Journal of Molecular Biology最新文献

In this article I tell the story of my career path and how I have come to focus my research on protein folding in the cell. My early fascination with protein folding began during my undergraduate research. My graduate work exploited reductionist approaches to explore structural features in proteins by using cyclic peptide models ofβ-turns. My career trajectory from these early days to present, described in the first section of this article, illustrates the importance of pursuing the scientific questions that one finds most exciting and seizing professional opportunities that enable these questions to be tackled productively. In addition, this trajectory shows how serendipity can shape a career path. The second section describes the extraordinary scientific discoveries I have witnessed in protein folding during my career. Here I explain how I was drawn into the world of protein folding in thecell. This turning point allowed me to participate in the explosion of research on molecular chaperones in the early 90's and to help elucidate the nature of chaperone-substrate recognition, a problem I continue to focus on. Examples of our research contributions are presented in the third section, with a perspective on major challenges for the future offered in the last section. Throughout my career I have engaged in many collaborations;each has opened new scientific doors. Importantly, I seek to instill in my trainees the same excitement about research that I feel and to foster their growth as scientists and their discovery of their own passions and talents.

The ribosome is a major cellular machine that converts genetic information into biological function. Emerging data show that the ribosome is not only a protein synthesis machine, but also participates in the maturation of the nascent protein into properly folded and active molecules. The ribosome surface near the opening of the polypeptide exit tunnel can interact directly with the newly synthesized protein and, more importantly, provides a platform where numerous protein biogenesis factors assemble, gain access to the nascent chain, and direct them into diverse biogenesis pathways. In this article, we review the current understanding of cotranslational protein maturation pathways, with an emphasis on systems in which biochemical studies provided a high-resolution molecular understanding and yielded generalizable mechanistic principles.

Being asked to write this "Reflections"-type article is a special honor offered at the end of one's career and I am grateful for the opportunity to think back over the more than 4-decade span of my career in the field of HIV/AIDS Retrovirology. Although at this point in time there is still no vaccine or curative therapy for HIV/AIDS, this invitation to reminiscence comes in the year of FDA approval of Lenacapavir, the first-in-class anti-HIV agent targeting the viral capsid. This triumph in the field is the culmination of efforts of many, including some in my own laboratory, who were among the first to produce recombinant capsid protein (CAp24) for structural and biochemical studies. A current drawback of this drug is its low genetic barrier to viral resistance. The research finding considered to be our most important contribution to the field of HIV research was framed by the challenge to suppress the emergence of resistant variants, and I describe the paths that drove me to risk exploration of the non-traditional for potential solutions. Finally, I share my views on what I consider to be an important open question for the future: how to achieve greater diversity and inclusion in Science.

I am a Professor of Biochemistry, Biophysics and Structural Biology and Plant and Microbial Biology at the University of California in Berkeley. I was born and raised in India, emigrated to the United States to attend university, earning a B.S. in Molecular Biology and a Ph.D. in Biochemistry at the University of Wisconsin in Madison. Following post-doctoral studies with Lawrence Bogorad at Harvard University where I became interested in genetic control of trace element quotas, I joined the department of Chemistry and Biochemistry at UCLA. One of the first to appreciate essential trace metals as potential regulators of gene expression, I articulated the details of the nutritional Cu regulon in Chlamydomonas. In parallel, I used genetic approaches to discover the genes governing missing steps in tetrapyrrole metabolism, including the attachment of heme to apocytochromes in the thylakoid lumen and the factors catalyzing the formation of ring V in chlorophyll. After biochemistry and classical genetics, I embraced genomics, taking a leadership role on the Joint Genome Institute's efforts on the Chlamydomonas genome and more recently, contributing to high quality assemblies of several genomes in the green algal radiation and large transcriptomic and proteomic datasets - focusing on the diel metabolic cycle in synchronized cultures and acclimation to key environmental and nutritional stressors -- that are well-used and appreciated by the community. A new venture in Berkeley is the promotion of Auxenochlorella protothecoides as the true "green yeast" and as a platform for engineering algae to produce useful bioproducts.

