Alfred L. Goldberg (1942–2023)

Alfred (Fred) Goldberg, a leader in the field of protein turnover and a mentor to hundreds of young scientists for a remarkable 50 years, died peacefully in his Brookline home on April 18th at the age of 80 after a long struggle against lymphoma and transthyretin cardiomyopathy. Fred was the husband of hematologist Joan Helpern Goldberg and the father of jazz pianist Aaron Goldberg and progressive political software engineer Julie Goldberg. Fred’s dedication to the study of protein turnover—and more specifically the proteasome—was legendary, and his impact on our understanding of the field has been so pervasive that it is difficult to imagine how it might have evolved were it not for his contributions. The trajectory of Fred’s career was unlikely in many respects, initially in that he was heedless enough to focus on the problem of protein turnover at all. Though reports of protein turnover had followed quickly upon the introduction of isotopic labeling methods, initially through the work of Schoenheimer, the field was poorly regarded when Fred decided to tackle the problem. Jacques Monod, who pioneered the field of gene expression with his operon model, had published that protein turnover was at best negligible in E. coli, and that prior work demonstrating the process in eukaryotes was so flawed as to be dismissed. However, Monod had generalized from the relative stability of ß-galactosidase to all proteins, whereas we now know that protein turnover rates in nature differ by 104 -fold or more, a signature of the diverse regulatory functions of such turnover. The protein turnover field had other problems as well—its paradigms were poised to be overturned. Based on stunning advances in electron microscopy after World War II, the discovery of the lysosome by Christian de Duve and colleagues led to the view that these protease-rich organelles would account for whatever protein turnover may occur in a eukaryotic cell. It later emerged from the work of Fred and others that the principal sites of rapid protein turnover are instead the cytoplasm and nucleus. Autophagic processes, which deliver proteins to the lysosome for degradation, are slower though also essential. Fred’s work also played a key role in bringing down another paradigm—that selective intracellular proteases would work much like trypsin and other intestinal proteases. For these enzymes, the exergonic nature of peptide bond cleavage provides a driving force for the reaction, and their specificity was understood to be determined by local sequence features and accessibility of the scissile bond. However, intracellular proteases such as the proteasome are driven by ATP hydrolysis, and their specificity is defined by substrate receptors, such as ubiquitin receptors, that are distant from and not coupled to their proteolytic sites. In the proteasome and related bacterial proteases, many of which Fred and his group discovered, the energy of ATP hydrolysis is converted into mechanical force, unfolding the substrate while injecting it into an otherwise inaccessible proteolytic chamber. When Fred joined Jim Watson’s laboratory as an undergraduate at Harvard in the early 60s, there was perhaps no better place to explore unsolved issues surrounding the central dogma of molecular biology, “DNA→RNA→protein.” Soon afterward, the atmosphere at Harvard was charged by the pioneering work on gene regulation by Walter Gilbert and Mark Ptashne, who discovered and isolated the lac and λ repressors, respectively. At this place and time, it must have seemed incongruous for a Watson-trained researcher to abandon central dogma work and turn to the barely existing field of protein turnover. Fred left Watson’s laboratory upon his graduation in 1963 and, after a year as a Churchill Fellow in Cambridge University, enrolled at Harvard Medical School. He struggled to sustain interest in his coursework, and after 2 years he baffled his parents in Rhode Island by suspending his medical studies and focusing solely on research. He transferred to the graduate program at Harvard Medical School and studied under H.M. Goodman, receiving his Ph.D. from the Department of Physiology. But he never actually withdrew from the medical program and later relished his imaginary dual existence as perpetual student and tenured faculty. At this point Fred began to study protein turnover. Among the most important aspects of his early work was the establishment of what were to become canonical model systems in the field of protein turnover: E. coli, muscle, and reticulocytes. In his studies on E. coli, initiated in the early 1970s, Fred found that structurally abnormal proteins, such