Multiprotein Assemblies Research Articles

The role and regulation of integrins in cell migration and invasion.

Integrin receptors are the main molecular link between cells and the extracellular matrix (ECM) as well as mediating cell-cell interactions. Integrin-ECM binding triggers the formation of heterogeneous multi-protein assemblies termed integrin adhesion complexes (IACs) that enable integrins to transform extracellular cues into intracellular signals that affect many cellular processes, especially cell motility. Cell migration is essential for diverse physiological and pathological processes and is dysregulated in cancer to favour cell invasion and metastasis. Here, we discuss recent findings on the role of integrins in cell migration with a focus on cancer cell dissemination. We review how integrins regulate the spatial distribution and dynamics of different IACs, covering classical focal adhesions, emerging adhesion types and adhesion regulation. We discuss the diverse roles integrins have during cancer progression from cell migration across varied ECM landscapes to breaching barriers such as the basement membrane, and eventual colonization of distant organs.

IrCLIP-RNP and Re-CLIP reveal patterns of dynamic protein associations on RNA.

RNA binding proteins ( RBPs ) control varied processes, including RNA splicing, stability, transport, and translation 1-3 . Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease 1,4,5 , however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation 6 was combined with mass spectrometry ( irCLIP-RNP ) to identify RNA-dependent associated proteins ( RDAPs ) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor ( EGF ), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP ( Re-CLIP ) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and detected simultaneous HNRNPC and UPF1 co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.

Multi-protein assemblies orchestrate co-translational enzymatic processing on the human ribosome

Nascent chains undergo co-translational enzymatic processing as soon as their N-terminus becomes accessible at the ribosomal polypeptide tunnel exit (PTE). In eukaryotes, N-terminal methionine excision (NME) by Methionine Aminopeptidases (MAP1 and MAP2), and N-terminal acetylation (NTA) by N-Acetyl-Transferase A (NatA), is the most common combination of subsequent modifications carried out on the 80S ribosome. How these enzymatic processes are coordinated in the context of a rapidly translating ribosome has remained elusive. Here, we report two cryo-EM structures of multi-enzyme complexes assembled on vacant human 80S ribosomes, indicating two routes for NME-NTA. Both assemblies form on the 80S independent of nascent chain substrates. Irrespective of the route, NatA occupies a non-intrusive ‘distal’ binding site on the ribosome which does not interfere with MAP1 or MAP2 binding nor with most other ribosome-associated factors (RAFs). NatA can partake in a coordinated, dynamic assembly with MAP1 through the hydra-like chaperoning function of the abundant Nascent Polypeptide-Associated Complex (NAC). In contrast to MAP1, MAP2 completely covers the PTE and is thus incompatible with NAC and MAP1 recruitment. Together, our data provide the structural framework for the coordinated orchestration of NME and NTA in protein biogenesis.

Mapping the Multiscale Proteomic Organization of Cellular and Disease Phenotypes.

While the primary sequences of human proteins have been cataloged for over a decade, determining how these are organized into a dynamic collection of multiprotein assemblies, with structures and functions spanning biological scales, is an ongoing venture. Systematic and data-driven analyses of these higher-order structures are emerging, facilitating the discovery and understanding of cellular phenotypes. At present, knowledge of protein localization and function has been primarily derived from manual annotation and curation in resources such as the Gene Ontology, which are biased toward richly annotated genes in the literature. Here, we envision a future powered by data-driven mapping of protein assemblies. These maps can capture and decode cellular functions through the integration of protein expression, localization, and interaction data across length scales and timescales. In this review, we focus on progress toward constructing integrated cell maps that accelerate the life sciences and translational research.

Comprehensive landscape of integrator complex subunits and their association with prognosis and tumor microenvironment in gastric cancer.

The integrator complex (INT) is a multiprotein assembly in gene transcription. Although several subunits of INT complex have been implicated in multiple cancers, the complex's role in gastric cancer (GC) is poorly understood. The gene expressions, prognostic values, and the associations with microsatellite instability (MSI) of INT subunits were confirmed by GEO and The Cancer Genome Atlas (TCGA) databases. cBioPortal, GeneMANIA, TISIDB, and MCPcounter algorithm were adopted to investigate the mutation frequency, protein-protein interaction network, and the association with immune cells of INT subunits in GC. Additionally, in vitro experiments were performed to confirm the role of INTS11 in pathogenesis of GC. The mRNA expression levels of INTS2/4/5/7/8/9/10/11/12/13/14 were significantly elevated both in GSE183904 and TCGA datasets. Through functional enrichment analysis, the functions of INT subunits were mainly associated with snRNA processing, INT, and DNA-directed 5'-3' RNA polymerase activity. Moreover, these INT subunit expressions were associated with tumor-infiltrating lymphocytes and MSI in GC. In vitro experiments demonstrated that knockdown of the catalytic core INTS11 in GC cells inhibits cell proliferation ability. INTS11 overexpression showed opposite effects. Our data demonstrate that the INT complex might act as an oncogene and can be used as a prognosis biomarker for GC.

