Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization

Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. The organization of the interface between the kinetochore and the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, remains incompletely understood. A 16-subunit complex, the constitutive centromere associated network (CCAN), bridges CENP-A to the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has also important implications for the mechanism of specific recognition of the CENP-A nucleosome. A supported model depicts the interaction as fuzzy and identifies the disordered CCAN subunit CENP-C as only determinant of specificity. A more speculative model identifies both CENP-C and CENP-N as specificity determinants, but implies CENP-A may be in a hemisome rather than in a classical octamer.

The Nucleocapsid of Paramyxoviruses: Structure and Function of an Encapsidated Template

Viruses of the Paramyxoviridae family share a common and complex molecular machinery for transcribing and replicating their genomes. Their non-segmented, negative-strand RNA genome is encased in a tight homopolymer of viral nucleoproteins (N). This ribonucleoprotein complex, termed a nucleocapsid, is the template of the viral polymerase complex made of the large protein (L) and its co-factor, the phosphoprotein (P). This review summarizes the current knowledge on several aspects of paramyxovirus transcription and replication, including structural and functional data on (1) the architecture of the nucleocapsid (structure of the nucleoprotein, interprotomer contacts, interaction with RNA, and organization of the disordered C-terminal tail of N), (2) the encapsidation of the genomic RNAs (structure of the nucleoprotein in complex with its chaperon P and kinetics of RNA encapsidation in vitro), and (3) the use of the nucleocapsid as a template for the polymerase complex (release of the encased RNA and interaction network allowing the progress of the polymerase complex). Finally, this review presents models of paramyxovirus transcription and replication.

Multivalent interactions make adherens junction–cytoskeletal linkage robust during morphogenesis

Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis, we define molecular mechanisms mediating junction–cytoskeletal linkage and explore the role of mechanosensing. We focus on the junction–cytoskeletal linker Canoe, a multidomain protein. We engineered the canoe locus to define how its domains mediate its mechanism of action. To our surprise, the PDZ and FAB domains, which we thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence, most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.

Structural organization of the C1b projection within the ciliary central apparatus

‘9+2’ motile cilia contain 9 doublet microtubules and a central apparatus (CA) composed of two singlet microtubules with associated projections. The CA plays crucial roles in regulating ciliary motility. Defects in CA assembly or function usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of most CA-projections remain largely unknown. Here, we combined genetic, proteomic, and cryo-electron tomographic approaches to compare the CA of wild-type Chlamydomonas with those of three CA-mutants. Our results show that two proteins, FAP42 and FAP246, are localized to the L-shaped C1b-projection of the CA, where they interact with the candidate CA-protein FAP413. FAP42 is a large protein that forms the peripheral ‘beam’ of the C1b-projection, and the FAP246-FAP413 subcomplex serves as the ‘bracket’ between the beam (FAP42) and the C1b ‘pillar’ that attaches the projection to the C1-microtubule. The FAP246-FAP413-FAP42 complex is essential for stable assembly of the C1b, C1f and C2b-projections, and loss of these proteins leads to ciliary motility defects.

Plectin in Skin Fragility Disorders

Plectin is a multi-faceted, 500 kDa-large protein, which due to its expression in different isoforms and distinct organs acts diversely as a cytoskeletal crosslinker and signaling scaffold. It functions as a mediator of keratinocyte mechanical stability in the skin, primarily through linking intermediate filaments to hemidesmosomes. Skin fragility may occur through the presence of mutations in the gene encoding for plectin, PLEC, or through the presence of autoantibodies against the molecule. Below, we review the cutaneous manifestations of plectinopathies as well as their systemic involvement in specific disease subtypes. We summarize the known roles of plectin in keratinocytes and fibroblasts and provide an outlook on future perspectives for plectin-associated skin disorders.

Immunogenicity Risk Assessment for Multi-specific Therapeutics

The objective of this manuscript is to provide the reader with a hypothetical case study to present an immunogenicity risk assessment for a multi-specific therapeutic as part of Investigational New Drug (IND) application. In order to provide context for the bioanalytical strategies used to support the multi-specific therapeutic presented herein, the introduction focuses on known immunogenicity risk factors. The subsequent hypothetical case study applies these principles to a specific example HC-12, based loosely on anti-TNFα and anti-IL-17A bispecific molecules previously in development, structured as an example immunogenicity risk assessment for submission to health authorities. The risk of higher incidence and safety impact of anti-drug antibodies (ADA) due to large protein complexes is explored in the context of multi-specificity and multi-valency of the therapeutic in combination with the oligomeric forms of the targets.

NLRP3 Inflammasome in Cardiovascular Disease: David`s Stone against Goliath?

