Publications search

Native mass spectrometry (MS) enables direct mass measurement of intact protein assemblies generating relevant subunit composition and stoichiometry information. Combined with cross-linking and structural data, native MS-derived information is crucial for elucidating the architecture of macromolecular assemblies by integrative structural methods. The exosome complex from budding yeast was among the first endogenous protein complexes to be affinity isolated and subsequently characterized by this technique, providing improved understanding of its composition and structure. We present a protocol that couples efficient affinity capture of yeast exosome complexes and sensitive native MS analysis, including rapid affinity isolation of the endogenous exosome complex from cryolysed yeast cells, elution in nondenaturing conditions by protease cleavage, depletion of the protease, buffer exchange, and native MS measurements using an Orbitrap-based instrument (Exactive Plus EMR).

The gamma-tubulin ring complex (gamma-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation, yet its structure is not known. Here, we present a cryo-EM reconstruction of the native human gamma-TuRC at similar to 3.8 angstrom resolution, revealing an asymmetric, cone-shaped structure. Pseudoatomic models indicate that GCP4, GCP5, and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes distal to the gamma-TuRC "seam.'' We also identify an unanticipated structural bridge that includes an actin-like protein and spans the gamma-TuRC lumen. Despite its asymmetric architecture, the gamma-TuRC arranges gamma-tubulins into a helical geometry poised to nucleate microtubules. Diversity in the gamma-TuRC subunits introduces large (>100,000 angstrom(2)) surfaces in the complex that allow for interactions with different regulatory factors. The observed compositional complexity of the gamma-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g., within biological condensates or alongside existing filaments.

Background: Hidradenitis suppurativa (HS) is characterized by recurrent, painful nodules in flexural areas. Objective: The objective of this study was to explore the placebo response in HS randomized clinical trials and to compare it briefly with the placebo response in psoriasis and atopic dermatitis. Methods: A Cochrane Review on interventions in HS was used as a starting point, and a systematic review was then undertaken by using the PubMed database, yielding 7 HS randomized clinical trials for inclusion in this study. Results: This review demonstrates that there is a robust placebo response in HS that is most marked in physical signs but also marked in pain responses. Limitations: Multiple outcome measures utilized in these studies and reporting bias limited this review. Conclusion: This large placebo response has implications for clinical trial design. This knowledge can also help deliver improved clinical care by forming the basis of nonpharmacologic treatments and help optimize current medication use to maximize the placebo effect.

In this study, we examined how channel-forming subunits of the nuclear pore complex (NPC) are assembled into a selective channel within a highly structured scaffold ring during postmitotic assembly. We focused on non-structured domains of the scaffold Nups and performed in vitro self-assembled particle assays with those derived from channel-forming FG-Nups. We found that non-structured domains of ELYS and Nup35N interacted with channel-forming FG-Nups to form a self-assembled particle. Sequential addition of FG-Nups into the scaffold particle revealed that ELYS, which initiates postmitotic NPC reassembly, interacts with early assembling FG-Nups (Nups98 and 153) but not middle stage-assembling FG-Nups (Nups58 and 62). Nup35, which assembles between the early and middle stages, facilitated the assembly of Nup62 into the early assembling Nups both in vitro and in vivo. These results demonstrate that ELYS and Nup35 have a role of facilitator in the ordered assembly of channel-forming FG-Nups during mitosis.

