During the period of my active research, I witnessed the introduction of new techniques and interdisciplinary approaches, which resulted in completely novel views of the structure and function of the extracellular matrix (ECM), which at this time was still called connective tissue. I memorize my shock and surprise when about 15 years ago my friend and colleague Darwin Prockop smuggled a few much older slides into an at this time modern research seminar, which I presented at his department. These slides originated from the time, when Darwin Prockop and I first met at one of the first ECM meeting in St. Margherita in 1966. The difference between the new research and the historical slides dramatically demonstrated the high pace of research. With these thoughts in mind I decided to write a chapter on the history of ECM research, which I experienced as a member of a group of scientists located in many different countries and disciplines, who all intensely collaborated with a pioneer in the field, Rupert Timpl. My personal review deals with the turn from a purely mechanical role of the connective tissue to the understanding of the importance of the ECM in cell differentiation and development. The discovery and characterization of a large repertoire of complex multifunctional proteins involved as major players in these events is described. Rupert passed away in October 2003 and this chapter is also a memorial to him.
Viktor Mutt was born on December 29, 1923, in Kidjärve, Estonia, and died on September 9, 1998, in Stockholm, Sweden. He came to Sweden via Finland as a refugee during the World War II and passed his thesis at Karolinska Institutet on the purification of secretin in 1959. So began a career that resulted in the discovery of over 50 naturally occurring biologically active peptides, including secretin, cholecystokinin, VIP, NPY, PYY, and many others. His scientific approach, which depended on the extraction of animal tissues on an industrial scale, permitted him to discover peptides present in low abundance, including hormone precursors and processing intermediates that could not have been elucidated by any other means with the technology available at the time. With him the era of large-scale purification from natural sources peaked and is now coming to an end, replaced by recombinant protein production. Viktor remained active to the end of his life, and only now, as this memorial is being written, have the many projects he directly initiated finally come to an end. His life was that of a devoted and successful scientist, a kind and benevolent man worthy to commemorate.
After studying at the Ludwig-Maximilian University in Munich and working for a PhD at Purdue University, USA, I started to do research in Munich on aspects of photophosphorylation and on the biosynthesis of raffinose type oligosaccharides. Later in Regensburg this led into detailed studies of an inducible active transport system of a eukaryotic cell (hexose/H+-symporter of Chlorella) as well as to the discovery of dolichol-phosphate-activated sugars and their role in glycoprotein biosynthesis. An excursion into the controversial field of plant transpiration and one into lateral plasma membrane compartmentation is included. Diversions from research by various administrative obligations are admitted.
Jacques Monod, a prominent figure of the golden age of Molecular Biology, is well known for his contributions to understanding the regulation of gene expression (the “operon” model) and the regulation of enzyme activities (the “allostery” model). Few people are aware of his deep interest in the mechanisms of protein folding, which he foresaw would become a problem of major importance. The author of the present chapter describes the meandring pathway which led him to join the laboratory of Jacques Monod and the way in which Jacques Monod triggered his interest for protein folding, inspired his first discoveries and influenced many of his contributions to an emerging area of research which now stands at the forefront of biological research.
Born 1932, I was brought up in Stettin. I was trained as an organic chemist at Cologne University, before I entered the Institute of Genetics there in 1962 to work on tRNA structure and function. From 1967 to my retirement in 1997 I worked at the Adolf-Butenandt-Institute (Munich University) on several aspects of molecular biology of the budding yeast, for example tRNA genes, Ty elements, the yeast genome project, and programmed proteolysis. I include facets of my engagement in national and international organizations.
The Table of Contents consists of personal recollections, historical and autobiographical perspectives of leading individuals who reflect on their careers and their impact on biochemistry, including Laurens L.M. van Deenen and Viktor Mutt.
I became a scientist in 1959 and continue to pursue that profession to this day. All these years I have worked at the same institute of the Academy of Sciences. Today the Institute of Molecular Biology is named after the great biochemist who was my teacher, Wladimir Engelhardt. The key words in my areas of research in biochemistry and molecular biology are, I would say, transfer RNAs, aminoacyl-tRNA synthetases, termination of translation in eukaryotes, reverse transcription, and tumor suppressor genes. The reader may not read every page in this chapter. He or she will gain an impression, nevertheless, of how biochemistry and molecular biology have developed and evolved during the last half century in the USSR and then in the Russian Federation. The brief description of a lifetime of scientific activities is supplemented by a quite extensive list of my published works.
The authors describe their lives as a scientist couple, extending over more than 40 years. They worked in related, but not identical fields of molecular cell biology and biomedicine, in many different places, and raised two sons. Although this life demanded frequent compromises, they consider this as a gain rather than a loss and conclude that they were granted a privileged life.
