SRCP1 Conveys Resistance to Polyglutamine Aggregation.

The polyglutamine (polyQ) diseases are a group of nine neurodegenerative diseases caused by the expansion of a polyQ tract that results in protein aggregation. Unlike other model organisms, Dictyostelium discoideum is a proteostatic outlier, naturally encoding long polyQ tracts yet resistant to polyQ aggregation. Here we identify serine-rich chaperone protein 1 (SRCP1) as a molecular chaperone that is necessary and sufficient to suppress polyQ aggregation. SRCP1 inhibits aggregation of polyQ-expanded proteins, allowing for their degradation via the proteasome, where SRCP1 is also degraded. SRCP1's C-terminal domain is essential for its activity in cells, and peptides that mimic this domain suppress polyQ aggregation in vitro. Together our results identify a novel type of molecular chaperone and reveal how nature has dealt with the problem of polyQ aggregation.

Off-the-shelf cryopreserved platelets for the detection of HIT and VITT antibodies.

Heparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.

Chemical Regulation of the Protein Quality Control E3 Ubiquitin Ligase C-Terminus of Hsc70 Interacting Protein (CHIP).

The ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) is an important regulator of proteostasis. Despite playing an important role in maintaining proteostasis, little progress has been made in developing small molecules that regulate ubiquitin transfer by CHIP. Here we used differential scanning fluorimetry to identify compounds that bound CHIP. Compounds that bound CHIP were then analyzed by quantitative ubiquitination assays to identify those that altered CHIP function. One compound, MS.001, inhibited both the chaperone binding and ubiquitin ligase activity of CHIP at low micromolar concentrations. Interestingly, we found that MS.001 did not have activity against isolated U-box or tetratricopeptide (TPR) domains, but instead only inhibited full-length CHIP. Using in silico docking we identified a potential MS.001 binding site on the linker domain of CHIP and mutation of this site rendered CHIP resistant to MS.001. Together our data identify an inhibitor of the E3 ligase CHIP and provides insight into the development of compounds that regulate CHIP activity.

Platelet and myeloid cell phenotypes in a rat model of Fabry disease.

Fabry disease results from a deficiency of the lysosomal enzyme ⍺-Galactosidase-A (⍺-Gal A) and is estimated to occur in approximately 1:4100 live births. Characteristic of the disease is the accumulation of α-Gal-A substrates, primarily the glycosphingolipids (GSLs) globotriaosylceramide and globotriaosylsphingosine. Thrombotic events are a significant concern for Fabry patients, with strokes contributing to a significant decrease in overall lifespan. Currently, the mechanisms underlying the increased risk of thrombotic events experienced by Fabry patients are incompletely defined. Using a rat model of Fabry disease, we provide an improved understanding of the mechanisms linking GSL accumulation to thrombotic risk. We found that ⍺-Gal A-deficient rats accumulate myeloid-derived leukocytes at sites of GSL accumulation, including in the bone marrow and circulation, and that myeloid-derived leukocyte and megakaryocyte populations were prominent among cell types that accumulated GSLs. In the circulation, ⍺-Gal A-deficient rats had increases in cytokine-producing cell types and a corresponding elevation of pro-inflammatory cytokines. Lastly, circulating platelets from ⍺-Gal A-deficient rats accumulated a similar set of ⍺-Galactosidase-A substrates as was observed in megakaryocytes in the bone marrow, and exhibited increased platelet binding to fibrinogen in microfluidic and flow cytometric assays.

Persistence of Ad26.COV2.S-associated vaccine-induced immune thrombotic thrombocytopenia (VITT) and specific detection of VITT antibodies.