KRAS GTPases are activated in one-third of cancers, and KRAS(G12C) is one of the most common activating alterations in lung adenocarcinoma(1,2). KRAS(G12C) inhibitors(3,4) are in phase-I clinical trials and early data show partial responses in nearly half of patients with lung cancer. How cancer cells bypass inhibition to prevent maximal response to therapy is not understood. Because KRAS(G12C) cycles between an active and inactive conformation(4-6), and the inhibitors bind only to the latter, we tested whether isogenic cell populations respond in a non-uniform manner by studying the effect of treatment at a single-cell resolution. Here we report that, shortly after treatment, some cancer cells are sequestered in a quiescent state with low KRAS activity, whereas others bypass this effect to resume proliferation. This rapid divergent response occurs because some quiescent cells produce new KRAS(G12C) in response to suppressed mitogen-activated protein kinase output. New KRAS(G12C) is maintained in its active, drug-insensitive state by epidermal growth factor receptor and aurora kinase signalling. Cells without these adaptive changes-or cells in which these changes are pharmacologically inhibited-remain sensitive to drug treatment, because new KRAS(G12C) is either not available or exists in its inactive, drug-sensitive state. The direct targeting of KRAS oncoproteins has been a longstanding objective in precision oncology. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome if we are to achieve complete and durable responses in the clinic.

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional 'on-bead' approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

Over the last 50 years, the concept of stress has evolved significantly, and our understanding of the underlying neurobiology has expanded dramatically. Rather than consider stress biology to be relevant only under unusual and threatening conditions, we conceive of it as an ongoing, adaptive process of assessing the environment, coping with it, and enabling the individual to anticipate and deal with future challenges. Though much remains to be discovered, the fundamental neurocircuitry that underlies these processes has been broadly delineated, key molecular players have been identified, and the impact of this system on neuroplasticity has been well established. More recently, we have come to appreciate the critical interaction between the brain and the rest of the body as it pertains to stress responsiveness. Importantly, this system can become overloaded due to ongoing environmental demands on the individual, be they physical, physiological, or psychosocial. The impact of this overload is deleterious to brain health, and it results in vulnerability to a range of brain disorders, including major depression and cognitive deficits. Thus, stress biology is one of the best understood systems in affective neuroscience and is an ideal target for addressing the pathophysiology of many brain-related diseases. The story we present began with the discovery of glucocorticoid receptors in hippocampus and has extended to other brain regions in both animal models and the human brain with the further discovery of structural and functional adaptive plasticity in response to stressful and other experiences.

We report the updated classification of Inborn Errors of Immunity/Primary Immunodeficiencies, compiled by the International Union of Immunological Societies Expert Committee. This report documents the key clinical and laboratory features of 416 inborn errors of immunity, including 64 gene defects that have either been discovered in the past 2 years since the previous update (published January 2018) or were characterized earlier but have since been confirmed or expanded upon in subsequent studies. The application of next-generation sequencing continues to expedite the rapid identification of novel gene defects, rare or common; broaden the immunological and clinical phenotypes of conditions arising from known gene defects and even known variants; and implement gene-specific therapies. These advances are contributing to greater understanding of the molecular, cellular, and immunological mechanisms of disease, thereby enhancing immunological knowledge while improving the management of patients and their families. This report serves as a valuable resource for the molecular diagnosis of individuals with heritable immunological disorders and also for the scientific dissection of cellular and molecular mechanisms underlying inborn errors of immunity and related human diseases.

Cellular functions are established through biological evolution, but are constrained by the laws of physics. For instance, the physics of protein folding limits the lengths of cellular polypeptide chains. Consequently, many cellular functions are carried out not by long, isolated proteins, but rather by multiprotein complexes. Protein complexes themselves do not escape physical constraints, one of the most important being the difficulty of assembling reliably in the presence of cellular noise. In order to lay the foundation for a theory of reliable protein complex assembly, we study here an equilibrium thermodynamic model of self-assembly that exhibits 4 distinct assembly behaviors: diluted protein solution, liquid mixture, "chimeric assembly," and "multifarious assembly." In the latter regime, different protein complexes can coexist without forming erroneous chimeric structures. We show that 2 conditions have to be fulfilled to attain this regime: 1) The composition of the complexes needs to be sufficiently heterogeneous, and 2) the use of the set of components by the complexes has to be sparse. Our analysis of publicly available databases of protein complexes indicates that cellular protein systems might have indeed evolved so as to satisfy both of these conditions.