Delineating redox cooperativity in water-soluble and membrane multiheme cytochromes through protein design.

Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to delineate the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.

Isolation and Partial Characterization of Novel, Structurally Uniform (Hfq6)n≥8 Assemblies Carrying Accessory Transcription and Translation Factors.

In growing E. coli cells, the transcription-translation complexes (TTCs) form characteristic foci; however, the exact molecular composition of these superstructures is not known with certainty. Herein, we report that, during our recently developed "fast" procedures for purification of E. coli RNA polymerase (RP), a fraction of the RP's α/RpoA subunits is displaced from the core RP complexes and copurifies with multiprotein superstructures carrying the nucleic acid-binding protein Hfq and the ribosomal protein S6. We show that the main components of these large multiprotein assemblies are fixed protein copy-number (Hfq6)n≥8 complexes; these complexes have a high level of structural uniformity and are distinctly unlike the previously described (Hfq6)n "head-to-tail" polymers. We describe purification of these novel, structurally uniform (Hfq6)n≥8 complexes to near homogeneity and show that they also contain small nonprotein molecules and accessory S6. We demonstrate that Hfq, S6, and RP have similar solubility profiles and present evidence pointing to a role of the Hfq C-termini in superstructure formation. Taken together, our data offer new insights into the composition of the macromolecular assemblies likely acting as scaffolds for transcription complexes and ribosomes during bacterial cells' active growth.

KAT8 beyond Acetylation: A Survey of Its Epigenetic Regulation, Genetic Variability, and Implications for Human Health.

Lysine acetyltransferase 8, also known as KAT8, is an enzyme involved in epigenetic regulation, primarily recognized for its ability to modulate histone acetylation. This review presents an overview of KAT8, emphasizing its biological functions, which impact many cellular processes and range from chromatin remodeling to genetic and epigenetic regulation. In many model systems, KAT8's acetylation of histone H4 lysine 16 (H4K16) is critical for chromatin structure modification, which influences gene expression, cell proliferation, differentiation, and apoptosis. Furthermore, this review summarizes the observed genetic variability within the KAT8 gene, underscoring the implications of various single nucleotide polymorphisms (SNPs) that affect its functional efficacy and are linked to diverse phenotypic outcomes, ranging from metabolic traits to neurological disorders. Advanced insights into the structural biology of KAT8 reveal its interaction with multiprotein assemblies, such as the male-specific lethal (MSL) and non-specific lethal (NSL) complexes, which regulate a wide range of transcriptional activities and developmental functions. Additionally, this review focuses on KAT8's roles in cellular homeostasis, stem cell identity, DNA damage repair, and immune response, highlighting its potential as a therapeutic target. The implications of KAT8 in health and disease, as evidenced by recent studies, affirm its importance in cellular physiology and human pathology.

Structure-Based Protein Assembly Simulations Including Various Binding Sites and Conformations.

Many biological functions are mediated by large complexes formed by multiple proteins and other cellular macromolecules. Recent progress in experimental structure determination, as well as in integrative modeling and protein structure prediction using deep learning approaches, has resulted in a rapid increase in the number of solved multiprotein assemblies. However, the assembly process of large complexes from their components is much less well-studied. We introduce a rapid computational structure-based (SB) model, GoCa, that allows to follow the assembly process of large multiprotein complexes based on a known native structure. Beyond existing SB Go̅-type models, it distinguishes between intra- and intersubunit interactions, allowing us to include coupled folding and binding. It accounts automatically for the permutation of identical subunits in a complex and allows the definition of multiple minima (native) structures in the case of proteins that undergo global transitions during assembly. The model is successfully tested on several multiprotein complexes. The source code of the GoCa program including a tutorial is publicly available on Github: https://github.com/ZachariasLab/GoCa. We also provide a web source that allows users to quickly generate the necessary input files for a GoCa simulation: https://goca.t38webservices.nat.tum.de.

How Artificial Intelligence Unravels the Complex Web of Cancer Drug Response.

