A revised AMS and tephra chronology for the Late Middle to Early Upper Paleolithic occupations of Ortvale Klde, Republic of Georgia.

The nature and timing of the shift from the Late Middle Paleolithic (LMP) to the Early Upper Paleolithic (EUP) varied geographically, temporally, and substantively across the Near East and Eurasia; however, the result of this process was the archaeological disappearance of Middle Paleolithic technologies across the length and breadth of their geographic distribution. Ortvale Klde rockshelter (Republic of Georgia) contains the most detailed LMP-EUP archaeological sequence in the Caucasus, an environmentally and topographically diverse region situated between southwest Asia and Europe. Tephrochronological investigations at the site reveal volcanic ash (tephra) from various volcanic sources and provide a tephrostratigraphy for the site that will facilitate future correlations in the region. We correlate one of the cryptotephra layers to the large, caldera-forming Nemrut Formation eruption (30,000 years ago) from Nemrut volcano in Turkey. We integrate this tephrochronological constraint with new radiocarbon dates and published ages in an OxCal Bayesian age model to produce a revised chronology for the site. This model increases the ages for the end of the LMP (∼47.5-44.2 ka cal BP) and appearance of the EUP (∼46.7-43.6 ka cal BP) at Ortvale Klde, which are earlier than those currently reported for other sites in the Caucasus but similar to estimates for specific sites in southwest Asia and eastern Europe. These data, coupled with archaeological, stratigraphic, and taphonomic observations, suggest that at Ortvale Klde, (1) the appearance of EUP technologies of bone and stone has no technological roots in the preceding LMP, (2) a LMP population vacuum likely preceded the appearance of these EUP technologies, and (3) the systematic combination of tephra correlations and absolute dating chronologies promises to substantially improve our inter-regional understanding of this critical time interval of human evolution and the potential interconnectedness of hominins at different sites.

Aggregation and Prion-Inducing Properties of the G-Protein Gamma Subunit Ste18 are Regulated by Membrane Association.

Yeast prions and mnemons are respectively transmissible and non-transmissible self-perpetuating protein assemblies, frequently based on cross-ß ordered detergent-resistant aggregates (amyloids). Prions cause devastating diseases in mammals and control heritable traits in yeast. It was shown that the de novo formation of the prion form [PSI+] of yeast release factor Sup35 is facilitated by aggregates of other proteins. Here we explore the mechanism of the promotion of [PSI+] formation by Ste18, an evolutionarily conserved gamma subunit of a G-protein coupled receptor, a key player in responses to extracellular stimuli. Ste18 forms detergent-resistant aggregates, some of which are colocalized with de novo generated Sup35 aggregates. Membrane association of Ste18 is required for both Ste18 aggregation and [PSI+] induction, while functional interactions involved in signal transduction are not essential for these processes. This emphasizes the significance of a specific location for the nucleation of protein aggregation. In contrast to typical prions, Ste18 aggregates do not show a pattern of heritability. Our finding that Ste18 levels are regulated by the ubiquitin-proteasome system, in conjunction with the previously reported increase in Ste18 levels upon the exposure to mating pheromone, suggests that the concentration-dependent Ste18 aggregation may mediate a mnemon-like response to physiological stimuli.

Geochemical Evidence for the Control of Fire by Middle Palaeolithic Hominins.

The use of fire played an important role in the social and technological development of the genus Homo. Most archaeologists agree that this was a multi-stage process, beginning with the exploitation of natural fires and ending with the ability to create fire from scratch. Some have argued that in the Middle Palaeolithic (MP) hominin fire use was limited by the availability of fire in the landscape. Here, we present a record of the abundance of polycyclic aromatic hydrocarbons (PAHs), organic compounds that are produced during the combustion of organic material, from Lusakert Cave, a MP site in Armenia. We find no correlation between the abundance of light PAHs (3-4 rings), which are a major component of wildfire PAH emissions and are shown to disperse widely during fire events, and heavy PAHs (5-6 rings), which are a major component of particulate emissions of burned wood. Instead, we find heavy PAHs correlate with MP artifact density at the site. Given that hPAH abundance correlates with occupation intensity rather than lPAH abundance, we argue that MP hominins were able to control fire and utilize it regardless of the variability of fires in the environment. Together with other studies on MP fire use, these results suggest that the ability of hominins to manipulate fire independent of exploitation of wildfires was spatially variable in the MP and may have developed multiple times in the genus Homo.

Yeast Models for Amyloids and Prions: Environmental Modulation and Drug Discovery.

