The mechanism of ubiquitination in the cullin-RING E3 ligase machinery: conformational control of substrate orientation.

In cullin-RING E3 ubiquitin ligases, substrate binding proteins, such as VHL-box, SOCS-box or the F-box proteins, recruit substrates for ubiquitination, accurately positioning and orienting the substrates for ubiquitin transfer. Yet, how the E3 machinery precisely positions the substrate is unknown. Here, we simulated nine substrate binding proteins: Skp2, Fbw7, beta-TrCP1, Cdc4, Fbs1, TIR1, pVHL, SOCS2, and SOCS4, in the unbound form and bound to Skp1, ASK1 or Elongin C. All nine proteins have two domains: one binds to the substrate; the other to E3 ligase modules Skp1/ASK1/Elongin C. We discovered that in all cases the flexible inter-domain linker serves as a hinge, rotating the substrate binding domain, optimally and accurately positioning it for ubiquitin transfer. We observed a conserved proline in the linker of all nine proteins. In all cases, the prolines pucker substantially and the pucker is associated with the backbone rotation toward the E2/ubiquitin. We further observed that the linker flexibility could be regulated allosterically by binding events associated with either domain. We conclude that the flexible linker in the substrate binding proteins orients the substrate for the ubiquitin transfer. Our findings provide a mechanism for ubiquitination and polyubiquitination, illustrating that these processes are under conformational control.

Molecular recognition of ubiquitin and Lys63-linked diubiquitin by STAM2 UIM-SH3 dual domain: the effect of its linker length and flexibility.

Multidomain proteins represent a broad spectrum of the protein landscape and are involved in various interactions. They could be considered as modular building blocks assembled in distinct fashion and connected by linkers of varying lengths and sequences. Due to their intrinsic flexibility, these linkers provide proteins a subtle way to modulate interactions and explore a wide range of conformational space. In the present study, we are seeking to understand the effect of the flexibility and dynamics of the linker involved in the STAM2 UIM-SH3 dual domain protein with respect to molecular recognition. We have engineered several constructs of UIM-SH3 with different length linkers or domain deletion. By means of SAXS and NMR experiments, we have shown that the modification of the linker modifies the flexibility and the dynamics of UIM-SH3. Indeed, the global tumbling of both the UIM and SH3 domain is different but not independent from each other while the length of the linker has an impact on the ps-ns time scale dynamics of the respective domains. Finally, the modification of the flexibility and dynamics of the linker has a drastic effect on the interaction of UIM-SH3 with Lys63-linked diubiquitin with a roughly eight-time weaker dissociation constant.

Autophagic and apoptotic features during programmed cell death in the fat body of the tobacco hornworm (Manduca sexta).

Two major pathways of programmed cell death (PCD)--the apoptotic and the autophagic cell death--were investigated in the decomposition process of the larval fat body during the 5th larval stage of Manduca sexta. Several basic aspects of apoptotic and autophagic cell death were analyzed by morphological and biochemical methods in order to disclose whether these mechanisms do have shared common regulatory steps. Morphological examination revealed the definite autophagic wave started on day 4 followed by DNA fragmentation as demonstrated by agarose gel electrophoresis and TUNEL assay. By the end of the wandering period the cells were filled with autophagic vacuoles and protein granules of heterophagic origin and the vast majority of the nuclei were TUNEL-positive. No evidence was found of other aspects of apoptosis, e.g. activation of executioner caspases. Close correlation was disclosed between the onset of autophagy and the nuclear accumulation of the ubiquitin-proteasome system.

Pattern of tau-1 and ubiquitin immunoreactivity in the white matter of temporal lobe in senile and with Alzheimer's disease brains.

Immunostaining pattern of the temporal white matter with anti-tau-1 and anti-ubiquitin was different in examined cases of Alzheimer's disease (AD) and normal aging. Tau-1 immunoreactivity was observed in the white matter of all AD brains, in loosely dispersed neuropil threads (NT), a few neurofibrillary tangles (NFT) and scattered glial cells, whereas in majority of senile brains the white matter was immunonegative. Ubiquitin immunoreactivity characterized by dot-like structures, evenly distributed throughout the white matter, was observed in all cases examined being more prominent in AD than in senile brains. The dot-like structures were unrelated to tau-1 immunostaining pattern, as neither NT and NFT nor glial cells were ubiquitin labeled. It was concluded, that different immunostaining with both antibodies used reflects variable pathological changes identified mostly in nerve fibers. There are neurofibrillary changes manifested by tau-1 labeled NT. However, they differed from cortical NT by lack of ubiquitin immunostaining. Non-filamentous ubiquitin-positive depots represent presumably nonspecific nerve fiber changes related to various pathological events, including AD and aging process.

Muscle fiber degeneration in distal myopathy with rimmed vacuole formation.

In 11 patients with distal myopathy with rimmed vacuole formation (DMRV), a well-known autosomal recessively inherited disorder, the rimmed vacuole formation appears to be the main pathological change accounting for the progressive muscle fiber degeneration. To gain a better understanding of the pathophysiology of the vacuole formation, we applied Congo red and immunohistochemical stains to muscle biopsies from these patients and the results were compared with those of patients with inclusion body myositis (IBM). The vacuoles in DMRV contained Congophilic amyloid material and deposits immunoreactive for beta-amyloid protein, both the NH2 and COOH termini of beta-amyloid protein precursor, ubiquitin, and tau protein. These results were similar to those seen in our present cases of IBM as well as in previously reported cases. Therefore, there may be no pathogenetic differences in the formation of rimmed vacuoles in DMRV and IBM. Nevertheless, the degenerative process involved in rimmed vacuole formation in various diseases may share a common pathogenetic mechanism with that in amyloid-plaque formation in Alzheimer's disease brain as has been proposed previously.