The Tissue-Specific Rep8/UBXD6 Tethers p97 to the Endoplasmic Reticulum Membrane for Degradation of Misfolded Proteins

The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded secretory proteins via the ER-associated degradation pathway. This functional diversity of p97 depends on its association with various cofactors, and to further our understanding of p97 function it is important that these cofactors are identified and analyzed. Here, we isolate and characterize the human protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8 is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p97 to the ER membrane for efficient ER-associated degradation

The potential of optical proteomic technologies to individualize prognosis and guide rational treatment for cancer patients

Genomics and proteomics will improve outcome prediction in cancer and have great potential to help in the discovery of unknown mechanisms of metastasis, ripe for therapeutic exploitation. Current methods of prognosis estimation rely on clinical data, anatomical staging and histopathological features. It is hoped that translational genomic and proteomic research will discriminate more accurately than is possible at present between patients with a good prognosis and those who carry a high risk of recurrence. Rational treatments, targeted to the specific molecular pathways of an individual’s high-risk tumor, are at the core of tailored therapy. The aim of targeted oncology is to select the right patient for the right drug at precisely the right point in their cancer journey. Optical proteomics uses advanced optical imaging technologies to quantify the activity states of and associations between signaling proteins by measuring energy transfer between fluorophores attached to specific proteins. Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) assays are suitable for use in cell line models of cancer, fresh human tissues and formalin-fixed paraffin-embedded tissue (FFPE). In animal models, dynamic deep tissue FLIM/FRET imaging of cancer cells in vivo is now also feasible. Analysis of protein expression and post-translational modifications such as phosphorylation and ubiquitination can be performed in cell lines and are remarkably efficiently in cancer tissue samples using tissue microarrays (TMAs). FRET assays can be performed to quantify protein-protein interactions within FFPE tissue, far beyond the spatial resolution conventionally associated with light or confocal laser microscopy. Multivariate optical parameters can be correlated with disease relapse for individual patients. FRET-FLIM assays allow rapid screening of target modifiers using high content drug screens. Specific protein-protein interactions conferring a poor prognosis identified by high content tissue screening will be perturbed with targeted therapeutics. Future targeted drugs will be identified using high content/throughput drug screens that are based on multivariate proteomic assays. Response to therapy at a molecular level can be monitored using these assays while the patient receives treatment: utilizing re-biopsy tumor tissue samples in the neoadjuvant setting or by examining surrogate tissues. These technologies will prove to be both prognostic of risk for individuals when applied to tumor tissue at first diagnosis and predictive of response to specifically selected targeted anticancer drugs. Advanced optical assays have great potential to be translated into real-life benefit for cancer patients

A study of the intermediate Early Bronze-Middle Bronze Age in Transjordan, Syria and Lebanon