After completing my post-doctoral training at the University of Alabama, Birmingham and a brief period on the faculty there, I joined the Department of Tumor Cell Biology at St. Jude Children's Research Hospital in 1987 as an Assistant Member and started my independent research program. For the following 37 years, I led a relatively small basic research group comprised at various times of post-doctoral fellows, graduate students, undergraduate students, and research technicians; many of whom I am still in contact. Last year I closed the lab and transitioned to an emeritus position at St. Jude. I continue to maintain several research collaborations covering areas of research that have long been dear to my heart. My post-doctoral studies on BiP revealed that it controlled immunoglobulin assembly and transport, and as such, played a critical role in fidelity of the immune response. My lab continued to define BiP's functions in protein folding and subunit assembly, as well as, in degradation using biochemical, cell-based, and biophysical analyses. Several ER localized co-factors that regulate the activity of BiP and allow it to contribute to its multiple ER functions were identified by our group. These include DnaJ family members and nucleotide change factors. Through a variety of collaborative studies, we pursued BiP's functions in maintaining the permeability barrier of the translocon, contributing to ER calcium stores, and regulating the up-stream transducers of the UPR, a stress response that is activated by the accumulation of unfolded proteins in the ER.

Charting future innovations is challenging. Yet, allosteric and orthosteric anticancer drugs are undergoing a revolution and taxing unresolved dilemmas await. Among the imaginative innovations, here we discuss cereblon and thalidomide derivatives as a means of recruiting neosubstrates and their degradation, allosteric heterogeneous bifunctional drugs like PROTACs, drugging phosphatases, inducers of targeted posttranslational protein modifications, antibody-drug conjugates, exploiting membrane interactions to increase local concentration, stabilizing the folded state, and more. These couple with harnessing allosteric cryptic pockets whose discovery offers more options to modulate the affinity of orthosteric, active site inhibitors. Added to these are strategies to counter drug resistance through drug combinations co-targeting pathways to bypass signaling blockades. Here, we discuss on the molecular and cellular levels, such inspiring advances, provide examples of their applications, their mechanisms and rational. We start with an overview on difficult to target proteins and their properties-rarely, if ever-conceptualized before, discuss emerging innovative drugs, and proceed to the increasingly popular allosteric cryptic pockets-their advantages-and critically, issues to be aware of. We follow with drug resistance and in-depth discussion of tumor heterogeneity. Heterogeneity is a hallmark of highly aggressive cancers, the core of drug resistance unresolved challenge. We discuss potential ways to target heterogeneity by predicting it. The increase in experimental and clinical data, computed (cell-type specific) interactomes, capturing transient cryptic pockets, learned drug resistance, workings of regulatory mechanisms, heterogeneity, and resistance-based cell signaling drug combinations, assisted by AI-driven reasoning and recognition, couple with creative allosteric drug discovery, charting future innovations.

The microtubule-associated protein tau aggregates into pathological β -sheet amyloid fibrils in Alzheimer's disease (AD) and other neurodegenerative diseases. In these aggregates, tau is chemically modified, including abnormal hyperphosphorylation and truncation. Truncation after D421 in the C-terminal domain occurs at early stages of AD. Here we investigate the structures of Δ D421-truncated 0N4R tau fibrils assembled in vitro in the absence of anionic cofactors. Using solid-state NMR spectroscopy and cryoelectron microscopy, we show that Δ D421-truncated 0N4R tau forms homogeneous fibrils whose rigid core adopts a three-layered β -sheet structure that spans R2, R3 and R4 repeats. This structure is essentially identical to that of full-length tau containing phospho-mimetic mutations at the PHF1 epitope in the C-terminal domain. In comparison, a Δ D421-truncated tau that additionally contains three phospho-mimetic mutations at the AT8 epitope in the proline-rich region forms a fibril core that includes the first half of the C-terminal domain, which is excluded from all known pathological tau fibril cores. These results indicate that the posttranslational modification code of tau contains redundancy: both charge modification and truncation of the C-terminal domain promote a three-layered β -sheet structure, which resembles pathological four-repeat tau structures in several tauopathies. In comparison, reducing the positive charges at the AT8 epitope in Δ D421-truncated tau promotes a fibril core that includes an immobilized C-terminal domain. The absence of this structure in tauopathy brains implies that Δ D421 truncation does not occur in conjunction with AT8 phosphorylation in diseased brains.