I was born in Stockton, California, U.S.A. in 1920 and soon went to Germany and lived in Berlin for about 2 years. Thereafter, I came back to Japan and graduated from Osaka University School of Medicine in 1942. After serving in the Japanese Navy as a medical officer, I returned to Osaka University to start my career as microbiologist. By the use of enrichment technique, I was able to isolate from the scorched soil, a strain of soil microorganism Pseudomonas that was able to grow on tryptophan as a sole source of carbon and nitrogen. Unlike in animals in which tryptophan is degraded to anthranilate and other intermediate metabolites and secreted in the urine, in this pseudomonas anthranilate was degraded to catechol and muconic acid, ultimately to CO2, NH3 and H2O. Furthermore, I was able to isolate a novel enzyme that catalyzed the oxidative cleavage of the aromatic structure of catechol yielding cis, cis-muconic acid as the reaction product. Molecular oxygen was obsolutely required for the reaction and could not be replaced by the known electron acceptor dyes or coenzymes. This finding together with various other properties of this enzyme indicated strongly the possibility that it could be an oxygen transferase rather than an oxidase. Therefore, it was named as “pyrocatechase” rather than catechol oxidase. However, the enzymatic incorporation of molecular oxygen into substrates had been completely ruled out in biological systems by the famous “dehydrogenation theory” by Heinrich Wieland, and my interpretation was not immediately accepted. In 1949, I went to U.S.A. as a postdoctoral fellow and then in 1954 became Chief of Section on Toxicology, NIH, Bethesda. In 1955, I was able to show by the use of 18O, a stable isotope of oxygen, that the oxygen atoms incorporated into the product, muconic acid, was completely derived from molecular oxygen and not from H2O. Thus, I proposed to name such an enzyme as “oxygenases.” In 1958, I came back from U.S.A. to become professor and chairman of the Department to Medical Chemistry, Kyoto University School of Medicine. Thereafter, I extended my work on tryptophan metabolism and oxygenases for almost 25 years. After retirement from Kyoto University, I switched my field of interest to the elucidation of the molecular mechanisms of sleep wake regulation and found that prostaglandin D2 is a sleep hormone which is involved in the regulation of both circadian and homeostatic regulation of non-REM sleep.
Research on the structural changes in glycerides associated with temperature-dependent phase changes in these lipids led Dennis Chapman onto the path of defining the dynamic behaviour of cell membranes. He developed the concept of membrane fluidity in which the lipid matrix of the membrane, represented by a bilayer of polar lipids, allowed diffusion of proteins and other membrane components in the plane of the membrane. It was also recognized that membrane fluidity could be modulated by cholesterol leading to the contemporary views of lateral phase separations and membrane rafts. He invented methods of mimicking membranes to coat prosthetic devices so as to render them haemocompatible. Dennis Chapman's career spanned both industrial and academic service but with a singular aim of transforming his scientific discoveries to the benefit of mankind.
Brief description of scientific life of Russian biochemist G.I. Abelev in tumor immunochemistry. Main attention is devoted to develop the system of analysis of complex system on the level of individual antigens. The discovery of alpha-fetoprotein and its use as tumor marker is described.
The contribution of Jakub Karol Parnas (1884–1949), a prominent Polish scientist, to the understanding of muscle biochemistry, including ammonia and carbohydrates sources and nucleotide metabolism, is described. Among his main achievements was the discovery of glycogen phosphorolysis, the first use of radioactive phosphorus in biological studies, and formulation and proof of phosphate transfer between glycolytic intermediates and ATP. The rewarding and successful life led by this man of great scientific and intellectual abilities up to the beginning of the Second World War, his dramatic fate during the war, and his tragic death in a Soviet prison reflects the turbulent history of the region for the 20th century.
