Stable Macromolecular Complexes Research Articles

An anti-CRISPR that pulls apart a CRISPR-Cas complex.

In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications1. Anti-CRISPRs-proteins that block the activity of CRISPR-Cas systems-are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms2-4. Here, we characterize the anti-CRISPR AcrIF25, and weshow that it inhibits the type I-F CRISPR-Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications.

A Co-based nitrogen-doped lignin carbon catalyst with high stability and wide operating window for rapid degradation of antibiotics

Co-based catalysts play a crucial role in the activation of peroxymonosulfate (PMS) for degradation contaminants. However, the practical application of such catalysts is hindered by challenges like the self-aggregation of Co nanoparticles and leaching of Co2+. In this study, the Co-based catalyst Co-N/C@CL was synthesized from carboxymethylated lignin obtained by grafting abundant carboxymethyl groups into alkali lignin, in which the presence of these carboxymethyl groups enhanced its water solubility and allowed the formation of stable macromolecular complexes with Co2+. This catalyst exhibited a high specific surface area (521.8 m2·g−1) and a uniform distribution of Co nanoparticles. Consequently, the Co-N/C@CL/PMS system could completely remove 20 ppm tetracycline (TC) in 2 min at a rate of 2.404 min−1. Experimental results and DFT calculations revealed that the synergistic effect of lignin carbon and Co NPs accelerated the cleavage and electron transfer of OO bonds, thus promoting the formation of 1O2, OH and SO4−, with 1O2 emerging as the predominant contributor. Moreover, Co-N/C@CL displayed excellent cycling stability and low Co2+ leaching. This work not only provides a feasible strategy for the preparation of highly active and stable Co-based carbon materials but also offers a promising catalyst for the efficient degradation of TC.

The molecular characteristics of DOMs derived from bio-stabilized wastewater activated sludge and its effect on alleviating Cd-stress in rice seedlings (Oryza sativa L.)

To recycle fertilizing contents in wastewater activated sludge (WAS) is attracting increasing interest. Dissolved organic matters (DOMs) in WAS with high content are biologically active. In this work, the molecular composition of DOMs derived from two typical bio-stabilized WAS (DOMBWS), aerobic composting (DOMACS) and anaerobic digestion (DOMADS), were analyzed. The mitigative effect and molecular mechanisms of DOMBWS on rice seedlings (Oryza sativa L.) under Cd-stress were investigated. Our study indicated that DOMBWS significantly alleviated Cd-stress and facilitated growth recovery of rice seedlings with distinct absorption mechanisms. DOMACS, primarily composed of CHO class with low molecular weight rich in carboxyl groups, forming labile Cd-DOM complexes, which promoted Cd-absorption of rice seedlings. While DOMADS comprised large molecular weight of CHON class interacted with Cd to produce stable macromolecular complexes in the form of microaggregates, consequently reducing Cd-absorption. At transcriptional level, DOMBWS restored auxin signal transduction and phenylpropanoid biosynthesis pathways in root cells, and got the expression of glutathione S-transferase well. Besides, DOMACS significantly promoted the metabolism of amino acids to alleviate phytotoxicity, while DOMADS improved the DNA repair function of rice seedlings. These findings provided novel insights into land-use of bio-stabilized WAS for remediation of heavy metals contaminated soils and food security.

The role of AGEs in pathogenesis of cartilage destruction in osteoarthritis.

Osteoarthritis (OA) is a degenerative disease resulting from progressive joint destruction caused by many factors. Its pathogenesis is complex and has not been elucidated to date. Advanced glycation end products (AGEs) are a series of irreversible and stable macromolecular complexes formed by reducing sugar with protein, lipid, and nucleic acid through a non-enzymatic glycosylation reaction (Maillard reaction). They are an important indicator of the degree of ageing. Currently, it is considered that AGEs accumulation in vivo is a molecular basis of age-induced OA, and AGEs production and accumulation in vivo is one of the important reasons for the induction and acceleration of the pathological changes of OA. In recent years, it has been found that AGEs are involved in a variety of pathological processes of OA, including extracellular matrix degradation, chondrocyte apoptosis, and autophagy. Clearly, AGEs play an important role in regulating the expression of OA-related genes and maintaining the chondrocyte phenotype and the stability of the intra-articular environment. This article reviews the latest research results of AGEs in a variety of pathological processes of OA, to provide a new direction for the study of OA pathogenesis and a new target for prevention and treatment.Cite this article: Bone Joint Res 2022;11(5):292–300.