Rare cases of COVID-19 vaccinated individuals develop anti-platelet factor 4 (PF4) antibodies that cause thrombocytopenia and thrombotic complications, a syndrome referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). Currently, information on the characteristics and persistence of anti-PF4 antibodies that cause VITT after Ad26.COV2.S vaccination is limited, and available diagnostic assays fail to differentiate Ad26.COV2.S and ChAdOx1 nCoV-19-associated VITT from similar clinical disorders, namely heparin-induced thrombocytopenia (HIT) and spontaneous HIT. Here we demonstrate that while Ad26.COV2.S-associated VITT patients are uniformly strongly positive in PF4-polyanion enzyme-linked immunosorbent assays (ELISAs); they are frequently negative in the serotonin release assay (SRA). The PF4-dependent p-selectin expression assay (PEA) that uses platelets treated with PF4 rather than heparin consistently diagnosed Ad26.COV2.S-associated VITT. Most Ad26.COV2.S-associated VITT antibodies persisted for >5 months in PF4-polyanion ELISAs, while the PEA became negative earlier. Two patients had otherwise unexplained mild persistent thrombocytopenia (140-150 x 103 /µL) 6 months after acute presentation. From an epidemiological perspective, differentiating VITT from spontaneous HIT, another entity that develops in the absence of proximate heparin exposure, and HIT is important, but currently available PF4-polyanion ELISAs and functional assay are non-specific and detect all three conditions. Here, we report that a novel un-complexed PF4 ELISA specifically differentiates VITT, secondary to both Ad26.COV2.S and ChAdOx1 nCoV-19, from both spontaneous HIT, HIT and commonly-encountered HIT-suspected patients who are PF4/polyanion ELISA-positive but negative in functional assays. In summary, Ad26.COV2.S-associated VITT antibodies are persistent, and the un-complexed PF4 ELISA appears to be both sensitive and specific for VITT diagnosis.

Most mutations that cause spinocerebellar ataxia autosomal recessive type 16 (SCAR16) destabilize the protein quality-control E3 ligase CHIP.

The accumulation of misfolded proteins promotes protein aggregation and neuronal death in many neurodegenerative diseases. To counteract misfolded protein accumulation, neurons have pathways that recognize and refold or degrade aggregation-prone proteins. One U-box-containing E3 ligase, C terminus of Hsc70-interacting protein (CHIP), plays a key role in this process, targeting misfolded proteins for proteasomal degradation. CHIP plays a protective role in mouse models of neurodegenerative disease, and in humans, mutations in CHIP cause spinocerebellar ataxia autosomal recessive type 16 (SCAR16), a fatal neurodegenerative disease characterized by truncal and limb ataxia that results in gait instability. Here, we systematically analyzed CHIP mutations that cause SCAR16 and found that most SCAR16 mutations destabilize CHIP. This destabilization caused mutation-specific defects in CHIP activity, including increased formation of soluble oligomers, decreased interactions with chaperones, diminished substrate ubiquitination, and reduced steady-state levels in cells. Consistent with decreased CHIP stability promoting its dysfunction in SCAR16, most mutant proteins recovered activity when the assays were performed below the mutants' melting temperature. Together, our results have uncovered the molecular basis of genetic defects in CHIP function that cause SCAR16. Our insights suggest that compounds that improve the thermostability of genetic CHIP variants may be beneficial for treating patients with SCAR16.

USP5 Is Dispensable for Monoubiquitin Maintenance in Drosophila.

Ubiquitination is a post-translational modification that regulates most cellular pathways and processes, including degradation of proteins by the proteasome. Substrate ubiquitination is controlled at various stages, including through its reversal by deubiquitinases (DUBs). A critical outcome of this process is the recycling of monoubiquitin. One DUB whose function has been proposed to include monoubiquitin recycling is USP5. Here, we investigated whether Drosophila USP5 is important for maintaining monoubiquitin in vivo We found that the fruit fly orthologue of USP5 has catalytic preferences similar to its human counterpart and that this DUB is necessary during fly development. Our biochemical and genetic experiments indicate that reduction of USP5 does not lead to monoubiquitin depletion in developing flies. Also, introduction of exogenous ubiquitin does not suppress developmental lethality caused by loss of endogenous USP5. Our work indicates that a primary physiological role of USP5 is not to recycle monoubiquitin for reutilization, but that it may involve disassembly of conjugated ubiquitin to maintain proteasome function.

Primary macrophages rely on histone deacetylase 1 and 2 expression to induce type I interferon in response to gammaherpesvirus infection.

