Cryo-EM structure of the Smc5/6 holo-complex

The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral integration. Here, we report the cryo-EM structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells --providing a full overview of the complex in its apo / non-liganded form, and revealing how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the "W-loop" of Smc4 or "F-loop" of Smc1, mediates an essential interaction with Nse1. Taken together, these data confirm a degree of functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.

Stabilization of the SARS-CoV-2 Receptor Binding Domain by Protein Core Redesign and Deep Mutational Scanning

Stabilizing antigenic proteins as vaccine immunogens or diagnostic reagents is a stringent case of protein engineering and design as the exterior surface must maintain recognition by receptor(s) and antigen specific antibodies at multiple distinct epitopes. This is a challenge, as stability-enhancing mutations must be focused on the protein core, whereas successful computational stabilization algorithms typically select mutations at solvent-facing positions. In this study we report the stabilization of SARS-CoV-2 Wuhan Hu-1 Spike receptor binding domain (S RBD) using a combination of deep mutational scanning and computational design, including the FuncLib algorithm. Our most successful design encodes I358F, Y365W, T430I, and I513L RBD mutations, maintains recognition by the receptor ACE2 and a panel of different anti-RBD monoclonal antibodies, is between 1-2°C more thermally stable than the original RBD using a thermal shift assay, and is less proteolytically sensitive to chymotrypsin and thermolysin than the original RBD. Our approach could be applied to the computational stabilization of a wide range of proteins without requiring detailed knowledge of active sites or binding epitopes, particularly powerful for cases when there are multiple or unknown binding sites.

Protein Core Fucosylation Regulates Planarian Head Regeneration via Neoblast Proliferation

Protein glycosylation is an important posttranslational modification that plays a crucial role in cellular function. However, its biological roles in tissue regeneration remain interesting and primarily ambiguous. In this study, we profiled protein glycosylation during head regeneration in planarian Dugesia japonica using a lectin microarray. We found that 6 kinds of lectins showed increased signals and 16 kinds showed decreased signals. Interestingly, we found that protein core fucosylation, manifested by Lens culinaris agglutinin (LCA) staining, was significantly upregulated during planarian head regeneration. Lectin histochemistry indicated that the LCA signal was intensified within the wound and blastemal areas. Furthermore, we found that treatment with a fucosylation inhibitor, 2F-peracetyl-fucose, significantly retarded planarian head regeneration, while supplement with L-fucose could improve DjFut8 expression and stimulate planarian head regeneration. In addition, 53 glycoproteins that bound to LCA were selectively isolated by LCA-magnetic particle conjugates and identified by LC-MS/MS, including the neoblast markers DjpiwiA, DjpiwiB, DjvlgA, and DjvlgB. Overall, our study provides direct evidence for the involvement of protein core fucosylation in planarian regeneration.

Protein structural consequences of DNA mutational signatures: A meta-analysis of somatic variants and deep mutational scanning data

Signatures of DNA motifs associated with distinct mutagenic exposures have been defined for somatic variants, but little is known about the consequences different mutational processes pose to the cancer cell, particularly the distribution of the resulting variants in the implied proteins and their structural regions (surface, core, interacting interface). Here we first compare the protein-level consequences of six mutational signatures (Aging, APOBEC, POLE, UV, 5-FU and Platinum) characterised by clear DNA motif preferences. By mapping individual substitution events observed in tumours to three-dimensional protein structures, we show that these common somatic mutational signatures are biased against the protein core, consistent with the lower tolerability of substitutions at such structurally important regions. On the other hand, deep mutational scanning (DMS) data allow us to probe the "dark matter" of somatic mutational landscape, exploring variants which are otherwise removed in purifying selection. A computational DMS analysis identifies mutational contexts (5'-G/C[T>G]A/G-3') which are associated with damaging mutations, by altering physicochemical characteristics of amino acids at the protein core. We argue that comprehensive DMS analysis can contribute to classification of variants according to their true impact to the stability/activity of the affected protein, decoupling this from pathogenicity prediction offered by conventional variant impact classifiers.