The title I have chosen for my personal recollections describes, in a nutshell, the direction my scientific endeavors from the time of my Ph.D. thesis, which I began in 1970, to the present day. Over the years, I have changed fields a number of times. There were periods when I was preoccupied with the development of methods; at other times, the focus was on biological problems. Science can be advanced by new hypotheses about how things work, which can be tested and proven right or wrong, and by new methods which enable us to tackle questions that we were unable to address with the existing methods. Or, as Richard Feynman put it, "Science means, sometimes, a special method of finding things out. Sometimes it means the body of knowledge arising from the things found out. It may also mean the new things you can do when you have found something out, or the actual doing of new things. This last field is usually called technology…." (R.P. Feynman in the John Danz Lectures, 1963 [Feynman 1998]). After graduating from the University of Bonn in late 1969, I joined the Institute of Biophysics and Electron Microscopy at the University of Düsseldorf in January 1970. The director of the Institute at the time was Helmut Ruska, who became my Ph.D. supervisor. Helmut Ruska, a medical doctor, was the younger brother of Ernst Ruska, the electrical engineer who, in 1932, at the age of 26, had published his calculations on the theoretical resolving power of an electron microscope and, in the face of strong skepticism, had completed the development of a commercial instrument by 1939 (Ruska 1979). Rarely has a scientific instrument had such an impact on so many branches of science, and yet it took more than 50 years before Ernst Ruska was rewarded with the Nobel Prize in Physics in 1986 for his fundamental work in electron optics and his design of the first electron microscope. Helmut Ruska, who was very close to his brother, realized immediately the potential of such an instrument for the biomedical sciences, in particular the visualization of hitherto invisible infectious agents and for ultrastructural studies of cells (Fig. 1). Helmut Ruska played a very important role in the early days of electron microscopy, not only by raising awareness and support—his clinical mentor at the Charité in Berlin, Richard Siebeck, became a decisive advocate at a critical time—but also by his achievements in the visualization of viruses, bacteria, and blood cells (for review, see Kruger et al. 2000; for relevant references, see also Ruska 1979). At the time I joined Helmut Ruska's laboratory, the performance of transmission electron microscopes had reached a level that allowed the imaging of single heavy atoms. Several groups in Europe, the United States, and Japan tried to take advantage of this capability and to use heavy atoms as site-specific labels, e.g., for mapping the bases in strands of DNA. In the same vein, Helmut Ruska gave me the task of exploring the use of heavy atom labels to study membrane topology. I decided to begin with well-defined model membranes before tackling membranes of biological relevance. I never got that far! Using Langmuir–Blodgett techniques, I prepared monomolecular layers at the water–air interface and transferred them under precisely controlled conditions to specimen supports, but when I exposed my carefully designed lipid layers to the electron beam, they faded away before I was able to take a picture. Occasionally, I obtained images of remnants of them with the heavy atoms coalesced into clusters. Eventually, with an unusually radiation-resistant organometallic compound of no relevance to biology, thorium-hexafluoracetylacetonate, I succeeded in obtaining images showing a heavy atom pattern that was consistent with my design plan (Baumeister and Hahn 1972). Helmut Ruska was preoccupied with administrative duties during my time as a graduate student in his laboratory and, as a consequence, his supervision of me was very casual. Nevertheless, he was very supportive and he gave me all the resources I needed for my work. In late August 1973, only a few months after receiving my Ph.D., Helmut Ruska died after a short illness. I had offers from other places, but decided to stay in Düsseldorf, and since it took several years until a successor for Helmut Ruska was found, I enjoyed complete freedom during my postdoctoral years. Thanks to benevolent reviewers, I obtained my first grant in 1974 and I began to work on radiation damage—the electron microscopist's greatest foe. I used a variety of methods for a quantitative assessment of radiation damage in lipids and proteins under the conditions encountered in electron microscopy (Baumeister et al. 1976; Hahn et al. 1976). My hope was that a better understanding of the underlying radiation chemistry might enable us to find a remedy—a vain hope as it turned out (Baumeister 1978). Having realized that I was on an unproductive path, I had to change direction. While the electron microscopy community in Germany with its strong tradition in electron optics was preoccupied with "high resolution," others, driven more strongly by their desire to obtain insights into biomolecular architectures, took more pragmatic approaches. Already in 1968, De Rosier and Klug had formulated the principles for the three-dimensional reconstruction of objects from projection images and applied them to the tail of bacteriophage T4, taking advantage of its helical symmetry (De Rosier and Klug 1968). When working with periodic or repetitive structures, one