Inflammation is involved in initiation, development and complications of the vast majority of non-communicable diseases. Recent research demonstrated that infl ammation is involved in pathogenesis of all major cardiovascular diseases. Different endogenous factors (LDL, nucleic acid strands, uric acid – collectively called „Damage Associated Molecular Patterns – DAMPs”) activate dedicated receptors („Pattern Recognition Receptors – PRR”) on monocytes, macrophages or dendritic cells responsible for the innate immunologic response. They have a major role in natural defense mechanisms against different pathogens and in normal conditions have a protective role. Among PRRs „NOD-like, leucin rich, pyrin containing (NLRP)” receptors are a 14-member family located in the cytoplasm. One of these is the NLRP3 resulting from nuclear transcription under the infl uence of NF-kB, a second messenger from membrane PRRs to the nucleus. Mostly the same factors responsible for NLRP3 intracellular expression stimulate its oligomerization resulting in a large protein complex, the NLRP3 infl ammasome. This activates caspase-1 responsible for IL-1b and IL-18 production and initiates an inflammatory reaction leading to various pathologic processes, such as atherosclerosis, hypertension, diabetes and heart failure. This is the current story as we know it of the NLRP3 infl ammasome, a small intracellular component that when inappropriately activated may does more harm than good.

PARROT is a flexible recurrent neural network framework for analysis of large protein datasets

The rise of high-throughput experiments has transformed how scientists approach biological questions. The ubiquity of large-scale assays that can test thousands of samples in a day has necessitated the development of new computational approaches to interpret this data. Among these tools, machine learning approaches are increasingly being utilized due to their ability to infer complex nonlinear patterns from high-dimensional data. Despite their effectiveness, machine learning (and in particular deep learning) approaches are not always accessible or easy to implement for those with limited computational expertise. Here we present PARROT, a general framework for training and applying deep learning-based predictors on large protein datasets. Using an internal recurrent neural network architecture, PARROT is capable of tackling both classification and regression tasks while only requiring raw protein sequences as input. We showcase the potential uses of PARROT on three diverse machine learning tasks: predicting phosphorylation sites, predicting transcriptional activation function of peptides generated by high-throughput reporter assays, and predicting the fibrillization propensity of amyloid beta with data generated by deep mutational scanning. Through these examples, we demonstrate that PARROT is easy to use, performs comparably to state-of-the-art computational tools, and is applicable for a wide array of biological problems.

Formulasi food bars berbahan baku koro pedang putih (Canavalia ensiformis) autoclaving - cooling

White jackbean is type of bean that grows in tropical and subtropical areas. White jackbean has large protein and complex carbohydrate content. The autoclaving - cooling process can be used to change starch into resistant starch in the white jackbe an flour through gelatinization and retrogradation processes. White jackbean flour treated with autoclaving – cooling process can be used as a raw material for the formulation of food bars. Currently food bars have been in great demand along with the needs to consume healthy and nutritious ready-to-eat food. This study aims to determine the appropriate formulation of white jack bean flour treated with autoclaving – cooling process to produce food bars with the best physical, chemical and organoleptic properties.The results showed that the best formulation is the formulation of white jackbean flour : wheat flour = 60 : 40 with 48.81% carbohydrate content, 14.87% protein content, 16.57% fat content, 17.07% moisture content, 2.68% ash content, 2269.3 gf breaking strength, 6.43 N texture, 79.59 color (L), 15.71 color (a), 17.21 color (b), 4.02 color organoleptic, 3.88 flavor organoleptic, 3.83 aroma organoleptic, 3.83 texture organoleptic. and 11,38% resistant starch content.

A Unique Gene Module in Thermococcales Archaea Centered on a Hypervariable Protein Containing Immunoglobulin Domains

Molecular mechanisms involved in biological conflicts and self vs nonself recognition in archaea remain poorly characterized. We apply phylogenomic analysis to identify a hypervariable gene module that is widespread among Thermococcales. These loci consist of an upstream gene coding for a large protein containing several immunoglobulin (Ig) domains and unique combinations of downstream genes, some of which also contain Ig domains. In the large Ig domain containing protein, the C-terminal Ig domain sequence is hypervariable, apparently, as a result of recombination between genes from different Thermococcales. To reflect the hypervariability, we denote this gene module VARTIG (VARiable Thermococcales IG). The overall organization of the VARTIG modules is similar to the organization of Polymorphic Toxin Systems (PTS). Archaeal genomes outside Thermococcales encode a variety of Ig domain proteins, but no counterparts to VARTIG and no Ig domains with comparable levels of variability. The specific functions of VARTIG remain unknown but the identified features of this system imply three testable hypotheses: (i) involvement in inter-microbial conflicts analogous to PTS, (ii) role in innate immunity analogous to the vertebrate complement system, and (iii) function in self vs nonself discrimination analogous to the vertebrate Major Histocompatibility Complex. The latter two hypotheses seem to be of particular interest given the apparent analogy to the vertebrate immunity.