Novel ARG1 variants identified in a patient with arginase 1 deficiency

We report a case of a 13-year-old boy with arginase 1 deficiency carrying a new variant in ARG1. Sanger sequencing identified the compound heterozygous variants: NM_000045.4: c.365G>A (p.Trp122*)/c.820G>A (p.Asp274Asn). Although not previously reported, the p.Asp274Asn variant is predicted to have strong pathogenicity because it is located in a highly conserved domain in the protein core and arginase activity in the patient was below measurement sensitivity.

Development of an M cell targeted nanocomposite system for effective oral protein delivery: preparation, in vitro and in vivo characterization

Background There is a strong need for non-invasive and patient-friendly delivery systems of protein drugs for long-term therapy. However, oral delivery of protein drugs is a big challenge due to many barriers including instability in the gastrointestinal (GI) tract and low permeability. To overcome the absorption barriers in GI tract and improve the patient compliance, this study aimed to develop an M cell targeted-nanocomposite delivery system of protein drugs. Results An aminoclay-protein core complex (AC-Ins) was prepared by using insulin as a model protein and then sequentially coated with Ulex europaeus agglutinin 1 (UEA-1) for M-cell targeting and the pH sensitive polymer, Eudragit® L100 (EUAC-Ins). All nanoparticles were obtained with a high entrapment efficiency (> 90%) and their structural characteristics were confirmed by Fourier transform-infrared spectroscopy, energy dispersive X-ray spectroscopy, and circular dichroism. Among the developed nanoparticles, EUAC-Ins effectively suppressed drug release at pH 1.2, while rapidly released drugs at pH 6.8 due to dissolution of the outer coating layer. The conformational stability of insulin entrapped in EUAC-Ins was well maintained in the presence of proteolytic enzymes. Compared to free insulin, EUAC-Ins increased the membrane transport of insulin by 4.4-fold in M cells. In parallel, oral administration of EUAC-Ins in mice enhanced insulin uptake by 4.1-fold in the intestinal Peyer’s patches and 2.6-fold in intestinal epithelium tissues with normal villi, compared to free insulin. Orally administered EUAC-Ins decreased significantly the blood glucose level in diabetic mice, while the effect of oral insulin solution was negligible. Conclusion An M cell targeted-ternary nanocomposite system obtained by dual coating of the aminoclay-protein core complex with UEA-1 and a pH dependent polymer is promising as an effective oral protein delivery carrier.

The Release of a Soluble Glycosylated Protein from Glycogen by Recombinant Lysosomal α-Glucosidase (rhGAA) In Vitro and Its Presence in Serum In Vivo

In studies on the degradation of glycogen by rhGAA, a glycosylated protein core material was found which consists of about 5–6% of the total starting glycogen. There was an additional 25% of the glycogen unaccounted for based on glucose released. After incubation of glycogen with rhGAA until no more glucose was released, no other carbohydrate was detected on HPAEC-PAD. Several oligosaccharides are then detectable if the medium is first boiled in 0.1 N HCl or incubated with trypsin. It is present in serum either in an HCl extract or in a trypsin digest. The characteristics of the in vivo serum material are identical to the material in the in vitro incubation medium. One oligosaccharide cannot be further degraded by rhGAA, from the incubation medium as well as from serum co-elute on HPAEC-PAD. Several masked oligosaccharides in serum contain m-inositol, e-inositol, and sorbitol as the major carbohydrates. The presence of this glycosylated protein in serum is a fraction of glycogen that is degraded outside the lysosome and the cell. The glycosylated protein in the serum is not present in the serum of Pompe mice not on ERT, but it is present in the serum of Pompe disease patients who are on ERT, so it is a biomarker of GAA degradation of lysosomal glycogen.