Directed evolution of and structural insights into antibody-mediated disruption of a stable receptor-ligand complex

Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional mechanism where antibody-mediated “disruption” of stable high-affinity macromolecular complexes can potentially enhance therapeutic efficacy. However, this mechanism is not well understood or utilized therapeutically. Here, we investigate and engineer the weak disruptive activity of an existing therapeutic antibody, omalizumab, which targets IgE antibodies to block the allergic response. We develop a yeast display approach to select for and engineer antibody disruptive efficiency and generate potent omalizumab variants that dissociate receptor-bound IgE. We determine a low resolution cryo-EM structure of a transient disruption intermediate containing the IgE-Fc, its partially dissociated receptor and an antibody inhibitor. Our results provide a conceptual framework for engineering disruptive inhibitors for other targets, insights into the failure in clinical trials of the previous high affinity omalizumab HAE variant and anti-IgE antibodies that safely and rapidly disarm allergic effector cells.

A Trans-Omics Comparison Reveals Common Gene Expression Strategies in Four Model Organisms and Exposes Similarities and Differences between Them

The ultimate goal of gene expression regulation is on the protein level. However, because the amounts of mRNAs and proteins are controlled by their synthesis and degradation rates, the cellular amount of a given protein can be attained by following different strategies. By studying omics data for six expression variables (mRNA and protein amounts, plus their synthesis and decay rates), we previously demonstrated the existence of common expression strategies (CESs) for functionally related genes in the yeast Saccharomyces cerevisiae. Here we extend that study to two other eukaryotes: the yeast Schizosaccharomyces pombe and cultured human HeLa cells. We also use genomic data from the model prokaryote Escherichia coli as an external reference. We show that six-variable profiles (6VPs) can be constructed for every gene and that these 6VPs are similar for genes with similar functions in all the studied organisms. The differences in 6VPs between organisms can be used to establish their phylogenetic relationships. The analysis of the correlations among the six variables supports the hypothesis that most gene expression control occurs in actively growing organisms at the transcription rate level, and that translation plays a minor role. We propose that living organisms use CESs for the genes acting on the same physiological pathways, especially for those belonging to stable macromolecular complexes, but CESs have been modeled by evolution to adapt to the specific life circumstances of each organism.

The MORN domain of Junctophilin2 regulates functional interactions with small-conductance Ca2+ -activated potassium channel subtype2 (SK2).

Small-conductance Ca2+ -activated K+ channel subtype2 (SK2) are stable macromolecular complexes that regulate myocardial excitability and Ca2+ homeostasis. Junctophilin-2 (JP2) is a membrane-binding protein, which provides functional crosstalk by physically linking with the cell-surface and intracellular ion channels. We previously demonstrated that the MORN domain of JP2 interacts with SK2 channels. However, the roles of the JP2 MORN domain in regulating the precise subcellular localization and molecular modulation of SK2 have not yet been incompletely understood. In the present study, in vitro and in vivo assays were used to confirm the physical interactions between the SK2 channel and JP2 in H9c2 and HEK293 cells, with a concentration on the association between the C-terminus of SK2 channels and the MORN domain of JP2. Furthermore, the membrane expression of SK2 were found to be significantly impaired by the mutation or knockdown of JP2. Using immunofluorescence staining along with Golgi/early endosome markers, we studied the mechanisms of JP2-regulated SK2 membrane trafficking, which indicates that the JP2 MORN domain is probably necessary for the retrograde trafficking of SK2 channels. The functional study demonstrates that whole cell SK2 current densities recorded from the HEK293 cells co-expressing the JP2-MORN domain with SK2 were significantly augmented, compared with cells expressing SK2 alone. Our findings suggest that the MORN domain of JP2 directly modulates SK2 channel current amplitude and trafficking, through its interaction with an overlapping region of the JP2 MORN domain on the SK2 C-terminus.