Type I interferon is induced shortly following viral infection and represents a first line of host defense against a majority of viral pathogens. Not surprisingly, both replication and latency of gammaherpesviruses, ubiquitous cancer-associated pathogens, are attenuated by type I interferon, although the mechanism of attenuation remains poorly characterized. Gammaherpesviruses also target histone deacetylases (HDACs), a family of pleiotropic enzymes that modify gene expression and several cell signaling pathways. Specifically, we have previously shown that a conserved gammaherpesvirus protein kinase interacts with HDAC1 and -2 to promote gammaherpesvirus replication in primary macrophages. In the current study, we have used genetic approaches to show that expression of HDAC1 and -2 is critical for induction of a type I interferon response following gammaherpesvirus infection of primary macrophages. Specifically, expression of HDAC1 and -2 was required for phosphorylation of interferon regulatory factor 3 (IRF3) and accumulation of IRF3 at the beta interferon promoter in gammaherpesvirus-infected primary macrophages. To our knowledge, this is the first demonstration of a specific role for HDAC1 and -2 in the induction of type I interferon responses in primary immune cells following virus infection. Furthermore, because HDAC1 and -2 are overexpressed in several types of cancer, our findings illuminate potential side effects of HDAC1- and -2-specific inhibitors that are currently under development as cancer therapy agents. IMPORTANCE Gammaherpesviruses establish chronic infection in a majority of the adult population and are associated with several malignancies. Infected cells counteract gammaherpesvirus infection via innate immune signaling mediated primarily through type I interferon. The induction of type I interferon expression proceeds through several stages using molecular mechanisms that are still incompletely characterized. In this study, we show that expression of HDAC1 and -2 by macrophages is required to mount a type I interferon response to incoming gammaherpesvirus. The involvement of HDAC1 and -2 in the type I interferon response highlights the pleiotropic roles of these enzymes in cellular signaling. Interestingly, HDAC1 and -2 are deregulated in cancer and are attractive targets of new cancer therapies. Due to the ubiquitous and chronic nature of gammaherpesvirus infection, the role of HDAC1 and -2 in the induction of type I interferon responses should be considered during the clinical development of HDAC1- and -2-specific inhibitors.

Transfusion-related acute lung injury-associated HNA-3a antibodies recognize complex determinants on choline transporter-like protein 2.

BACKGROUND: HNA-3a-specific antibodies can cause severe, sometimes fatal, transfusion-related acute lung injury when present in transfused blood. The HNA3-a/b antigens are determined by an R154Q polymorphism in the first of five extracellular (EC) loops of the 10-membrane-spanning choline transporter-like protein 2 (CTL2) expressed on neutrophils, lymphocytes, and other tissues. Approximately 50% of HNA-3a antibodies (Type 1) can be detected using CTL2 Loop 1 peptides containing R154; the remaining 50% (Type 2) fail to recognize this target. Understanding the basis for this difference could guide efforts to develop practical assays to screen blood donors for HNA-3 antibodies. STUDY DESIGN AND METHODS: Reactions of HNA-3a antibodies against recombinant versions of human, mouse, and human/mouse (chimeric) CTL2 were characterized using flow cytometry and various solid-phase assays. RESULTS: The findings show that, for binding to CTL2, Type 2 HNA-3a antibodies require nonpolymorphic amino acid residues in the third, and possibly the second, EC loops of CTL2 to be in a configuration comparable to that found naturally in the cell membrane. In contrast, Type 1 antibodies require only peptides from the first EC loop that contain R154 for recognition. CONCLUSION: Although Type 1 HNA-3a antibodies can readily be detected in solid-phase assays that use a CTL2 peptide containing R154 as a target, development of a practical test to screen blood donors for Type 2 antibodies will pose a serious technical challenge because of the complex nature of the epitope(s) recognized by this antibody subgroup.

Full-length recombinant choline transporter-like protein 2 containing arginine 154 reconstitutes the epitope recognized by HNA-3a antibodies.