can minimize radiation damage by underexposing the samples; the information is retrieved from the statistically noisy images by averaging over many identical structures. Averaging can be performed by direct superposition (see, e.g., Markham et al. 1964) or by Fourier transform filtering (see, e.g., De Rosier and Klug 1972) using both optical and digital methods (Aebi et al. 1973). Using the aforementioned stratagem and applying it to unstained, glucose-embedded purple membrane, Henderson and Un-win succeeded in 1975 in obtaining a 7 Å structure of bacteriorhodopsin (Henderson and Unwin 1975), which became the paradigm of a membrane protein structure. Having read about a bacterium of legendary radiation resistance, Micrococcus radiodurans (now Deinococcus radiodurans), and knowing from the literature that a regular protein layer was a component of its cell wall, I focused my work on this structure. Before long, I obtained decent micrographs of this structure (Fig. 2A), which I called the hexagonally-packed-intermediate (HPI)-layer, but I ran into a dilemma with the image processing. After having done some initial experiments with optical filtration, I became convinced that computer methods were the future; however, with the notable exception of Walter Hoppe in Munich, optical methods were preferred to computer methods in Germany at the time. The arguments in favor of optical image processing (averaging and correction of contrast transfer function) were the size of the images that could be processed and the speed. The downside was lack of flexibility, and the fabrication of suitable masks became a serious bottleneck. Since I had neither access to the necessary infrastructure nor the know-how for computer-based processing, I started a collaboration with Olaf Kübler at the ETH in Zürich who was in an inverse position: He had the software and the hardware that was needed but no data. In the following years, we made substantial progress in elucidating the molecular architecture of the D. radiodurans cell envelope (Baumeister and Kübler 1978; Kübler and Baumeister 1978; Baumeister et al. 1981, 1982). In 1979, I organized a meeting entitled "Electron Microscopy at Molecular Dimensions" held at Burg Gemen near Münster, Germany, which in retrospect became quite influential (see, e.g., Deisenhofer and Michel 1989). Besides state-of-the-art applications, it covered many developments in technology from image recording and processing to low-temperature electron microscopy (EM), or strategies for making regular 2-D arrays (Baumeister and Vogell 1980). The intrinsic disorder in the 2-D protein arrays limits the resolution one can attain by Fourier filtering, and during the Gemen meeting, I became convinced that there are better ways of dealing with imperfect 2-D crystals. The emerging methods for averaging of single molecules offered an alternative, and I decided to join forces with Joachim Frank. A few months later when I visited him in Albany, we explored the application of correlation-based averaging to the micrographs of the HPI-layer, but to our disappointment we failed to obtain meaningful results during this short period of time. A year later, when I spent several months at the Cavendish Laboratory in Cambridge, England, working with Owen Saxton, we were able to overcome the problems and obtained the first correlation-averaged images of the HPI-layer with a significantly improved resolution. In trying to get this work published, we faced unusual problems; it took several rounds of reviewing and several steps down the ladder of (journal) prestige, until our manuscript was finally published (Saxton and Baumeister 1982). In retrospect, however, it is gratifying to see that more than 20 years later, this paper is still cited frequently and, in the guise of "lattice unbending" (Baldwin et al. 1988) our strategem for overcoming the limitations due to lattice disorder became part of the standard repertoire used for processing images of 2-D crystals. Shortly thereafter, we applied correlation averaging to Scanning Transmission Electron Microscopy (STEM) images of unstained preparations of the HPI-layer and obtained the first quantitative mass maps (Engel et al. 1982), the beginning of a long-standing and successful collaboration with Andreas Engel at the Biocenter in Basel, Switzerland. At the beginning of 1982, I moved to the Max-Planck-Institute of Biochemistry where, after a short overlap period, I was appointed successor of Walter Hoppe. Hoppe was a microscopist-turned-X-ray crystallographer of remarkable originality and theoretical ability but with limited interest in the practical aspects of structural biology. No humble man, he insisted that his "nonconventional" approach to the structural analysis of individual macromolecules, as he liked to call it, was superior to other strategies (for review, see Hoppe 1983). I remained unconvinced, and felt that the clever tactics of "single particle analysis" as pioneered by Joachim Frank, a former student of Hoppe, and Marin van Heel at the Fritz-Haber-Institute in Berlin held greater promise. Not only did their approach greatly simplify data acquisition, the combination of intelligent image classification procedures and extensive averaging had the great advantage of yielding significant and interpretable structural data (for a recent review, see Frank 2002). In all fairness, I must add that in spite of a fierce public dispute I had with Walter Hoppe a few years earlier (Baumeister and Hahn 1975; Hoppe et al. 1975) and divergent