The intersection of precision medicine and artificial intelligence (AI) holds profound implications for cancer treatment, with the potential to significantly advance our understanding of drug responses based on the intricate architecture of tumor cells. A recent study by Park and colleagues titled "A Deep Learning Model of Tumor Cell Architecture Elucidates Response and Resistance to CDK4/6 Inhibitors" epitomizes this intersection by leveraging an interpretable deep learning model grounded in a comprehensive map of multiprotein assemblies in cancer, known as Nested Systems in Tumors. This study not only elucidates mechanisms underlying the response to CDK4/6 inhibitors in breast cancer therapy but also highlights the critical role of model interpretability leading to new mechanistic insights.

ZMIZ1 enhances ERα-dependent expression of E2F2 in breast cancer.

The estrogen receptor-α (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1-2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 h. Gene set enrichment analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1-ER-E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumour cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER-ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance.

A deep learning model of tumor cell architecture elucidates response and resistance to CDK4/6 inhibitors

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6is) have revolutionized breast cancer therapy. However, <50% of patients have an objective response, and nearly all patients develop resistance during therapy. To elucidate the underlying mechanisms, we constructed an interpretable deep learning model of the response to palbociclib, a CDK4/6i, based on a reference map of multiprotein assemblies in cancer. The model identifies eight core assemblies that integrate rare and common alterations across 90 genes to stratify palbociclib-sensitive versus palbociclib-resistant cell lines. Predictions translate to patients and patient-derived xenografts, whereas single-gene biomarkers do not. Most predictive assemblies can be shown by CRISPR–Cas9 genetic disruption to regulate the CDK4/6i response. Validated assemblies relate to cell-cycle control, growth factor signaling and a histone regulatory complex that we show promotes S-phase entry through the activation of the histone modifiers KAT6A and TBL1XR1 and the transcription factor RUNX1. This study enables an integrated assessment of how a tumor’s genetic profile modulates CDK4/6i resistance.

MIMAS is a new giant multifunctional player in the mitochondrial megacomplex playground

Mitochondria are rich in multi-protein assemblies that are usually dedicated to one function. In this issue of Cell Reports, Horten etal.1 describe a 3-nanometer megacomplex in the mitochondrial inner membrane, which serves multiple functions integrating mitochondria biogenesis and metabolism.

Self-lysis microbial consortia for predictable multi-proteins assembly

The scope of bioengineering is expanding from the design of single strain to the microbial communities, allowing for the division-of-labor in synthesizing the multi-protein systems. Predicting the composition of the final product during the biomanufacturing process, however, can be difficult. Consortia-based manufacturing has the potential to boost production efficiency, but this benefit primarily holds in the upstream. The current format of downstream process heavily relies on the centralized facility, and is not economical and flexible to address the demands in small-scale. Here, we present a concise and manageable platform to enable the multi-protein system assembly. We engineer a self-lysis microbial consortium, where each strain lyses autonomously at high densities and produces a single protein component. The product fraction can be precisely tuned by varying the inoculation ratio. Utilizing this platform, we assemble a classical 34-component PURE (protein synthesis using recombinant elements) system. We have further optimized the downstream process of the biomanufacturing by incorporating the porous structure of polymeric materials. The encapsulated autolysis consortium can produce and release the proteins while maintaining the cell factories enclosed in the materials by exporting the multi-protein system for collection. Our research provides a novel approach to the flexible and controllable production of multi-protein systems, opening up new possibilities for pathway assembly and portable biomanufacturing.

Evolution of techniques and tools for replication fork proteome and protein interaction studies.

Understanding the complex network of protein-protein interactions (PPI) that govern cellular functions is essential for unraveling the molecular basis of biological processes and diseases. Mass spectrometry (MS) has emerged as a powerful tool for studying protein dynamics, enabling comprehensive analysis of protein function, structure, post-translational modifications, interactions, and localization. This article provides an overview of MS techniques and their applications in proteomics studies, with a focus on the replication fork proteome. The replication fork is a multi-protein assembly involved in DNA replication, and its proper functioning is crucial for maintaining genomic integrity. By combining quantitative MS labeling techniques with various data acquisition methods, researchers have made significant strides in elucidating the complex processes and molecular mechanisms at the replication fork. Overall, MS has revolutionized our understanding of protein dynamics, offering valuable insights into cellular processes and potential targets for therapeutic interventions.

Trabid patient mutations impede the axonal trafficking of adenomatous polyposis coli to disrupt neurite growth

ZRANB1 (human Trabid) missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterized these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organization and neural development. Zranb1 knock-in mice harboring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalized causing impaired organization of the cytoskeleton that underlie the neuronal and developmental phenotypes.

