body protein (CHMP) family—cytosolic proteins that are targeted to membranes via yet unknown signals. Membrane targeting is thought to result in a membrane-associated protein network that presumably acts at a late budding step. Here we provide structural evidence based on small-angle X-ray scattering data that ESCRT-III CHMP3 can adopt two conformations in solution: a closed globular form that most likely represents the cytosolic conformation and an open extended conformation that might represent the activated form of CHMP3. Both the closed and open conformations of CHMP3 interact with AMSH with high affinity. Although the C-terminal region of CHMP3 is required for AMSH interaction, a peptide thereof reveals only weak binding to AMSH, suggesting that other regions of CHMP3 contribute to the high-affinity interaction. Thus, AMSH, including its MIT (microtubule interacting and transport) domain, interacts with ESCRT-III CHMP3 differently from reported Vps4 MIT domain-CHMP protein interactions.     

Identification of AMSH-LP containing a Jab1/MPN domain metalloenzyme motif

We have isolated a cDNA clone encoding a new AMSH (associated molecule with the SH3 domain of STAM) family protein, termed AMSH-like protein (AMSH-LP). AMSH-LP has similar characteristics to AMSH; both AMSH-LP and AMSH are expressed ubiquitously in various human tissues, contain a putative nuclear localization signal (NLS), an Mpr/Pad1/N-terminal (MPN) domain, and a Jab1/MPN domain metalloenzyme (JAMM) motif in their structures, and are excluded from the nucleus when lacking either the NLS or MPN domain. Moreover, we observed an enhancement of interleukin 2 (IL-2)-mediated c-myc induction in AMSH-LP-transfected cells similar to that seen in AMSH-transfected cells, suggesting a functional similarity between AMSH-LP and AMSH. However, the present study demonstrated that AMSH-LP, unlike AMSH, fails to bind to the SH3 domains of STAM1 (signal transducing adaptor molecule 1) and Grb2. These results suggest that AMSH-LP and AMSH may have different functions.     

Endogenous opiates and behavior: 2010

This paper is the thirty-third consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2010 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration (Section 16); and immunological responses (Section 17).     

AMSH is required to degrade ubiquitinated proteins in the central nervous system

Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. AMSH (associated molecule with the SH3 domain of STAM) is a deubiquitination enzyme that participates in the endosomal sorting of several cell-surface molecules. AMSH impairment results in missorted ubiquitinated cargoes in vitro and severe neurodegeneration in vivo, but it is not known how AMSH deficiency causes neuronal damage in the brain. Here, we demonstrate that AMSH^−/− mice developed ubiquitinated protein accumulations as early as embryonic day 10 (E10), and that severe deposits were present in the brain at postnatal day 8 (P8) and P18. Interestingly, TDP-43 was found to accumulate and colocalize with glial marker-positive cells in the brain. Glutamate receptor and p62 accumulations were also found; these molecules colocalized with ubiquitinated aggregates in the brain. These data suggest that AMSH plays an important role in degrading ubiquitinated proteins and glutamate receptors in vivo. AMSH^−/− mice provide an animal model for neurodegenerative diseases, which are commonly characterized by the generation of proteinaceous aggregates.