views on the course to take, he was, in general, supportive when I arrived in Martinsried and began to set up my laboratory. We continued our work with the HPI-layer; a 3-D model was generated in due course and, using cryomicroscopy, an 8 Å projection map was also obtained (Baumeister et al. 1986; Rachel et al. 1986; Fig. 2B). With the plentiful resources now at our disposal, we not only extended our structural studies to several other bacterial surface layers, we also widened our repertoire of methods. Our comparative structural studies revealed some common architectural principles (Baumeister et al. 1986, 1988) and sequence analyses led to the identification of new motifs (Peters et al. 1987, 1989; Lupas et al. 1994) such as the S-layer homology domain (for a recent review, see Engelhardt and Peters 1998) but the biological function of Slayers remained an enigma. Intuitively, I still feel that there must be some function beyond mediating adhesion to animate or inanimate surfaces or protecting underlying components of the cell envelope, but this remains pure speculation. Colleagues in Martinsried (Wolfram Zillig) and in Regensburg (Karl-Otto Stetter) introduced me to the exciting world of extremophiles. Most hyperthermophiles belong to the archaeal domain of life where (glyco)protein surface layers are common. They represent the main macromolecular component of the cell envelope and are intimately associated with the plasma membrane. Some show a high degree of order and have a role in maintaining and possibly determining cell shape (Wildhaber and Baumeister 1987; Phipps et al. 1991b) while others form poorly ordered and flexible surface networks on pleomorphic cells (Wildhaber et al. 1987; Peters et al. 1995). In spite of their apparent diversity, archaeal surface layers have some common structural principles: A stalk of variable length (10–70 nm) emanates from a membrane-anchoring domain and connects to a highly variable (filiform or bulky) domain that forms a canopy-like layer by means of end-to-end contacts enclosing a quasi-periplasmic space (Baumeister and Lembcke 1992). A periplasmic space of unusual width and maintained by a rod-shaped spacer protein (Omp α) is also found in the hyperthermophilic ancestral bacterium Thermotoga maritima (Engel et al. 1992; Lupas et al. 1995). The structural principles of archaeal surface layer proteins is exemplified particularly clearly by tetrabrachion, the giant glycoprotein found on the surface of Staphylothermus marinus, where it forms a poorly ordered, branched network (Peters et al. 1995). This filiform molecule is anchored in the cell membrane at the C-terminal end of a 70-nm-long stalk and branches at the other end into four arms, each of 24 nm length, which form the canopy-like meshwork. A hybrid approach, which used EM and biochemical data as well as molecular biology and bioinformatics, led to a very detailed model structure (Fig. 3; Peters et al. 1996), the salient features of which, in the meantime, have been confirmed by X-ray crystallography (Stetefeld et al. 2000). The C-terminal part is formed by a right-handed, coiled coil of four α-helices; the almost flawless pattern of aliphatic residues, mainly leucine and isoleucine, throughout the hydrophobic core of the stalk provides an explanation for its exceptional stability. At a proline residue, the stalk switches from a right-handed supercoil to a left-handed one. At a flexible glycine-rich hinge region, the stalk branches into four arms, each formed by a "heavy chain" and a "light chain", which in turn are each derived from the translated 1524-residue polypeptide by internal proteolytic cleavage. The most likely topology of the arms is a three-stranded coil of antiparallel β-sheets. There is a patch of negative charges on the outer face of the coiled coil near the middle of the stalk, which serves as an anchoring device for a large, hyperthermostable protease of the subtilisin family; in the stalk-bound form the protease is resistant to heat inactivation up to a temperature of 125°C (Mayr et al. 1996), while the stalk withstands heating up to 130°C. Obviously, one function of the Staphylothermus surface layer is to provide an extracellular holding compartment for a protease that could otherwise cause havoc. In 1989, my laboratory became interested in studying the structure and function of a large (20S) protein complex, at the time known as the multicatalytic proteinase (Dahlmann et al. 1989). Already in 1980, a large, multisubunit protease had been isolated and characterized (Hase et al. 1980; Wilk and Orlowski 1980; Orlowski and Wilk 1981). Initially, the multicatalytic proteinase was believed to be composed of 3–5 subunits, ranging from 24 kDa to 28 kDa in size; it displayed three distinct proteolytic activities (trypsin-like, chymotrypsin-like, and peptidylglutamylpeptide-hydrolyzing) when assays were performed with small synthetic peptides, and it was noted that the integrity of the 20S complex was essential for all proteolytic activities. Attempts were made to assign specific activities to distinct subunits, but in spite of the efforts of many groups, the nature of the active sites remained enigmatic. Along a different line, a particle named "prosome" was under intensive investigation in the mid-1980's (for review, see Scherrer et al. 1990). Reminiscent in size and subunit composition of the multicatalytic protease complex, it appeared to be associated with RNA and it was suggested to have a role in the regulation of gene expression. In 1988, it was