as mutant proteins, products of biosynthetic errors, and oxidatively damaged proteins, were major targets of degradation pathways. We now refer to this as quality control degradation, and it is currently recognized as one of the most complex and important aspects of the ubiquitin-proteasome system. Decades later, as the etiological agents of various neurodegenerative diseases were progressively identified, and their pathological forms were found to be misfolded and moreover ubiquitinated, Fred’s early advocacy of quality control degradation was vindicated, and the field shifted in his direction. In his studies of skeletal muscle, Fred established a central paradigm of muscle physiology—that the size of the muscle can be altered in different settings by manipulating the ratio of protein synthesis and protein catabolism. In sequential single-author papers in the late 1960s, he documented that in settings of skeletal muscle hypertrophy, where the muscle is induced to become larger due to increased load on the muscle, protein turnover decreases while protein synthesis increases. These findings established skeletal muscle atrophy and hypertrophy as model systems to understand mechanisms of protein catabolism and anabolism. Unlike the skeletal muscle cell, the reticulocyte—the immediate precursor of the red blood cell—undergoes proteome-wide protein turnover as a differentiative program (which recalls Fred’s most memorable typo, having once claimed to have raised reticulocytes in phenylhydrazine-treated rabbis). The remarkable nature of protein turnover in this cell type was noted by Samuel Rapoport, but it was Fred and his trainee Joe Etlinger who in 1977 established reticulocyte lysates—essentially a cytoplasmic extract—as a faithful and highly active in vitro system that could allow the dissection of protein degradation pathways.1Etlinger J.D. Goldberg A.L. A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes.Proc. Natl. Acad. Sci. USA. 1977; 74: 54-58Crossref PubMed Google Scholar Biochemical fractionation of these lysates quickly led to the discovery of protein ubiquitination by Hershko and Ciechanover, which was foundational for the field. Following this, regulated protein turnover was seen as governed by ubiquitin ligases, and it gradually dawned on the field that there were in fact not one but many hundreds of these enzymes, each functioning as a specificity factor. Returning to muscle in 2001, and applying this paradigm, Fred and his trainees Marcelo Gomes, Stuart Lecker, and Marco Sandri helped elucidate a class of genes that they called “atrogenes,” which are important for skeletal muscle atrophy. One of these atrogenes, known as Atrogin1 or MAFbx, is a ubiquitin ligase whose expression is in current worldwide use as a clinical marker of acute muscle atrophy, since it has been found to be upregulated in almost every setting where such atrophy takes place. Beyond this pioneering work, Fred is best known today for his work on the proteasome, which his and Marty Rechsteiner’s groups discovered independently.2Hough R. Pratt G. Rechsteiner M. Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates.J. Biol. Chem. 1986; 261: 2400-2408Abstract Full Text PDF PubMed Google Scholar,3Waxman L. Fagan J.M. Goldberg A.L. Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes, one of which degrades ubiquitin conjugates.J. Biol. Chem. 1987; 262: 2451-2457Abstract Full Text PDF PubMed Google Scholar The proteasome is abundant, stable, and highly active, and several groups had tracked its activity beforehand, but it remained an obscure and anonymous peptidolytic activity until the mid-80s, when Fred, Marty, and their coworkers showed that it was specific for ubiquitin-protein conjugates. Fred’s group found that the proteasome came in two forms, the 20S and 26S complex. The 20S complex is taken up into the 26S complex along with a 19S complex that carries specificity for ubiquitin and contains six distinct ATPase enzymes that translocate substrates into the proteolytic chamber of the 20S complex. The physiological roles of the proteasome, which were to prove so interesting, were still completely unclear at that time. These insights only emerged from the discovery of proteasome inhibitors in the early 1990s, which Fred spearheaded. The initiative evolved not just from the increasing refinement of proteasome biochemistry but also from Fred’s longstanding studies of muscle biology, and it hinged on a string of lucky breaks. At the time too little was known about the proteasome to predict what putative applications might work out—even the mechanism of peptide bond cleavage was a mystery at the time. Adding to these liabilities, it