Jason E. Gestwicki

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Maintaining proteostasis is the province of the proteostasis network, which consists of the cellular components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental functional unit in biology with relevance to many diseases. Despite its importance, the human proteostasis network has until now lacked a precise definition, limiting our ability to interrogate proteostasis, integrate multiomic datasets, compare results across studies, or model proteostasis dysfunction in disease. To address this limitation, we have operationally defined the human proteostasis network by providing a comprehensive, annotated, and manually curated list of its components classified into nine Branches based on their function and localization - cytonuclear proteostasis, ER proteostasis, mitochondrial proteostasis, nuclear proteostasis, proteostasis network regulation, translation, extracellular proteostasis, the autophagy-lysosome pathway, and the ubiquitin-proteasome system - which we present in a series of three manuscripts of which this is the first. Overall, the proteostasis network annotation encompasses 3,151 components described by 4,043 annotations. Here, we present the 1,190 components that make up the first seven of these Branches, which comprise the compartment-based proteostasis network (cytonuclear, ER, mitochondrial, nuclear, and extracellular proteostasis), proteostasis network regulation, and translation. These Branches include chaperones, folding enzymes, systems for trafficking proteins, organelle-specific degradation systems, and the cell's translation machinery. This work provides a framework that will facilitate insights into how proteostasis is preserved in health, lost in aging and disease, and how it could be restored through pharmacological intervention.

Tau filaments are a hallmark of neurodegenerative tauopathies, such as Alzheimer’s disease (AD). Structural studies have revealed that patient-derived tau fibrils adopt distinct folds in different tauopathies; however, it is unclear what forces guide this process. To explore this question, we investigated the assembly of a tau fragment containing four disease-associated phospho-mimetics (termed Tau(297–407)-4D) in vitro. Under cofactor-free and quiescent conditions, Tau(297–407)-4D forms fibrils with a core structure that partially resembles the AD fold after about 7 days. Strikingly, we noticed that this filament behaves as a hydrogel and evolves into two new polymorphs as it ages over the next 35 days. Thus, tau fibrils formed under cofactor-free conditions are dynamic, exhibiting substantial nonequilibrium behavior. To probe what types of perturbations might stabilize these structures, we applied mechanical agitation, which drove the filaments toward thermodynamic equilibrium in a mechanism consistent with Ostwald ripening into solid-phase, micrometer-sized particles. Likewise, the addition of polyanionic cofactors to preformed Tau(297–407)-4D fibrils significantly stabilized them, as judged by solubility equilibria and chemical denaturation experiments. A subset of the polyanions also remodeled the fibril structure and tuned the extent of fibril–fibril interactions (i.e., “clumping”). We conclude that environmental factors, such as mechanical stress and/or polyanions, play an important role in promoting the thermodynamic stability of otherwise dynamic tau fibrils. We speculate that, in patients, such factors might contribute to the maturation of disease-specific conformers.

The remarkable and problematic capacity of Mycobacterium tuberculosis to persist in the host is dependent on a functional 20S proteasome that is structurally and functionally analogous to its mammalian counterpart. It has been proposed that this enzyme is critical for the bacterium’s adaptive response to a variety of stressors in the host. Here, we report the structure of a rationally designed and species-selective inhibitor of the Mtb 20S proteasome in complex with the enzyme. The inhibitor was inspired by the syringolin natural products—covalent proteasome inhibitors that mimic a substrate. Structure elucidation of the Mtb 20S proteasome-inhibitor adduct via cryo-electron microscopy revealed that the inhibitor mimics a substrate yet engages the active site in a different fashion than the syringolin natural products. Corroboration of this model via the synthesis of hypothetical inhibitors led to the discovery of a more potent inhibitor, as evidenced in kinetic assays for enzyme inhibition. Both inhibitors are permeable to and active in M. tuberculosis. They sensitize the bacterium to chemically and pharmacologically induced nitrosative stress in vitro. Importantly, they suppress the persistence of live mycobacteria in murine and human macrophages.