established beyond doubt that the prosome and the multicatalytic proteinase complex were one and the same particle (Arrigo et al. 1988; Falkenburg et al. 1988) and the name "proteasome" was coined by Alfred L. Goldberg (Harvard Medical School) to highlight its only established function, the proteolytic one, and its complex structure. In the following years, evidence began to accumulate that the 20S proteasome was part of an even larger complex, the 26S proteasome, which was implicated in the ATP-dependent degradation of ubiquitin-conjugated proteins (Eytan et al. 1989; Driscoll and Goldberg 1990; Rechsteiner et al. 1993). By 1990, the 20S proteasome was structurally rather featureless and its subunit composition and stoichiometry were ill-defined. Reports that proteasomes could undergo changes in subunit composition during development (Haass and Kloetzel 1989) made its structural analysis a daunting challenge, since structural methods rely, in one guise or another, on averaging and, therefore, on homogeneous preparations of molecules. This led us to search for proteasomes of hopefully simpler subunit composition in prokaryotic cells. While our initial attempts to find proteasomes in bacteria were unsuccessful, we found them in the archaeon Thermoplasma acidophilum (Dahlmann et al. 1989). The Thermoplasma proteasome turned out to be very similar in size and shape to proteasomes from eukaryotic cells, but much simpler in subunit composition; it comprises only two subunits, α (25.8 kDa) and β (22.3 kDa). The two subunits have significant sequence similarity, suggesting that they arose from a common ancestor via gene duplication (Zwickl et al. 1991, 1992a). Due to its relative simplicity, the ensuing years saw the Thermoplasma proteasome play a pivotal role in elucidating the structure and enzymatic mechanism of this intriguing protein degradation machine. In 1991, a first, three-dimensional structure of the Thermoplasma proteasome was obtained by EM single particle analysis, showing with remarkable clarity the organization of the barrel-shaped complex with its tripartite inner compartment (Hegerl et al. 1991). Immunoelectron microscopy studies allowed us to assign the α-subunits to the two outer rings of the barrel, and the β-subunits to the inner rings (Grziwa et al. 1991). Mass measurements by STEM helped us to establish the stoichiometry (α7β7β7α7), and metal decoration studies of proteasome crystals (not yet good enough for high resolution X-ray crystallography) clearly revealed the symmetry of the 20S complex. The structural model we put forward on the basis of these data stood the test of time and it recurred in all proteasomes, eukaryotic and prokaryotic (Pühler et al. 1992). Another important advance was the expression of fully assembled and functional 20S proteasomes in Escherichia coli (Zwickl et al. 1992b; Fig. 4A). It not only allowed us to perform systematic mutagenesis studies aimed at identifying the active site, it also greatly facilitated the growth of crystals diffracting to high resolution (Jap et al. 1993). In 1995, the crystal structure analysis was completed in a collaboration with the group of Robert Huber (Löwe et al. 1995; Fig. 4B). The long-sought catalytic nucleophile of the 20S proteasome, the N-terminal threonine of the mature β-subunit was identified independently and almost simultaneously by site-directed mutagenesis and crystal structure analysis (Löwe et al. 1995; Seemüller et al. 1995). As anticipated from their sequence similarity the (noncatalytic) α-and the (catalytic) β-type subunits showed the same fold: a four-layer α + β structure with two antiparallel five-stranded β-sheets, flanked on one side by two, and on the other side by three α-helices. In the β-type subunits, the β-sheet sandwich is closed at one end by four hairpin loops and opens at the opposite end to form the active-site cleft; the cleft is oriented toward the inner surface of the central cavity. In the α-type subunits, an additional helix formed by an N-terminal extension crosses the top of the β-sheet sandwich and fills this cleft. Initially, the proteasome fold was believed to be unique; however, it turned out to be common to a new superfamily of proteins referred to as Ntn (N-terminal nucleophile) hydrolases (Brannigan et al. 1995). Beyond the common fold, members of this family share the mechanisms of the nucleophilic attack and self-processing (for reviews, see Baumeister et al. 1998; Dodson and Wlodawer 1998; Seemüller et al. 2001; Zwickl et al. 2002). The crystal structure revealed that access to the inner cavity that harbors the active sites is controlled by four constrictions. The constrictions in the α-rings which give access to the two "antechambers" are narrow and partially obstructed, while the constrictions which regulate access to the central cavity are wider. We were able to show with Nanogold-labeled substrates, visible in electron micrographs, that polypeptides indeed enter the proteasome via the orifice at the center of the α-rings. Bulky additions to the polypeptide chain, such as a gold cluster, prevent passage into the interior, suggesting that the discrimination between folded and unfolded substrates is based on a size-exclusion mechanism (Wenzel and Baumeister 1995). Thus the 20S proteasome is a molecular nano-compartment that confines the proteolytic reaction to its interior and sequesters it from the crowded environment of the cell. Interestingly, formation of the active sites by the posttranslational removal of the propeptides of the