The disordered protein SERF promotes α-Synuclein aggregation through liquid-liquid phase separation.

The aggregation of α-Synuclein (α-Syn) into amyloid fibrils is the hallmark of Parkinson's disease. Under stress or other pathological conditions, the accumulation of α-Syn oligomers is the main contributor to the cytotoxicity. A potential approach for treating Parkinson's disease involves preventing the accumulation of these α-Syn oligomers. In this study, we present a novel mechanism involving a conserved group of disorderly proteins known as small EDRK-rich factor (SERF), which promotes the aggregation of α-Syn through a cophase separation process. Using diverse methods like confocal microscopy, fluorescence recovery after photobleaching assays, solution-state NMR spectroscopy, and Western blot, we determined that the N-terminal domain of SERF1a plays a role in the interactions that occur during cophase separation. Within these droplets, α-Syn undergoes a gradual transformation from solid condensates to amyloid fibrils, while SERF1a is excluded from the condensates and dissolves into the solution. Notably, in vivo experiments show that SERF1a cophase separation with α-Syn significantly reduces the deposition of α-Syn oligomers and decreases its cellular toxicity under stress. These findings suggest that SERF1a accelerates the conversion of α-Syn from highly toxic oligomers to less toxic fibrils through cophase separation, thereby mitigating the biological damage of α-Syn aggregation.

Mechanistic basis for receptor-mediated pathological α-synuclein fibril cell-to-cell transmission in Parkinson's disease.

The spread of pathological α-synuclein (α-syn) is a crucial event in the progression of Parkinson's disease (PD). Cell surface receptors such as lymphocyte activation gene 3 (LAG3) and amyloid precursor-like protein 1 (APLP1) can preferentially bind α-syn in the amyloid over monomeric state to initiate cell-to-cell transmission. However, the molecular mechanism underlying this selective binding is unknown. Here, we perform an array of biophysical experiments and reveal that LAG3 D1 and APLP1 E1 domains commonly use an alkaline surface to bind the acidic C terminus, especially residues 118 to 140, of α-syn. The formation of amyloid fibrils not only can disrupt the intramolecular interactions between the C terminus and the amyloid-forming core of α-syn but can also condense the C terminus on fibril surface, which remarkably increase the binding affinity of α-syn to the receptors. Based on this mechanism, we find that phosphorylation at serine 129 (pS129), a hallmark modification of pathological α-syn, can further enhance the interaction between α-syn fibrils and the receptors. This finding is further confirmed by the higher efficiency of pS129 fibrils in cellular internalization, seeding, and inducing PD-like α-syn pathology in transgenic mice. Our work illuminates the mechanistic understanding on the spread of pathological α-syn and provides structural information for therapeutic targeting on the interaction of α-syn fibrils and receptors as a potential treatment for PD.

A Hydroxylamine-Mediated Amidination of Lysine Residues That Retains the Protein's Positive Charge.

Lysine-specific peptide and protein modification strategies are widely used to study charge-related functions and applications. However, these strategies often result in the loss of the positive charge on lysine, significantly impacting the charge-related properties of proteins. Herein, we report a strategy to preserve the positive charge and selectively convert amines in lysine side chains to amidines using nitriles and hydroxylamine under aqueous conditions. Various unprotected peptides and proteins were successfully modified with a high conversion rate. Moreover, the reactive amidine moiety and derived modification site enable subsequent secondary modifications. Notably, positive charges were retained during the modification. Therefore, positive charge-related protein properties, such as liquid-liquid phase separation behaviour of α-synuclein, were not affected. This strategy was subsequently applied to a lysine rich protein to develop an amidine-containing coacervate DNA complex with outstanding mechanical properties. Overall, our innovative strategy provides a new avenue to explore the characteristics of positively charged proteins.

piSTING: A Pocket-Independent Agonist Based on Multivalency-Driven STING Oligomerization.

The stimulator of interferon genes (STING) pathway is a potent therapeutic target for innate immunity. Despite the efforts to develop pocket-dependent small-molecule STING agonists that mimic the endogenous STING ligand, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), most of these agonists showed disappointing results in clinical trials owing to the limitations of the STING pocket. In this study, we developed novel pocket-independent STING-activating agonists (piSTINGs), which act through multivalency-driven oligomerization to activate STING. Additionally, a piSTING-adjuvanted vaccine elicited a significant antibody response and inhibited tumour growth in therapeutic models. Moreover, a piSTING-based vaccine combination with aPD-1 showed remarkable potential to enhance the effectiveness of immune checkpoint blockade (ICB) immunotherapy. In particular, piSTING can strengthen the impact of STING pathway in immunotherapy and accelerate the clinical translation of STING agonists.

A rapid and selective methionine oxidative modification strategy.

Considering the fact that site-selective late-stage diversification of peptides and proteins remains a challenge for biochemistry, strategies targeting low-abundance natural amino acids need to be further developed. As an extremely oxidation-sensitive and low-abundance amino acid, methionine emerges as a promising target for chemo- and site-selective modification. Herein we report an efficient and highly selective modification on methionine residues by one-pot O- and N-transfer reaction, generating sulfoximine-modified peptides with near-perfect conversion within 10 min. Moreover, the great tolerance to other natural amino acids has been demonstrated in reactions with various peptide substrates. To demonstrate the generality of this protocol, we have modified natural peptides and obtained sulfoximination products with high conversion rates. This methodology provides a novel strategy as the expansion of the methionine-based peptide functionalization toolbox.

Parkinson's disease-related phosphorylation at Tyr39 rearranges α-synuclein amyloid fibril structure revealed by cryo-EM.