Integration of macromolecular complex data into the Saccharomyces Genome Database.

Proteins seldom function individually. Instead, they interact with other proteins or nucleic acids to form stable macromolecular complexes that play key roles in important cellular processes and pathways. One of the goals of Saccharomyces Genome Database (SGD; www.yeastgenome.org) is to provide a complete picture of budding yeast biological processes. To this end, we have collaborated with the Molecular Interactions team that provides the Complex Portal database at EMBL-EBI to manually curate the complete yeast complexome. These data, from a total of 589 complexes, were previously available only in SGD’s YeastMine data warehouse (yeastmine.yeastgenome.org) and the Complex Portal (www.ebi.ac.uk/complexportal). We have now incorporated these macromolecular complex data into the SGD core database and designed complex-specific reports to make these data easily available to researchers. These web pages contain referenced summaries focused on the composition and function of individual complexes. In addition, detailed information about how subunits interact within the complex, their stoichiometry and the physical structure are displayed when such information is available. Finally, we generate network diagrams displaying subunits and Gene Ontology annotations that are shared between complexes. Information on macromolecular complexes will continue to be updated in collaboration with the Complex Portal team and curated as more data become available.

Critical role of HLA-DRβ* binding peptides' peripheral flanking residues in fully-protective malaria vaccine development

A vaccine candidate component must fit perfectly into the antigen presenting HLA-DRβ* molecule's groove (or canonical nonapeptide) peptide binding region (PBR) during antigen presentation to the T-cell receptor (TCR), conforming a specific and stable macromolecular complex and induce an appropriate immune response.Antigen's peripheral flanking residues (PFR, positions (p) -p2 and p10) must thus establish strong interactions with the HLA-DRβ* - TCR complex. These amino acids (aa) have specific physico-chemical characteristics enabling differentiation between non-protective but antibody-inducer (NPAI), short-lived protection inducer (SLPI) and long-lasting protection inducer (LLPI) peptides when used as an antimalarial vaccine component. Their identification (through 1H-NMR and Aotus monkey immunization) and proper modification contributes to a logical and rational methodology for long-lasting and protective immunological memory.

The effect of stable macromolecular complexes of ionic polyphosphazene on HIV Gag antigen and on activation of human dendritic cells and presentation to T-cells

Neonates and infants are susceptible to infection due to distinct immune responses in early life. Therefore, development of vaccine formulation and delivery systems capable of activating human newborn leukocytes is of global health importance. Poly[di(carboxylatophenoxy)phosphazene] (PCPP) belongs to a family of ionic synthetic polyphosphazene polyelectrolyte compounds that can form non-covalent interactions with protein antigens and demonstrate adjuvant activity in animals and in human clinical trials. However, little is known about their ability to activate human immune cells. In this study, we characterized the effects of PCPP alone or in combination with a model antigen (recombinant HIV-Gag (Gag)), on the maturation, activation and antigen presentation by human adult and newborn dendritic cells (DCs) in vitro. PCPP treatment induced DC activation as assessed by upregulation of co-stimulatory molecules and cytokine production. Studies benchmarking PCPP to Alum, the most commonly used vaccine adjuvant, demonstrated that both triggered cell death and release of danger signals in adult and newborn DCs. When complexed with Gag antigen, PCPP maintained its immunostimulatory characteristics while permitting internalization and presentation of Gag by DCs to HIV-Gag-specific CD4+ T cell clones. The PCPP vaccine formulation outlined here has intrinsic adjuvant activity, can facilitate effective delivery of antigen to DCs, and may be advantageous for induction of beneficial T cell-mediated immunity. Moreover, polyphosphazenes can further reduce cost of vaccine production and distribution through their dose-sparing and antigen-stabilizing properties, thus potentially eliminating the need for cold chain distribution.