β-subunits (Seemüller et al. 1996) is coupled to the assembly of the 20S proteasome in such a manner that activation is delayed until the assembly is complete (for review, see Seemüller et al. 2001). This led us to propose the concept of self-compartmentalization as a regulatory principle (Lupas et al. 1997; Baumeister et al. 1998). As mentioned earlier, it began to transpire in the early 1990s that the 20S proteasome of eukaryotes associates with regulatory complexes, in an ATP-dependent manner, to form the 26S proteasome. Now it is firmly established that this 2.5 MDa complex altogether comprising more than 30 different subunits acts downstream in the ubiquitin–proteasome pathway and is the central player in intracellular proteolysis. Proteins destined for degradation are marked by covalent attachment of Ub chains, which mediate recognition by the 26S proteasome (for recent reviews, see Hershko and Ciechanover 1998; Voges et al. 1999). In 1993, we were able to provide the first detailed description of the 26S complex, based on electron microscopy and image analysis (Peters et al. 1993). The averages showing the regulatory (19S) particles attached to one or both ends of the 20S proteasome core particle (the "dragon-head" or "double dragon-head" motif) became the classical textbook images of the 26S proteasome. Since then, however, progress has been embarrassingly slow; the notorious instability of the complex and its dynamics have made it very difficult to achieve more than gradual improvements of the structural model (Glickmann et al. 1998; Walz et al. 1998; Hölzl et al. 2000). While it is clear that the role of the 19S regulatory complexes is the preparation of substrates for degradation in the 20S core particle—involving the recognition of ubiquitinated substrates, the removal of the polyubiquitin chains, the unfolding of substrates, and assistance in translocation across the gates of the 20S complex—the precise topology and role of the 19S subunits is hitherto only dimly understood (Zwickl et al. 1999). In 1991, we found, in a serendipitous manner, a novel ATPase complex. During the lysis of accidentally heat-shocked Pyridictium cells on electron microscopy grids, a massive release of toroidal particles composed of the stacked octameric rings was observed (Phipps et al. 1991a). Not only the shape, but also the heat-shock induction of this complex were reminiscent of the GroEL/Hsp60 family, and therefore raised the possibility that it represented an archaeal chaperonin. Subsequently, we named it "thermosome" to highlight its heat induction and extreme thermostability (Phipps et al. 1993). Independently, a closely related complex (TF55) was discovered in the laboratory of Art Horwich in Yale (Trent et al. 1991). The thermosome or TF55 were the first representatives of the Group II chaperonins found in archaea and in the eukaryotic cytosol. The main structural feature distinguishing the Group II from the Group I chaperonins is, in the absence of a co-chaperonin, a built-in lid provided by the protrusions of the apical domains which can seal the folding chamber by an iris-type closure mechanism (Klumpp et al. 1997; Gutsche et al. 1999). In 1996, in our quest for a more comprehensive understanding of the protein quality control machinery in Thermoplasma we found a fascinating, large proteolytic complex that works in conjunction with an array of aminopeptidases (Tamura et al. 1996). In view of the shape of the hexamer, we named it "tricorn protease"; soon thereafter we were able to show that tricorn protease exists in the cell as a giant icosahedral complex of approximately 15 MDa, which in addition to its peptide-cleaving activity, appears to serve as an organizing center for the more downstream elements of the protein degradation pathway (Walz et al. 1997). Tricorn protease converts the oligo-peptides (typically about 8 amino acid residues) released by the proteasome into smaller (2–4 residue) peptides which are degraded further by aminopeptidases (Tamura et al. 1998). These findings stimulated the search for "functional homologs" of tricorn protease in eukaryotic cells; one of the candidates is tripeptidylpeptidase (TPP) II, another giant protein complex with an intriguing structure (Geier et al. 1999; Rockel et al. 2002). In 2000, we completed the sequencing of the genome of Thermoplasma acidophilum, an endeavor we had undertaken with modest resources (Ruepp et al. 2000). It not only served to further establish Thermoplasma as a model system for studying cellular protein quality control, it also provided the platform for a very ambitious project, namely the mapping of its cellular proteome by cryoelectron tomography; this, in turn, can be expected to shed new light on the pathways of intracellular protein quality control (Fig. 5). The foundations of electron tomography were laid already in the late 1960s. In their landmark paper, De Rosier and Klug outlined very clearly and in general terms the principles of 3D reconstruction from electron micrographs (De Rosier and Klug 1968). Being aware of the practical problems in recording 3-D data sets, they took advantage of the helical symmetry of the bacteriophage T4 tail in a very pragmatic manner. Walter Hoppe, guided by his background in X-ray crystallography, also realized the potential of 3-D electron microscopy. Diverging from the approaches taken by most others, he focused on the development of methods suitable for studying individual structures ("Crystallography of crystals consisting of a single unit cell," Hoppe 