Amyloids are self-perpetuating protein aggregates causing neurodegenerative diseases in mammals. Prions are transmissible protein isoforms (usually of amyloid nature). Prion features were recently reported for various proteins involved in amyloid and neural inclusion disorders. Heritable yeast prions share molecular properties (and in the case of polyglutamines, amino acid composition) with human disease-related amyloids. Fundamental protein quality control pathways, including chaperones, the ubiquitin proteasome system and autophagy are highly conserved between yeast and human cells. Crucial cellular proteins and conditions influencing amyloids and prions were uncovered in the yeast model. The treatments available for neurodegenerative amyloid-associated diseases are few and their efficiency is limited. Yeast models of amyloid-related neurodegenerative diseases have become powerful tools for high-throughput screening for chemical compounds and FDA-approved drugs that reduce aggregation and toxicity of amyloids. Although some environmental agents have been linked to certain amyloid diseases, the molecular basis of their action remains unclear. Environmental stresses trigger amyloid formation and loss, acting either via influencing intracellular concentrations of the amyloidogenic proteins or via heterologous inducers of prions. Studies of environmental and physiological regulation of yeast prions open new possibilities for pharmacological intervention and/or prophylactic procedures aiming on common cellular systems rather than the properties of specific amyloids.

Dietary Supplementation With Cyplexinol for Amelioration of Osteoporosis: A Case Study.

A 50-y-old male in otherwise good health was diagnosed with severe osteoporosis with a lumbar spinal t score of -3.8. Further testing revealed no underlying causes other than a family history of the disease. He was placed on a trial regimen of 450 mg Cyplexinol twice daily for 4 mo. Repeat DEXA scans after 4 mo of this therapy showed an improved lumbar spinal t score of -3.3, the first time that improvement in t scores has been demonstrated in this short amount of time. Cyplexinol, the first orally consumable demineralized bone matrix consisting of a naturally derived bone morphogenetic protein complex, may be a beneficial alternative to conventional treatments for osteoporosis with an ability to reverse bone mineral density loss in as little as 4 mo.

Prion-based memory of heat stress in yeast.

Amyloids and amyloid-based prions are self-perpetuating protein aggregates which can spread by converting a normal protein of the same sequence into a prion form. They are associated with diseases in humans and mammals, and control heritable traits in yeast and other fungi. Some amyloids are implicated in biologically beneficial processes. As prion formation generates reproducible memory of a conformational change, prions can be considered as molecular memory devices.  We have demonstrated that in yeast, stress-inducible cytoskeleton-associated protein Lsb2 forms a metastable prion in response to high temperature. This prion promotes conversion of other proteins into prions and can persist in a fraction of cells for a significant number of cell generations after stress, thus maintaining the memory of stress in a population of surviving cells. Acquisition of an amino acid substitution required for Lsb2 to form a prion coincides with acquisition of increased thermotolerance in the evolution of Saccharomyces yeast. Thus the ability to form an Lsb2 prion in response to stress coincides with yeast adaptation to growth at higher temperatures. These findings intimately connect prion formation to the cellular response to environmental stresses.

Yeast Short-Lived Actin-Associated Protein Forms a Metastable Prion in Response to Thermal Stress.

Self-perpetuating ordered protein aggregates (amyloids and prions) are associated with a variety of neurodegenerative disorders. Although environmental agents have been linked to certain amyloid diseases, the molecular basis of their action remains unclear. We have employed endogenous yeast prions as a model system to study environmental control of amyloid formation. A short-lived actin-associated yeast protein Lsb2 can trigger prion formation by other proteins in a mode regulated by the cytoskeleton and ubiquitin-dependent processes. Here, we show that such a heterologous prion induction is due to the ability of Lsb2 to form a transient prion state, generated in response to thermal stress. Evolutionary acquisition of prion-inducing activity by Lsb2 is traced to a single amino acid change, coinciding with the acquisition of thermotolerance in the Saccharomyces yeast lineage. This raises the intriguing possibility that the transient prion formation could aid in functioning of Lsb2 at higher temperatures.

Prions, Chaperones, and Proteostasis in Yeast.

Prions are alternatively folded, self-perpetuating protein isoforms involved in a variety of biological and pathological processes. Yeast prions are protein-based heritable elements that serve as an excellent experimental system for studying prion biology. The propagation of yeast prions is controlled by the same Hsp104/70/40 chaperone machinery that is involved in the protection of yeast cells against proteotoxic stress. Ribosome-associated chaperones, proteolytic pathways, cellular quality-control compartments, and cytoskeletal networks influence prion formation, maintenance, and toxicity. Environmental stresses lead to asymmetric prion distribution in cell divisions. Chaperones and cytoskeletal proteins mediate this effect. Overall, this is an intimate relationship with the protein quality-control machinery of the cell, which enables prions to be maintained and reproduced. The presence of many of these same mechanisms in higher eukaryotes has implications for the diagnosis and treatment of mammalian amyloid diseases.