CIITA: A Master Regulator of Adaptive Immunity Shows Its Innate Side in the Bone

CIITA (the Major Histocompatibility Class (MHC) II transactivator) has long been known as the master regulator of MHC Class II genes, which are critical for normal immune function (reviewed in (1, 2)). CIITA is a non-DNA binding co-activator that specifically regulates expression of Major Histocompatibility Class II molecules, with CIITA deficiency leading to the rare human immunodeficiency disease termed Bare Lymphocyte Syndrome (3). CIITA also regulates expression of genes encoding accessory proteins required for MHCII-restricted antigen presentation, thus CIITA is central to controlling the response to foreign antigens and the maintenance of tolerance (1, 2). In this issue of the Journal of Bone and Mineral Research, Benasciutti and colleagues demonstrate that CIITA is also a major regulator of osteoclast differentiation and bone homeostasis (4).

A-type lamins maintain the positional stability of DNA damage repair foci in mammalian nuclei.

A-type lamins encoded by LMNA form a structural fibrillar meshwork within the mammalian nucleus. How this nuclear organization may influence the execution of biological processes involving DNA transactions remains unclear. Here, we characterize changes in the dynamics and biochemical interactions of lamin A/C after DNA damage. We find that DNA breakage reduces the mobility of nucleoplasmic GFP-lamin A throughout the nucleus as measured by dynamic fluorescence imaging and spectroscopy in living cells, suggestive of incorporation into stable macromolecular complexes, but does not induce the focal accumulation of GFP-lamin A at damage sites. Using a proximity ligation assay and biochemical analyses, we show that lamin A engages chromatin via histone H2AX and its phosphorylated form (γH2AX) induced by DNA damage, and that these interactions are enhanced after DNA damage. Finally, we use three-dimensional time-lapse imaging to show that LMNA inactivation significantly reduces the positional stability of DNA repair foci in living cells. This defect is partially rescued by the stable expression of GFP-lamin A. Thus collectively, our findings suggest that the dynamic structural meshwork formed by A-type lamins anchors sites of DNA repair in mammalian nuclei, providing fresh insight into the control of DNA transactions by nuclear structural organization.

A proteolytic cleavage to separate the sarcolemma/T‐tubule from the sarcoplasmic reticulum

A proteolytic cleavage to separate the sarcolemma/T‐tubule from the sarcoplasmic reticulum

Small-Conductance Ca2+-Activated K+ Channels: Form and Function

Small-conductance Ca(2+)-activated K(+) channels (SK channels) are widely expressed throughout the central nervous system. These channels are activated solely by increases in intracellular Ca(2+). SK channels are stable macromolecular complexes of the ion pore-forming subunits with calmodulin, which serves as the intrinsic Ca(2+) gating subunit, as well as with protein kinase CK2 and protein phosphatase 2A, which modulate Ca(2+) sensitivity. Well-known for their roles in regulating somatic excitability in central neurons, SK channels are also expressed in the postsynaptic membrane of glutamatergic synapses, where their activation and regulated trafficking modulate synaptic transmission and the induction and expression of synaptic plasticity, thereby affecting learning and memory. In this review we discuss the molecular and functional properties of SK channels and their physiological roles in central neurons.

Biosynthesis, Localization, and Macromolecular Arrangement of the Plasmodium falciparum Translocon of Exported Proteins (PTEX)

To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA(+) ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ∼600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.

Ca2+-Activated K+Channels: From Protein Complexes to Function

Molecular research on ion channels has demonstrated that many of these integral membrane proteins associate with partner proteins, often versatile in their function, or even assemble into stable macromolecular complexes that ensure specificity and proper rate of the channel-mediated signal transduction. Calcium-activated potassium (K(Ca)) channels that link excitability and intracellular calcium concentration are responsible for a wide variety of cellular processes ranging from regulation of smooth muscle tone to modulation of neurotransmission and control of neuronal firing pattern. Most of these functions are brought about by interaction of the channels' pore-forming subunits with distinct partner proteins. In this review we summarize recent insights into protein complexes associated with K(Ca) channels as revealed by proteomic research and discuss the results available on structure and function of these complexes and on the underlying protein-protein interactions. Finally, the results are related to their significance for the function of K(Ca) channels under cellular conditions.