1978). In fact, his group presented as early as 1974 a 3-D reconstruction of single fatty acid synthetase molecules obtained by tomography (Hoppe et al. 1974). As mentioned earlier, the "brute force" approach they used provoked some criticism. Besides doubts that negative staining can portray details of the underlying structure to the resolution they claimed, the main concern was the enormous electron dose to which the specimen was exposed during recording of the data. There was much discussion in the following years as to whether it might ultimately be possible to do electron tomography with acceptable electron doses. Also in 1968, R.G. Hart at the Lawrence Livermore Laboratory published a paper entitled "Electron microscopy of unstained biological material: The polytropic montage" (Hart 1968). Despite its vision, the Hart paper had negligible impact. For a vision to materialize, timing is a crucial element; if it is too early, the necessary technologies might not yet exist. The key problem in electron tomography, which for many years was a formidable obstacle and a deterrent, is to reconcile two requirements that are in conflict with each other: To obtain a reconstruction that is detailed and largely undistorted, one has to collect data over as wide a tilt range as possible with increments as small as possible (for review, see Baumeister et al. 1999). At the same time, the electron dose must be minimized. Above a critical dose, the specimen undergoes structural degradation that, in the worst case, can render a reconstruction meaningless. In principle, one could fractionate the dose over as many projections as an optimized tilt geometry might require. However, there is a practical limitation; the signal-to-noise ratio of the 2-D images has to be sufficient to permit their accurate alignment by cross-correlation. This problem is further aggravated by the far-from-perfect mechanical accuracy of the tilting devices that causes image shifts and changes of focus. Therefore, following each change of tilt angle, the specimen (or its image) has to be realigned and refocused. Doing this manually and with minimal exposure to the elect
The primary focus of this review is the research that resulted in the discovery of a new factor (von Willebrand factor, VWF) in the coagulation of blood; the prime motor in initial haemostasis. Further research that lead to the identification of VWF as a multimeric protein that interacts with vessel wall and platelets is also considered, as is the regulation of this factor's activity in blood, an important aspect of both haemostasis and thrombosis.
I had the great opportunity to start my scientific career at the time of the onset of molecular biology and my initial work laid the foundation for later contributions made in the fields of archaeal and bacterial protein synthesis and its regulation. I recount my academic life in two German universities and the role, which mentors played in the development of my career and how the excellence of my students contributed to the achievements.
Besides directing the MD-PhD program at the University of Zurich and chairing the Department of Pathology, Adriano Aguzzi runs a laboratory which is entirely dedicated to the study of prion diseases. In this autobiographical essay, he traces the motifs behind his tortuous trajectory which started in Italy and led him to Switzerland after two decades of stints in Germany, Austria, and the United States.
Born on July 14, 1931 I passed my childhood and the war in Strasbourg. In 1944 my parents, my brother and I were arrested by the Gestapo and sent to a SS camp in Germany. We were liberated by the American army in 1945. After studying pharmacy, physiology and biochemistry at Strasbourg University I became a researcher in 1955 at the Centre National de la Recherche Scientifique (CNRS) in J.‐P. Ebel's laboratory. I first worked on the structure and function of linear polyphosphates in yeast and bacteria. This was interrupted by my 28 months military service at a research laboratory of the health service in Lyon where I isolated and studied a soluble substrate of lysozyme. I got a PhD in pharmacy in 1961 and in sciences in 1964 and became assistant professor in 1964, then full professor (1969) at the Faculty of Pharmacy of which I was dean in 1969–1970. There I lectured biochemistry, molecular biology and toxicology. After a post-doc in R.W. Holley's laboratory in Ithaca (USA) in 1965, I turned to the study of tRNAs, which became my main research topic for 35 years. I was concerned with their isolation, primary structure and their rare nucleotides. I discovered their specific cleavage by lead, and studied several aminoacyl-tRNA synthetases. In parallel, I also did research in toxicology and discovered the mode of action of ricin and of ochratoxin A on protein synthesis. My last research was concerned with the genotoxicity of mycotoxins and the formation of DNA adducts. This was possible, thanks to a very active laboratory and many gifted and hard working co-workers as well as many international collaborations. I worked at the CNRS Institute of Molecular and Cellular Biology of which I became director after the retirement of J.‐P. Ebel. I was involved in many scientific organisations: President of the French Biochemical Society and of the French Toxicological Society (1979–1980), I was Secretary general of FEBS (1984–1989) and President of FEBS (1999–2003). I was also president of EUROTOX (1990–1992). I am member of the French National Academy of Medicine since 1988.