Middle Palaeolithic toolstone procurement behaviors at Lusakert Cave 1, Hrazdan valley, Armenia.

Strategies employed by Middle Palaeolithic hominins to acquire lithic raw materials often play key roles in assessing their movements through the landscape, relationships with neighboring groups, and cognitive abilities. It has been argued that a dependence on local resources is a widespread characteristic of the Middle Palaeolithic, but how such behaviors were manifested on the landscape remains unclear. Does an abundance of local toolstone reflect frequent encounters with different outcrops while foraging, or was a particular outcrop favored and preferentially quarried? This study examines such behaviors at a finer geospatial scale than is usually possible, allowing us to investigate hominin movements through the landscape surrounding Lusakert Cave 1 in Armenia. Using our newly developed approach to obsidian magnetic characterization, we test a series of hypotheses regarding the locations where hominins procured toolstone from a volcanic complex adjacent to the site. Our goal is to establish whether the cave's occupants procured local obsidian from preferred outcrops or quarries, secondary deposits of obsidian nodules along a river, or a variety of exposures as encountered while moving through the river valley or across the wider volcanic landscape during the course of foraging activities. As we demonstrate here, it is not the case that one particular outcrop or deposit attracted the cave occupants during the studied time intervals. Nor did they acquire obsidian at random across the landscape. Instead, our analyses support the hypothesis that these hominins collected obsidian from outcrops and exposures throughout the adjacent river valley, reflecting the spatial scale of their day-to-day foraging activities. The coincidence of such behaviors within the resource-rich river valley suggests efficient exploitation of a diverse biome during a time interval immediately preceding the Middle to Upper Palaeolithic "transition," the nature and timing of which has yet to be determined for the region.

Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates.

Sumoylation during genotoxic stress regulates the composition of DNA repair complexes. The yeast metalloprotease Wss1 clears chromatin-bound sumoylated proteins. Wss1 and its mammalian analog, DVC1/Spartan, belong to minigluzincins family of proteases. Wss1 proteolytic activity is regulated by a cysteine switch mechanism activated by chemical stress and/or DNA binding. Wss1 is required for cell survival following UV irradiation, the smt3-331 mutation and Camptothecin-induced formation of covalent topoisomerase 1 complexes (Top1cc). Wss1 forms a SUMO-specific ternary complex with the AAA ATPase Cdc48 and an adaptor, Doa1. Upon DNA damage Wss1/Cdc48/Doa1 is recruited to sumoylated targets and catalyzes SUMO chain extension through a newly recognized SUMO ligase activity. Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins. In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent. Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.

Quantitative analysis of protein-protein interactions.

Numerous authors, including contributors to this volume, have described methods to detect protein-protein interactions. Many of these approaches are now accessible to the inexperienced investigator thanks to core facilities and/or affordable instrumentation. This chapter discusses some common design considerations that are necessary to obtain valid measurements, as well as the assumptions and analytical methods that are relevant to the quantitation of these interactions.

Satsurblia: new insights of human response and survival across the Last Glacial Maximum in the southern Caucasus.

The region of western Georgia (Imereti) has been a major geographic corridor for human migrations during the Middle and Upper Palaeolithic (MP/UP). Knowledge of the MP and UP in this region, however, stems mostly from a small number of recent excavations at the sites of Ortvale Klde, Dzudzuana, Bondi, and Kotias Klde. These provide an absolute chronology for the Late MP and MP-UP transition, but only a partial perspective on the nature and timing of UP occupations, and limited data on how human groups in this region responded to the harsh climatic oscillations between 37,000-11,500 years before present. Here we report new UP archaeological sequences from fieldwork in Satsurblia cavein the same region. A series of living surfaces with combustion features, faunal remains, stone and bone tools, and ornaments provide new information about human occupations in this region (a) prior to the Last Glacial Maximum (LGM) at 25.5-24.4 ka cal. BP and (b) after the LGM at 17.9-16.2 ka cal. BP. The latter provides new evidence in the southern Caucasus for human occupation immediately after the LGM. The results of the campaigns in Satsurblia and Dzudzuana suggest that at present the most plausible scenario is one of a hiatus in the occupation of this region during the LGM (between 24.4-17.9 ka cal. BP). Analysis of the living surfaces at Satsurblia offers information about human activities such as the production and utilisation of lithics and bone tools, butchering, cooking and consumption of meat and wild cereals, the utilisation of fibers, and the use of certain woods. Microfaunal and palynological analyses point to fluctuations in the climate with consequent shifts in vegetation and the faunal spectrum not only before and after the LGM, but also during the two millennia following the end of the LGM.