Direct, single molecule measurement of RNA by long-read transcriptome sequencing (LRTS) enables the reliable detection of transcripts and alternative splicing events, thus contributing to the identification of splicing mechanisms, improvement of current gene models, as well as to the prediction of more reliable protein isoforms. LRTS data comes from either PacBio's single-molecule real time sequencing or from Oxford Nanopore's nanopore sequencing. Previously, we developed IsoTools, a software originally designed for processing and analyzing PacBio data. IsoTools copes with the complexity of LRTS data and offers multiple functionality for transcript identification and quantification as well as the analysis of differential isoform usage and local differential splicing events. Here, we report an update of the software, IsoTools 2.0, and demonstrate its additional performance on Oxford Nanopore data from multiple experimental protocols. We present the IsoTools 2.0 workflow, highlighting novel functionalities with respect to reliable transcript detection as well as transcription start site prediction. Additionally, we show novel metrics for structural description and quantification of gene model variability based on the gene's transcripts. We demonstrate the performance of IsoTools 2.0 on a variety of experimental protocols for library construction from a recent LRTS challenge. We show that IsoTools 2.0 is able to cope with the inherent complexity of LRTS data and that the workflow generates meaningful hypotheses on biomarkers for alternative splicing. The software is available from https://github.com/HerwigLab/IsoTools2/.

α-Synuclein (αSyn) is a key culprit in the pathogenesis of synucleinopathies such as Parkinson's Disease (PD), in which it forms not only insoluble aggregates called amyloid fibrils but also smaller, likely more detrimental species termed oligomers. This property is shared with other amyloidogenic proteins such as the Alzheimer's Disease-associated amyloid-β (Aβ). We previously found an intriguing interplay between off-pathway Aβ oligomers and Aβ fibrils, in which the oligomers interfere with fibril formation via inhibition of secondary nucleation by blocking secondary nucleation sites on the fibril surface. Here, using ThT aggregation kinetics and atomic force microscopy (AFM), we tested if the same interplay applies to αSyn fibrils. Both homotypic (i.e. αSyn) and heterotypic (i.e. Aβ) off-pathway oligomers inhibited αSyn aggregation in kinetic assays of secondary nucleation. Initially soluble, kinetically trapped Aβ oligomers co-precipitated with αSyn(1-108) fibrils. The resulting co-assemblies were imaged as clusters of curvilinear oligomers by AFM. The results indicate that off-pathway oligomers have a general tendency to bind amyloid fibril surfaces, also in the absence of sequence homology between fibril and oligomer. The interplay between off-pathway oligomers and amyloid fibrils adds another level of complexity to the homo- and hetero-assembly processes of amyloidogenic proteins.

My focus shifted over the years. In 1978 I developed an efficient O(n³) dynamic programming algorithm for the-then open problem of RNA secondary structure prediction. This algorithm, now dubbed the "Nussinov algorithm", "Nussinov plots", and "Nussinov diagrams", is still taught in classes across Europe and the U.S. As sequences started coming out in the 1980s, I started seeking genome-encoded functional signals, later becoming a bioinformatics trend. In the early 1990s I transited to proteins, co-developing a powerful computer vision-based docking algorithm. In the late 1990s, I proposed the foundational role of conformational ensembles in molecular recognition and allostery. At the time, conformational ensembles and free energy landscapes were viewed as physical properties of protein molecules but were not associated with function. The classical view of molecular recognition and binding was based on only two conformations captured by crystallography: open and closed. I proposed that all conformational states preexist. Proteins always have not one folded form- nor two-but many folded forms. Thus, rather than inducing fit, binding can work by shifting the ensembles between states, and this shifting, or redistributing the ensembles to maintain equilibrium, is the origin of the allosteric effect and protein, thus cell, function. This new paradigm impacted community views in allosteric drug design, catalysis, and regulation. Dynamic conformational ensemble shifts are now acknowledged as the origin of recognition, allostery, and signaling, underscoring that conformational ensembles-not proteins-are the workhorses of the cell, pioneering the fundamental idea that dynamic ensembles are the driving force behind cellular processes.