The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.

The Ubiquitin System

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis

The crystal structure of the 20S proteasome from the yeast Saccharomyces cerevisiae shows that its 28 protein subunits are arranged as an (α1...α7, β1...β7)2 complex in four stacked rings and occupy unique locations. The interior of the particle, which harbours the active sites, is only accessible by some very narrow side entrances. The β-type subunits are synthesized as proproteins before being proteolytically processed for assembly into the particle. The proforms of three of the seven different β-type subunits, (β1/PRE3, β2/PUP1 and β5/PRE2, are cleaved between the threonine at position 1 and the last glycine of the pro-sequence, with release of the active-site residue Thr 1. These three β-type subunits have inhibitor-binding sites, indicating that PRE2 has a chymotrypsin-like and a trypsin-like activity and that PRE3 has peptidylglutamyl peptide hydrolytic specificity. Other β-type subunits are processed to an intermediate form, indicating that an additional nonspecific endopeptidase activity may exist which is important for peptide hydrolysis and for the generation of ligands for class I molecules of the major histocompatibility complex.

Structure of 20S proteasome from yeast at 2.4 A resolution.

▪ Abstract A growing number of cellular regulatory mechanisms are being linked to protein modification by the polypeptide ubiquitin. These include key transitions in the cell cycle, class I antigen processing, signal transduction pathways, and receptor-mediated endocytosis. In most, but not all, of these examples, ubiquitination of a protein leads to its degradation by the 26S proteasome. Following attachment of ubiquitin to a substrate and binding of the ubiquitinated protein to the proteasome, the bound substrate must be unfolded (and eventually deubiquitinated) and translocated through a narrow set of channels that leads to the proteasome interior, where the polypeptide is cleaved into short peptides. Protein ubiquitination and deubiquitination are both mediated by large enzyme families, and the proteasome itself comprises a family of related but functionally distinct particles. This diversity underlies both the high substrate specificity of the ubiquitin system and the variety of regulatory mechanisms...

Ubiquitin-dependent protein degradation

Mice Lacking p27Kip1 Display Increased Body Size, Multiple Organ Hyperplasia, Retinal Dysplasia, and Pituitary Tumors

Proteasomes degrade endogenous proteins. Two subunits, LMP-2 and LMP-7, are encoded in a region of the major histocompatibility complex (MHC) that is critical for class I-restricted antigen presentation. Mice with a targeted deletion of the gene encoding LMP-7 have reduced levels of MHC class I cell-surface expression and present the endogenous antigen HY inefficiently; addition of peptides to splenocytes deficient in LMP-7 restores wild-type class I expression levels. This demonstrates the involvement of LMP-7 in the MHC class I presentation pathway and suggests that LMP-7 functions as an integral part of the peptide supply machinery.

MHC class I expression in mice lacking the proteasome subunit LMP-7

In T-cell precursors, the T-cell-receptor beta chain is expressed before the T-cell-receptor alpha chain and is sufficient to advance T-cell development in the absence of T-cell receptor alpha chains. In immature T cells, the T-cell-receptor beta protein can form disulphide-linked heterodimers with the pre-T-cell-receptor alpha chain and associate with signal-transducing CD3 molecules. The recently cloned pre-T-cell-receptor alpha gene encodes a transmembrane protein that is expressed in immature but not mature T cells. Here we show that alpha beta, but not gamma delta, cell development is severely hampered in pre-T-cell-receptor alpha-gene-deficient mice, which establishes a crucial role for the pre-T-cell receptor in early thymocyte development.

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Crucial role of the pre-T-cell receptor alpha gene in development of alpha beta but not gamma delta T cells.

The T cell antigen receptor (TCR) beta chain regulates early T cell development in the absence of the TCR alpha chain. The developmentally controlled gene described here encodes the pre-TCR alpha (pT alpha) chain, which covalently associates with TCR beta and with the CD3 proteins forms a pre-TCR complex that transduces signals in immature thymocytes. Unlike the lambda 5 pre-B cell receptor protein, the pT alpha chain is a type I transmembrane protein whose cytoplasmic tail contains two potential phosphorylation sites and a Src homology 3 (SH3)-domain binding sequence. Pre-TCR alpha transfection experiments indicated that surface expression of the pre-TCR is controlled by additional developmentally regulated proteins. Identification of the pT alpha gene represents an essential step in the structure-function analysis of the pre-TCR complex.

/pdf/analysis-and-expression-of-a-cloned-pre-t-cell-receptor-gene-1a91flhk1g.pdf

Analysis and expression of a cloned pre-T cell receptor gene.

▪ Abstract The pre-T cell receptor (pre-TCR) that minimally consists of the TCRβ chain and the disulfide-linked pre-T cell receptor alpha (pTα) chain in association with signal-transducing CD3 molecules rescues from programmed cell death cells with productive TCRβ rearrangements. The pre-TCR induces expansion and differentiation of these cells such that they become TCRαβ bearing CD4+8+ thymocytes, which express only a single TCRβ chain and then either die of neglect or—upon TCR-ligand interaction—undergo either positive or negative selection. The newly discovered pTα gene encodes a transmembrane protein that belongs to the Ig superfamily and contains a cytoplasmic tail that, however, has no essential function in signal transduction, which is mediated by CD3 molecules and most likely p56lck. Experiments in pTα gene–deficient mice show that the pre-TCR has a crucial role in maturation as well as allelic exclusion of αβ T cells but is not required for the development of γδ-expressing cells. The function of t...

Structure and function of the pre-t cell receptor

A transgenic reporter mouse strain, which expressed the human CD25 (hCD25) surface marker as a reporter under the control of the pre-T cell receptor α(pTα) promoter, was used to identify lymphoid precursors that expressed pTα intracellularly. The hCD25 reporter marked intra- and extrathymic precursors of lymphocytes but not myeloid cells. The earliest intrathymic precursors were CD4loCD8−CD25−CD44+c-Kit+ cells that expressed elevated levels of Notch-1 mRNA. Clonogenic assays showed that the extrathymic precursors were common lymphoid progenitors (CLPs) that included CD19−, B220+, Thy1+ and CD4+ cells. Thus, the pTα reporter can be used to trace lymphopoiesis between CLPs and αβ T cells. The slower extinction of the hCD25 reporter compared to pTα enabled us to define points at which pTα− lineages branched off.

Fehling Hj

Papers

MHC class I expression in mice lacking the proteasome subunit LMP-7

Crucial role of the pre-T-cell receptor alpha gene in development of alpha beta but not gamma delta T cells.

Analysis and expression of a cloned pre-T cell receptor gene.

Structure and function of the pre-t cell receptor

Tracing lymphopoiesis with the aid of a pTalpha-controlled reporter gene.