The general homogeneity of artifacts of the Intermediate Early Bronze - Middle Bronze Age in Palestine has been acknowledged for some years, but the evidence for a relative chronology and typology of objects within the period is based largely on the contents of tombs. Since the surveys of Nelson Glueck in Transjordan it has been apparent that in this region there exist a very large number of EB.MB settlement sites with artifacts directly comparable to those of Contemporary Palestine. It was hoped therefore that some evidence for development within the period might be more readily discernible in Transjordan than in Palestine, where regional differences between cemeteries were sufficiently marked to make any chronological succession very difficult to establish. If the order of succession could be achieved in Transjordan, it would therefore elucidate some of the most difficult problems of chronology in Palestine, as well as shedding light on an area that was in itself insufficiently well known. Subsequently the scope of this research was widened to include all areas within which this homogeneous culture was discernible. This study is based therefore on the field work of several seasons in Syria, Lebanon and particularly Transjordan. It is primarily a collation of most aspects of archaeological evidence in the areas where objects typical of the EB.MB period have been found, and a discussion of the inferences which may be drawn from this evidence. The period is defined chronoigically c. 2350 - 1900 B.C., and at the outset the general basis of terminology and chronology accepted in this study are defined, as there are a multiplicity of terms currently in use. The first section attempts a very short summary of the literary and historical evidence from Mesopotamia, Egypt and Syria-Palestine which bears on the questions of the ethnic identity of the occupants of Palestine - Syria in the late third - early second millennia and what evidence there is for population movements. Although there is no direct historical solution to these problems, such historical evidence as there is must provide a framework within which the archaeological evidence can be more accurately assessed. The second section is concerned with the geographical evidence. The first topic is the limits of settlement of EB.MB peoples based on the evidence of archaeological survey, only in terms of people using artifacts directly comparable to those in Palestine. Sources and methods are described. The discussion which follows suggests topographical and climatic factors which appear to have influenced settlement at this time, and finally some tentative suggestions are made concerning political and social organization, and direction of settlement. Distribution of sites and the surface evidence suggest that new influences are most likely to have come from the north-east, penetrating Palestine and TransJordan by way of the major river valleys. Section three, dealing with the pottery, contains the weight of the evidence for the conclusions. It deals first with a description of the excavations and pottery from Tell Iktanu, which provided the basis for the pottery typology and relative chronology of the EB.MB in Transjordan. There follows a discussion of the comparative material from surface survey and excavation in Transjordan which suggests where this material should be placed in relation to regional and chronological variations. The main inferences which can be drawn from this pottery evidence are that much of the pottery is directly derived from that of the local EBA, and secondly that there are definable "innovating features" as opposed to mere typological developments from EBA to EB.MB; and that therefore if the origins of these innovations can be traced, a clear case for the origins of the new population element which intermingled with that of the EBA should also be indicated. A brief survey of the contemporary archaeology of areas to the north indicates that apart from the south Lebanon cemeteries no direct contacts for either EB.MB pottery or the "innovating features" are found in Lebanon. The central Syrian pottery shows greater complexity, but the conclusion is that a sequence Kama K - Kama J - Qatna Tomb IV -r Hama H excludes the "innovating features" observed in TransJordan, and that contemporary Hama J develops from three major urban traditions a) late Early Dynastic III of north Syria indicated mainly in Hama J8-7, b) Sargonid of north Syria from Hama J7 onwards, and c) Levantine EBA throughout. The direct links for the "innovating features", dated c.2350 B.C. in Transjordan, are found in the ED III traditions of the central Euphrates region, from Mari, Til Barsip, Harran, Amarna and Hammam, and it is concluded that people who were associated, but not perhaps directly part of that urban tradition, migrated south from their homelands following the campaigns of Lugalzagesi, the Akkadian expansion in north Mesopotamia and campaigns of Sargon. A secondary influence is traced c.2100-2000 B.C. affecting Palestine more considerably than TransJordan, of which the origins are not clearly discernible, but which are probably due to new influences rather than influxes of new people. The degree of continuity in population and pottery typology is however stressed for the whole period. Sections four and five deal with the stone and metal work. The flints in particular indicate continuity of "Canaanean" traditions from EBA through EB.MB to MBA. There are not many metal objects on which to base a relative typology, but it is also suggested that throughout the period the pin types in particular indicate greater continuity of early inland Syrian traditions than has previously been recognised. Section six shows that there is considerable diversity of grave types, but again tentatively suggests there is a clear, and probably early, north Syrian influence on a basically EBA tradition, and concludes by suggesting that traditions of burial are one of the very few factors by which the assimilation of the EB.MB population in MBA society post-1900 B.C. can be detected archaeologically. Section seven is concerned with the evidence of habitation. It is generally agreed that the EB.MB period is one of nomadic or semi-nomadic occupation, but in some respects the evidence for sedentary occupation has been denigrated. The evidence for both ways of life is reviewed and it is concluded that there are two major processes involved, i) the decline of EBA urban civilization which nonetheless has a lingering influence in the EB.MB period and ii) the introduction of a semi-nomadic element in the population which gradually shows a tendency to settle permanently in fertile areas. In the concluding chapter a few other aspects of the EB.MB period are discussed, including the suggestion that cattle played an important part in pastoralism, and that there was sufficient agriculture to indicate not more than partial nomadism. A brief summary of other views of typology and the origins of the people of the EB.MB period is then followed by conclusions based on the evidence of the previous chapters. These conclusions are that c. 2350 B.C. semi-nomadic people arrived in Transjordan from north Syria and intermingled with the probably West Semitic EB III population. These newcomers may also have been West Semites, but there is no definite evidence that they were Amorites though the evidence does not exclude the possibility. This population remained largely unchanged, and in considerable isolation, until c. 1900 B.C. There are new influences apparent c. 2100 B.C. but the basic continuity of artifacts and social order is clear, and the changes probably do not indicate a large influx of new people. At the end of the archaeological period, this rather primitively organized society was destroyed by new urban traditions brougjht by fresh influxes of people from Syria, which probably absorbed the older population in the building of new towns.</p