BACKGROUND: Recent reports have shown that the HNA-3a leukocyte antigen, a target for antibodies that cause severe transfusion-related acute lung injury, correlates with an arginine 154 (rather than glutamine) polymorphism in choline transporter-like protein 2 (CTL2) but did not show directly that R154 determines HNA-3a. CTL2 peptides containing R154 are recognized by only half of HNA-3a antibodies studied to date. Constructs that react with all HNA-3a antibodies are needed to fully define the HNA-3a epitope. STUDY DESIGN AND METHODS: HEK293 cells were transfected with cDNA encoding full-length CTL2 linked to green fluorescent protein (GFP). Transfectants were selected for GFP expression and tested with antibodies specific for HNA-3a and -3b. RESULTS: Each of 20 HNA-3a antibodies reacted preferentially with HEK293 cells expressing the R154 CTL2 construct. An HNA-3b antibody reacted only with CTL2 (Q154). CONCLUSIONS: These findings provide direct evidence that R154 in the context of full-length CTL2 is both necessary and sufficient to create the HNA-3a epitope but suggest that posttranslational modifications of the protein, for example, S-S bonds or addition of glycans, are necessary for recognition of HNA-3a by many antibodies. This could complicate development of an assay for large-scale screening of blood donors to detect anti-HNA-3a.

Vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is primarily a complication of adenoviral vector-based covid-19 vaccination. In VITT, thrombocytopenia and thrombosis mediated by anti-platelet factor 4 (PF4) antibodies can be severe, often characterized by thrombosis at unusual sites such as the cerebral venous sinus and splanchnic circulation. Like in heparin-induced thrombocytopenia (HIT) and spontaneous HIT, VITT antibodies recognize PF4-polyanion complexes and activate PF4-treated platelets but additionally bind to un-complexed PF4, a critical finding that could be leveraged for more specific detection of VITT. Intravenous immunoglobulin and non-heparin-based anticoagulation remain the mainstay of treatment. Second dose/boosters of mRNA covid-19 vaccines appear safe in patients with adenoviral vector-associated VITT. Emerging data is consistent with the possibility that ultra-rare cases of VITT may be seen in the setting of mRNA and virus-like particle (VLP) technology-based vaccinations and until more data is available, it is prudent to consider VITT in the differential diagnosis of all post-vaccine thrombosis and thrombocytopenia reactions.

Progress in the understanding and treatment of Fabry disease.

BACKGROUND: Fabry disease is caused by α-galactosidase A deficiency. Substrates of this lysosomal enzyme accumulate, resulting in cellular dysfunction. Patients experience neuropathic pain, kidney failure, heart disease, and strokes. SCOPE OF REVIEW: The clinical picture and molecular features of Fabry disease are described, along with updates on disease mechanisms, animal models, and therapies. MAJOR CONCLUSIONS: How the accumulation of α-galactosidase A substrates, mainly glycosphingolipids, leads to organ damage is incompletely understood. Enzyme replacement and chaperone therapies are clinically available to patients, while substrate reduction, mRNA-based, and gene therapies are on the horizon. Animal models exist to optimize these therapies and elucidate disease mechanisms for novel treatments. GENERAL SIGNIFICANCE: Recent newborn screening studies demonstrate that Fabry disease is the most common lysosomal storage disease. As many countries now include Fabry disease in their screening panels, the number of identified patients is expected to increase significantly. Better knowledge of disease pathogenesis is needed to improve treatment options.

Fine specificity of drug-dependent antibodies reactive with a restricted domain of platelet GPIIIA.

Drug-induced immune thrombocytopenia is caused by drug-dependent antibodies (DDAbs) that bind tightly to platelet glycoproteins only when drug is present. How drugs mediate this interaction is not yet resolved. Several studies indicate that sites recognized by DDAbs tend to cluster in specific structural domains, suggesting they may recognize a limited number of distinct epitopes. To address this issue, we characterized the binding sites for 16 quinine-dependent antibodies thought on the basis of preliminary studies to be possibly specific for a single epitope on glycoprotein IIIa (GPIIIa). Fourteen of the antibodies reacted with a 29-kDa GPIIIa fragment comprising only the GPIIIa hybrid and plextrin-semaphorin-integrin homology domains. However, studies with mutant GPIIIa and the blocking monoclonal antibody AP3 showed that the 14 DDAbs recognize at least 6 and possibly more distinct, but overlapping, structures involving GPIIIa residues 50 to 66. The findings suggest that even antibodies specific for restricted domains on a target glycoprotein may each have a slightly different fine specificity; ie, "unique" epitopes recognized by DDAbs may be rare or nonexistent. The observations are consistent with a recently proposed model in which drug reacts noncovalently with both target protein and antibody to promote binding of an otherwise nonreactive immunoglobulin.