Inference of Macromolecular Assemblies from Crystalline State

We discuss basic physical–chemical principles underlying the formation of stable macromolecular complexes, which in many cases are likely to be the biological units performing a certain physiological function. We also consider available theoretical approaches to the calculation of macromolecular affinity and entropy of complexation. The latter is shown to play an important role and make a major effect on complex size and symmetry. We develop a new method, based on chemical thermodynamics, for automatic detection of macromolecular assemblies in the Protein Data Bank (PDB) entries that are the results of X-ray diffraction experiments. As found, biological units may be recovered at 80–90% success rate, which makes X-ray crystallography an important source of experimental data on macromolecular complexes and protein–protein interactions. The method is implemented as a public WWW service †. † http://www.ebi.ac.uk/msd-srv/prot int/pistart.html

Carbohydrate-appended curdlans as a new family of glycoclusters with binding properties both for a polynucleotide and lectins

Beta-1,3-glucans having carbohydrate-appendages (alpha-D-mannoside, N-acetyl-beta-D-glucosaminide and beta-lactoside) at the C6-position of every repeating unit can be readily prepared from curdlan (a linear beta-1,3-glucan) through regioselective bromination/azidation to afford 6-azido-6-deoxycurdlan followed by chemo-selective Cu(i)-catalyzed [3 + 2]-cycloaddition with various carbohydrate modules having a terminal alkyne. The resultant carbohydrate-appended curdlans can interact with polycytosine to form stable macromolecular complexes consistent with two polysaccharide strands and one polycytosine strand. Furthermore, these macromolecular complexes show strong and specific affinity toward carbohydrate-binding proteins (lectins). Therefore, one can utilize these carbohydrate-appended curdlans as a new family of glycoclusters.

Water-Soluble Complexes Formed by Natural Polyphenols and Bovine Serum Albumin: Evidence from Gel Electrophoresis

The formation of water-soluble complexes from combinations of various polyphenols and bovine serum albumin (BSA) was analyzed by gel electrophoresis. Sodium dodecylsulfate (SDS) and native polyacrylamide gel electrophoretic (PAGE) analyses clearly showed complexes formed from BSA with oenothein B, corilagin, (+)-catechin, procyanidin B3, and gallic acid derivatives; however, it was difficult to distinguish between the complexes containing a single BSA molecule and the BSA molecule itself by using size-exclusion chromatography (SEC). Combining SDS and native PAGE analyses showed instability in the macromolecular complexes formed from pentagalloylglucose (PGG) and BSA. Research also revealed that the oxidation of (-)-epigallocatechin gallate (EGCG) contributed to the formation of more stable macromolecular complexes from EGCG and BSA.

Poly(N-substituted-acrylamide)-branched Schizophyllans Are Useful for Selective Recovery of Homopolynucleotides through Convenient and Quick Precipitation Procedures

Schizophyllans carrying poly(N-substituted-acrylamide)-branches were synthesized from native schizophyllan through Ce(IV)-initiated radical homo- or co-polymerization of N-iso-propyl- and N-sec-butyl-acrylamide. A series of structural analyses including elemental analysis, gel permeation chromatography, 1H NMR, and enzymatic degradation using β-1,3-glucanase revealed that schizophyllans (150 kDa) bearing long poly(N-substituted-acrylamide)-branches (ca. 290 kDa) are obtained. These SPG-conjugates form macromolecular complexes with certain homopolynucleotides such as poly(C), poly(dA), and poly(A) and then efficiently co-precipitate them on heating. For example, schizophyllan carrying poly(N-iso-propylacrylamide)-branches can selectively recover poly(C) from a mixture of poly(C) and poly(U) by simple heating (36 °C) followed by centrifugation. HPLC analysis of the recovered polynucleotide revealed the high poly(C)-selectivity (poly(C):poly(U) = 95:5). Similarly, schizophyllan carrying more hydrophobic poly(N-iso-propylacrylamide-co-N-sec-butylacrylamide)-branches precipitates at lower temperature (ca. 20 °C) and is applicable to isolation of poly(A) that forms less stable macromolecular complexes with SPG and SPG-derivatives (the dissociation temperatures are around 30 °C).