Trabid patient mutations impede the axonal trafficking of adenomatous polyposis coli to disrupt neurite growth.

ZRANB1 (human Trabid) missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterized these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organization and neural development. Zranb1 knock-in mice harboring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalized causing impaired organization of the cytoskeleton that underlie the neuronal and developmental phenotypes.

Cysteine desulfurase (IscS)-mediated fine-tuning of bioenergetics and SUF expression prevents Mycobacterium tuberculosis hypervirulence.

Iron-sulfur (Fe-S) biogenesis requires multiprotein assembly systems, SUF and ISC, in most prokaryotes. M. tuberculosis (Mtb) encodes a complete SUF system, the depletion of which was bactericidal. The ISC operon is truncated to a single gene iscS (cysteine desulfurase), whose function remains uncertain. Here, we show that MtbΔiscS is bioenergetically deficient and hypersensitive to oxidative stress, antibiotics, and hypoxia. MtbΔiscS resisted killing by nitric oxide (NO). RNA sequencing indicates that IscS is important for expressing regulons of DosR and Fe-S-containing transcription factors, WhiB3 and SufR. Unlike wild-type Mtb, MtbΔiscS could not enter a stable persistent state, continued replicating in mice, and showed hypervirulence. The suf operon was overexpressed in MtbΔiscS during infection in a NO-dependent manner. Suppressing suf expression in MtbΔiscS either by CRISPR interference or upon infection in inducible NO-deficient mice arrests hypervirulence. Together, Mtb redesigned the ISC system to "fine-tune" the expression of SUF machinery for establishing persistence without causing detrimental disease in the host.

Maturation of the [FeFe]‐Hydrogenase: Direct Transfer of the (κ3‐cysteinate)FeII(CN)(CO)2 Complex B from HydG to HydE

Abstract[FeFe]‐hydrogenases efficiently catalyze the reversible oxidation of molecular hydrogen. Their prowess stems from the intricate H‐cluster, combining a [Fe4S4] center with a binuclear iron center ([2Fe]H). In the latter, each iron atom is coordinated by a CO and CN ligand, connected by a CO and an azadithiolate ligand. The synthesis of this active site involves a unique multiprotein assembly, featuring radical SAM proteins HydG and HydE. HydG initiates the transformation of L‐tyrosine into cyanide and carbon monoxide to generate complex B, which is subsequently transferred to HydE to continue the biosynthesis of the [2Fe]H‐subcluster. Due to its instability, complex B isolation for structural or spectroscopic characterization has been elusive thus far. Nevertheless, the use of a biomimetic analogue of complex B allowed circumvention of the need for the HydG protein during in vitro functional investigations, implying a similar structure for complex B. Herein, we used the HydE protein as a nanocage to encapsulate and stabilize the complex B product generated by HydG. Using X‐ray crystallography, we successfully determined its structure at 1.3 Å resolution. Furthermore, we demonstrated that complex B is directly transferred from HydG to HydE, thus not being released into the solution post‐synthesis, highlighting a transient interaction between the two proteins.

Maturation of the [FeFe]-Hydrogenase: Direct Transfer of the (κ3 -cysteinate)FeII (CN)(CO)2 Complex B from HydG to HydE.

[FeFe]-hydrogenases efficiently catalyze the reversible oxidation of molecular hydrogen. Their prowess stems from the intricate H-cluster, combining a [Fe4 S4 ] center with a binuclear iron center ([2Fe]H ). In the latter, each iron atom is coordinated by a CO and CN ligand, connected by a CO and an azadithiolate ligand. The synthesis of this active site involves a unique multiprotein assembly, featuring radical SAM proteins HydG and HydE. HydG initiates the transformation of L-tyrosine into cyanide and carbon monoxide to generate complex B, which is subsequently transferred to HydE to continue the biosynthesis of the [2Fe]H -subcluster. Due to its instability, complex B isolation for structural or spectroscopic characterization has been elusive thus far. Nevertheless, the use of a biomimetic analogue of complex B allowed circumvention of the need for the HydG protein during in vitro functional investigations, implying a similar structure for complex B. Herein, we used the HydE protein as a nanocage to encapsulate and stabilize the complex B product generated by HydG. Using X-ray crystallography, we successfully determined its structure at 1.3 Å resolution. Furthermore, we demonstrated that complex B is directly transferred from HydG to HydE, thus not being released into the solution post-synthesis, highlighting a transient interaction between the two proteins.