From 2000 to 2019, the global malaria death toll fell from 864,000 to 576,000 deaths; however, progress on reducing this toll has since slowed, with the COVID-19 pandemic contributing to an increase in the mortality rate since 2020. The emergence of widespread resistance in the major malaria parasite (Plasmodium falciparum) to first-line treatment drugs has highlighted the need for new antimalarials and smarter drug delivery strategies. Nevertheless, recent successes of the first malaria vaccines (RTS,S/AS01 and R21/MM) have renewed the promise of widely applicable vaccines in the future. Since 2015, the Eastern Mediterranean Region has experienced an overall increase in malaria cases and deaths. In the Arabian Peninsula, while most of the Gulf Cooperation Council (GCC) countries have been declared free of indigenous malaria (Bahrain, Kuwait, Qatar, and the United Arab Emirates), malaria is still endemic in two GCC countries (Saudi Arabia and Oman) and their neighbors (Yemen). Furthermore, a number of factors threaten malaria control in this region, especially antimalarial drug resistance, the emergence of highly invasive mosquito species, increasing average temperatures, and imported malaria. In this review, we assess the current worldwide status of malaria before providing a critical evaluation of the malaria situation in the GCC countries.

Tau hyperphosphorylation is linked to tauopathy aggregates, but the effects of individual sites on tau assembly remain unclear. Here, we used protein semisynthesis to generate defined Tau(297–407) proteoforms containing specific combinations of phosphorylation within the PHF1 epitope. Spontaneous aggregation revealed that phosphorylation of S396, and S400 to a lesser extent, promoted nucleation, while phosphorylation of T403 and S404 suppressed assembly. A similar reactivity trend of pS396 > pS400 > WT > pT403 > pS404 was observed for seeded assembly, and all proteoforms, even anti-aggregation ones, were incorporated. The fibrils have similar thermodynamic stability, suggesting that phosphorylation selectively tunes reactivity and not thermodynamics. Cryo-EM revealed that pS400 produces a chronic traumatic encephalopathy (CTE) protofilament conformation. Strikingly, one of the structures observed in the pS400 sample appeared to capture a secondary nucleation step. Together, these studies reveal the importance of positional effects of phosphorylation on tau self-assembly.

Binding and catalysis play central roles in living systems. While natural proteins have finely tuned affinities for their primary ligands, they also bind weakly and promiscuously to other molecules, which serve as starting points for the incremental evolution of different specificities. Thus, modern proteins have emerged from the joint exploration of sequence and structural space. Interactions between natural proteins and small molecules can be systematically profiled by crystallographic fragment screening in defined geometries, yet this approach has not been applied to highly designable de novo proteins. Here we apply this method to explore the binding specificity of a de novo small-molecule-binding protein, apixaban-binding helical bundle. As in nature, we found that it formed weak complexes, which were excellent starting points for the design of entirely distinct functions, including a turn-on fluorophore binder and a highly efficient Kemp eliminase with a catalytic efficiency of 3,200,000 M⁻¹ s⁻¹, approaching the diffusion limit. This work illustrates how simultaneous consideration of sequence and chemical structure diversity can guide the emergence of different functions in designed proteins. The alternative text for this image may have been generated using AI.

Hsp70s are essential molecular chaperones that are increasingly recognized to be regulated by post-translational modifications. Here, we show that phosphorylation of a conserved threonine (T495), previously shown to be exploited by a Legionella pneumophila kinase to inhibit Hsp70, occurs endogenously in human cells in response to DNA damage, particularly when base excision repair is overburdened. This modification is cell cycle dependent, and in yeast, phosphomimetic or phosphonull Hsp70 variants disrupt G1/S progression under normal and DNA-damaging conditions. Biochemically, the phosphomimetic T495E mutation locks Hsp70 in an open-like conformation without blocking substrate engagement. Together, our results reveal a conserved mechanism by which dynamic Hsp70 phosphorylation regulates the G1/S transition, and delays cell cycle progression during DNA damage, highlighting how pathogen-derived insights can uncover fundamental cell biology principles.