seemed that proteasome inhibitors would be pretty toxic. Nonetheless, adequate capital was raised by 1993 to form a small company in Cambridge with the objective of finding proteasome inhibitors. It was named MyoGenics since the projected clinical application was to suppress disease-associated muscle wasting. Subsequently, Tom Maniatis joined the board, bringing his interest in the regulation of the activation of the transcription factor NF-kB and gene regulation. With revised objectives the company was renamed ProScript. Finding proteasome inhibitors did not require large-scale chemical screens or vast DNA-encoded libraries. By the time the company was formed, proteasome inhibitors were already in the literature, but they were not well characterized and were in key cases misassigned as inhibitors of other proteases. That is how MG101 was found, which led to the research compound MG132, which has been widely used by cell biologists.4Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules.Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2212) Google Scholar,5Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B.Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1924) Google Scholar However, even with good inhibitors in hand, ProScript was running out of gas when the National Cancer Institute was induced to check them for anti-cancer activity, and they showed a surprisingly strong activity in assays based on cancer-derived cell lines. There were still few takers among the investment community, but the company was saved by being acquired by another small biotech, LeukoSite. Even then the project was not out the woods, because LeukoSite itself was quickly acquired by Millennium, where proteasome inhibitors were regarded if anything as a liability in the deal. But through the persistence of Julian Adams, formerly CSO at ProScript, Millennium was coaxed to bring PS-341 forward, and powerful effects on multiple myeloma were immediately found in clinical trials. PS-341 proved to revolutionize the treatment of multiple myeloma and was approved by the FDA in 2003 as bortezomib (a.k.a. Velcade); 20 years later it remains the standard of care for multiple myeloma. Toxicity proved to be less of a problem than anticipated because interestingly the inhibitors are already therapeutically effective at doses where inhibition is far from complete. Another disease now treated by proteasome inhibition is light-chain amyloidosis. The inhibitors attenuate amyloid formation by killing the transformed cells that secrete the amyloidogenic light chain. To date hundreds of thousands of patients have been treated with proteasome inhibitors, a testimony to the tremendous impact of Fred’s work. Even to his scientific “competitors,” Fred was a generous scientist. He often exchanged ideas and data before they were published. He wanted to test his ideas through discussion, and he was happy to hear conflicting or complicating results. One of us had his office just down the hall, and Fred would drop in on the way home to mull over recent data and papers, whether momentous or not—he just liked to chat. He was all science but rarely strung together more than a few sentences without stopping for some quip that just struck him or an ironic aside. During the COVID-19 pandemic his physical isolation had to be extreme, as he was immunocompromised, but even when hospitalized he managed to reliably Zoom into journal clubs and lectures. He would explain the newest details of his treatments, which were indeed often experimental, no differently than if an interesting paper had just appeared. At least at Harvard, Fred had no equal in the composition of light verse. While they came out like limericks, he never seemed to observe the rules of limerick construction. If a departmental chair stepped down, Fred would have a poem for it, whipped out of his pocket late in the evening when the crowd had loosened up, somehow working in bawdy passages however dry the actual subject matter. At Fred’s funeral, his children Julie and Aaron paid tribute to him with their own verse, referring to Fred’s many poems about his medical problems. In Aaron’s words:So let’s spare you the science encomiumsFred’s output more polished than chromiumI’ll save that for rhyming ChatGPTMy aim now is his humanity. Ever more generous in facing the endFred turned his brave gift of verse to legendMary was right that she’d birthed a bardThose 500 papers they weren’t near as hard.Rhyme was his sword and shield, with pure wit heBattled and found grace. No self-pity. The authors thank all those who assisted in the writing of this piece, especially Larry Dick, Tom Maniatis, and J. Wade Harper.