Stress-dependent proteolytic processing of the actin assembly protein Lsb1 modulates a yeast prion.

Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI(+)] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI(+)] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI(+)] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments.

Analysis of collagen preservation in bones recovered in archaeological contexts using NIR Hyperspectral Imaging.

The scope of this article is to propose an innovative method based on Near Infrared Hyperspectral Chemical Imaging (NIR-HCI) to rapidly and non-destructively evaluate the relative degree of collagen preservation in bones recovered from archaeological contexts. This preliminary study has allowed the evaluation of the potential of the method using bone samples from the Early Upper Palaeolithic, Mesolithic and Neolithic periods at the site of Trou Al'Wesse in Belgium. NIR-HCI, combined with chemometric tools, has identified specific spectral bands characteristic of collagen. A chemometric model has been built using Partial Least Squares Discriminant Analysis (PLS-DA) to identify bones with and without collagen. This enables the evaluation of the degree of collagen preservation and homogeneity in bones within and between different strata, which has direct implications for archaeological applications (e.g., taphonomic analyses, assemblage integrity) and sample selection for subsequent analyses requiring collagen. Two archaeological applications are presented: comparison between sub-layers in an Early Upper Palaeolithic unit, and evaluation of the range of variability in collagen preservation within a single Holocene stratum.

Physiological and environmental control of yeast prions.

Prions are self-perpetuating protein isoforms that cause fatal and incurable neurodegenerative disease in mammals. Recent evidence indicates that a majority of human proteins involved in amyloid and neural inclusion disorders possess at least some prion properties. In lower eukaryotes, such as yeast, prions act as epigenetic elements, which increase phenotypic diversity by altering a range of cellular processes. While some yeast prions are clearly pathogenic, it is also postulated that prion formation could be beneficial in variable environmental conditions. Yeast and mammalian prions have similar molecular properties. Crucial cellular factors and conditions influencing prion formation and propagation were uncovered in the yeast models. Stress-related chaperones, protein quality control deposits, degradation pathways, and cytoskeletal networks control prion formation and propagation in yeast. Environmental stresses trigger prion formation and loss, supposedly acting via influencing intracellular concentrations of the prion-inducing proteins, and/or by localizing prionogenic proteins to the prion induction sites via heterologous ancillary helpers. Physiological and environmental modulation of yeast prions points to new opportunities for pharmacological intervention and/or prophylactic measures targeting general cellular systems rather than the properties of individual amyloids and prions.

Regulation of proteolysis by human deubiquitinating enzymes.

The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USPs) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

BAP1 is phosphorylated at serine 592 in S-phase following DNA damage.

The human BAP1 deubiquitinating enzyme is a chromatin-bound transcriptional regulator and tumor suppressor. BAP1 functions in suppressing cell proliferation, yet its role in the DNA damage response pathway is less understood. In this study we characterized DNA damage-induced phosphorylation of BAP1 at serine 592 (pS592) and the cellular outcomes of this modification. In contrast to the majority of BAP1, pS592-BAP1 is predominantly dissociated from chromatin. Our findings support a model whereby stress induced phosphorylation functions to displace BAP1 from specific promoters. We hypothesize that this regulates the transcription of a subset of genes involved in the response to DNA damage.

Two ZnF-UBP domains in isopeptidase T (USP5).

Human ubiquitin-specific cysteine protease 5 (USP5, also known as ISOT and isopeptidase T), an 835-residue multidomain enzyme, recycles ubiquitin by hydrolyzing isopeptide bonds in a variety of unanchored polyubiquitin substrates. Activation of the enzyme's hydrolytic activity toward ubiquitin-AMC (7-amino-4-methylcoumarin), a fluorogenic substrate, by the addition of free, unanchored monoubiquitin suggested an allosteric mechanism of activation by the ZnF-UBP domain (residues 163-291), which binds the substrate's unanchored diglycine carboxyl tail. By determining the structure of full-length USP5, we discovered the existence of a cryptic ZnF-UBP domain (residues 1-156), which was tightly bound to the catalytic core and was indispensable for catalytic activity. In contrast, the previously characterized ZnF-UBP domain did not contribute directly to the active site; a paucity of interactions suggested flexibility between these two domains consistent with an ability by the enzyme to hydrolyze a variety of different polyubiquitin chain linkages. Deletion of the known ZnF-UBP domain did not significantly affect rate of hydrolysis of ubiquitin-AMC and suggested that it is likely associated mainly with substrate targeting and specificity. Together, our findings show that USP5 uses multiple ZnF-UBP domains for substrate targeting and core catalytic function.