Posttranslational modifications (PTMs) of α-synuclein (α-syn), e.g., phosphorylation, play an important role in modulating α-syn pathology in Parkinson's disease (PD) and α-synucleinopathies. Accumulation of phosphorylated α-syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to α-syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous α-syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-electron microscopy (cryo-EM) structure of the pY39 α-syn fibril, which reveals a fold of α-syn with pY39 in the center of the fibril core forming an electrostatic interaction network with eight charged residues in the N-terminal region of α-syn. This structure composed of residues 1 to 100 represents the largest α-syn fibril core determined so far. This work provides structural understanding on the pathology of the pY39 α-syn fibril and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.

Chemical modifications of tryptophan residues in peptides and proteins.

Chemical protein modifications facilitate the investigation of natural posttranslational protein modifications and allow the design of proteins with new functions. Proteins can be modified at a late stage on amino acid side chains by chemical methods. The indole moiety of tryptophan residues is an emerging target of such chemical modification strategies because of its unique reactivity and low abundance. This review provides an overview of the recently developed methods of tryptophan modification at the peptide and protein levels.

Phosphorylation and O-GlcNAcylation at the same α-synuclein site generate distinct fibril structures.

α-Synuclein forms amyloid fibrils that are critical in the progression of Parkinson's disease and serves as the pathological hallmark of this condition. Different posttranslational modifications have been identified at multiple sites of α-synuclein, influencing its conformation, aggregation and function. Here, we investigate how disease-related phosphorylation and O-GlcNAcylation at the same α-synuclein site (S87) affect fibril structure and neuropathology. Using semi-synthesis, we obtained homogenous α-synuclein monomer with site-specific phosphorylation (pS87) and O-GlcNAcylation (gS87) at S87, respectively. Cryo-EM revealed that pS87 and gS87 α-synuclein form two distinct fibril structures. The GlcNAc situated at S87 establishes interactions with K80 and E61, inducing a unique iron-like fold with the GlcNAc molecule on the iron handle. Phosphorylation at the same site prevents a lengthy C-terminal region including residues 73 to 140 from incorporating into the fibril core due to electrostatic repulsion. Instead, the N-terminal half of the fibril (1-72) takes on an arch-like fibril structure. We further show that both pS87 and gS87 α-synuclein fibrils display reduced neurotoxicity and propagation activity compared with unmodified α-synuclein fibrils. Our findings demonstrate that different posttranslational modifications at the same site can produce distinct fibril structures, which emphasizes link between posttranslational modifications and amyloid fibril formation and pathology.

14-3-3ζ Participates in the Phase Separation of Phosphorylated and Glycated Tau and Modulates the Physiological and Pathological Functions of Tau.

Tau plays a major role in Alzheimer's disease (AD) and several other neurodegenerative diseases. Tau undergoing liquid-liquid phase separation (LLPS) performs specific physiological functions, induces pathological processes, and contributes to neurodegeneration. Regulating Tau phase separation helps maintain physiological functions of Tau and inhibits pathological aggregation. Here, we show that the 14-3-3 zeta isoform (14-3-3ζ) participates in Tau LLPS. 14-3-3ζ can undergo co-phase separation with WT Tau, participate in and stabilize Tau droplets, and inhibit Tau droplet-driven tubulin assembly. On the other hand, 14-3-3ζ disrupts the LLPS of phosphorylated and glycated Tau, thereby inhibiting the amyloid aggregation initiated by LLPS.

Assessment of the value of 3D-DSA combined with neurointerventional thrombolysis in the treatment of senile cerebrovascular occlusion.

Assessment of the value of three-dimensional digital subtraction angiography (3D-DSA) combined with neurointerventional thrombolysis in the treatment of senile cerebrovascular occlusion was investigated. A total of 129 patients with senile cerebrovascular occlusion admitted to the Affiliated Hospital of Zunyi Medical University from August 2015 to September 2017 were collected. Among them, 69 patients who underwent neurointerventional catheter thrombolysis under 3D-DSA were included in the study group, and 60 patients treated with neurointerventional thrombolysis were the control group. The levels of inflammatory cytokines IL-6, IL-1ß and IL-8 in the two groups were measured by enzyme linked immunosorbent assay (ELISA) before treatment (T0), 7 days (7d) after treatment (T1) and 14 days (14d) after treatment (T2). The score of the National Institute of Health Stroke Scale and the clinical efficacy of patients in the two groups were compared before and after treatment, and Barthel index (BI) was used for investigation before and after treatment. The recurrence rate of disease in the two groups within 1 year was recorded. At T1, IL-6, IL-1ß and IL-8 in the study group were significantly lower than those in the control group (P<0.05). The NIHSS score in the study group was lower than that in the control group after treatment (P<0.05). The BI score in the study group was significantly higher than that in the control group after treatment (P<0.05). After the prognostic follow-up, the disease recurrence rate of the study group was significantly lower than that of the control group (P<0.05). In conclusion, 3D-DSA combined with neurointerventional thrombolysis can significantly reduce the expression of inflammatory cytokines and improve the quality of life in patients with cerebrovascular occlusion, which has a high clinical value.

Late-stage peptide and protein modifications through phospha-Michael addition reaction.

We developed a late-stage modification strategy by a phospha-Michael addition reaction between various functional phosphines and unprotected dehydroalanine (Dha) peptides and proteins under mild conditions. This strategy was applied to generate a staple peptide to enhance its cell membrane penetrability, and it was also able to regulate α-synuclein aggregation properties and morphological characteristics with the addition of different charges.