Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS.

N-Linked glycosylation in hemagglutinin and neuraminidase glycoproteins of influenza viruses affects antigenic and receptor binding properties, and precise analyses of site-specific glycoforms in these proteins are critical in understanding the antigenic and immunogenic properties of influenza viruses. In this study, we developed a glycoproteomic approach by using a timsTOF Pro mass spectrometer (MS) to determine the abundance and heterogeneity of site-specific glycosylation for influenza glycoproteins. Compared with a Q Exactive HF MS, the timsTOF Pro MS method without the hydrophilic interaction liquid chromatography column enrichment achieved similar glycopeptide coverage and quantities but was more effective in identifying low-abundance glycopeptides. We quantified the distributions of intact site-specific glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9) and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans at N411 and, however, that the type and quantities of glycans were distinct between these viruses. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. In summary, timsTOF Pro MS method can quantify intact site-specific glycans for influenza glycoproteins without enrichment and thus facilitate influenza vaccine development and production.

A Comprehensive Study of Gradient Conditions for Deep Proteome Discovery in a Complex Protein Matrix.

Bottom-up mass-spectrometry-based proteomics is a well-developed technology based on complex peptide mixtures from proteolytic cleavage of proteins and is widely applied in protein identification, characterization, and quantitation. A tims-ToF mass spectrometer is an excellent platform for bottom-up proteomics studies due to its rapid acquisition with high sensitivity. It remains challenging for bottom-up proteomics approaches to achieve 100% proteome coverage. Liquid chromatography (LC) is commonly used prior to mass spectrometry (MS) analysis to fractionate peptide mixtures, and the LC gradient can affect the peptide fractionation and proteome coverage. We investigated the effects of gradient type and time duration to find optimal gradient conditions. Five gradient types (linear, logarithm-like, exponent-like, stepwise, and step-linear), three different gradient lengths (22 min, 44 min, and 66 min), two sample loading amounts (100 ng and 200 ng), and two loading conditions (the use of trap column and no trap column) were studied. The effect of these chromatography variables on protein groups, peptides, and spectral counts using HeLa cell digests was explored. The results indicate that (1) a step-linear gradient performs best among the five gradient types studied; (2) the optimal gradient duration depends on protein sample loading amount; (3) the use of a trap column helps to enhance protein identification, especially low-abundance proteins; (4) MSFragger and PEAKS Studio have high similarity in protein group identification; (5) MSFragger identified more protein groups among the different gradient conditions compared to PEAKS Studio; and (6) combining results from both database search engines can expand identified protein groups by 9-11%.

Identification and quantification of bioactive compounds suppressing SARS-CoV-2 signals in wastewater-based epidemiology surveillance.

Recent SARS-CoV-2 wastewater-based epidemiology (WBE) surveillance have documented a positive correlation between the number of COVID-19 patients in a sewershed and the level of viral genetic material in the wastewater. Efforts have been made to use the wastewater SARS-CoV-2 viral load to predict the infected population within each sewershed using a multivariable regression approach. However, reported clear and sustained variability in SARS-CoV-2 viral load among treatment facilities receiving industrial wastewater have made clinical prediction challenging. Several classes of molecules released by regional industries and manufacturing facilities, particularly the food processing industry, can significantly suppress the SARS-CoV-2 signals in wastewater by breaking down the lipid-bilayer of the membranes. Therefore, a systematic ranking process in conjugation with metabolomic analysis was developed to identify the wastewater treatment facilities exhibiting SARS-CoV-2 suppression and identify and quantify the chemicals suppressing the SARS-COV-2 signals. By ranking the viral load per diagnosed case among the sewersheds, we successfully identified the wastewater treatment facilities in Missouri, USA that exhibit SARS-CoV-2 suppression (significantly lower than 5 × 1011 gene copies/reported case) and determined their suppression rates. Through both untargeted global chemical profiling and targeted analysis of wastewater samples, 40 compounds were identified as candidates of SARS-CoV-2 signal suppressors. Among these compounds, 14 had higher concentrations in wastewater treatment facilities that exhibited SARS-CoV-2 signal suppression compared to the unsuppressed control facilities. Stepwise regression analyses indicated that 4-nonylphenol, palmitelaidic acid, sodium oleate, and polyethylene glycol dioleate are positively correlated with SARS-CoV-2 signal suppression rates. Suppression activities were further confirmed by incubation studies, and the suppression kinetics for each bioactive compound were determined. According to the results of these experiments, bioactive molecules in wastewater can significantly reduce the stability of SARS-CoV-2 genetic marker signals. Based on the concentrations of these chemical suppressors, a correction factor could be developed to achieve more reliable and unbiased surveillance results for wastewater treatment facilities that receive wastewater from similar industries.

Impact of Web-Based Cognitive Behavioral Therapy for Insomnia on Stress, Health, Mood, Cognitive, Inflammatory, and Neurodegenerative Outcomes in Rural Dementia Caregivers: Protocol for the NiteCAPP CARES and NiteCAPP SHARES Randomized Controlled Trial.

BACKGROUND: Chronic insomnia affects up to 63% of family dementia caregivers. Research suggests that chronic insomnia prompts changes in central stress processing that have downstream negative effects on health and mood, as well as on cognitive, inflammatory, and neurodegenerative functioning. We hypothesize that cognitive behavioral therapy for insomnia (CBT-I) will reverse those downstream effects by improving insomnia and restoring healthy central stress processing. Rural caregivers are particularly vulnerable, but they have limited access to CBT-I; therefore, we developed an accessible digital version using community input (NiteCAPP CARES). OBJECTIVE: This trial will evaluate the acceptability, feasibility, and short-term and long-term effects of NiteCAPP CARES on the sleep and stress mechanisms underlying poor caregiver health and functioning. METHODS: Dyads (n=100) consisting of caregivers with chronic insomnia and their coresiding persons with dementia will be recruited from Columbia and surrounding areas in Missouri, United States. Participant dyads will be randomized to 4 weeks (plus 4 bimonthly booster sessions) of NiteCAPP CARES or a web-based sleep hygiene control (NiteCAPP SHARES). Participants will be assessed at baseline, after treatment, and 6- and 12-month follow-ups. The following assessments will be completed by caregivers: 1 week of actigraphy and daily diaries measuring sleep, Insomnia Severity Index, arousal (heart rate variability), inflammation (blood-derived biomarkers: interleukin-6 and C-reactive protein), neurodegeneration (blood-derived biomarkers: plasma amyloid beta [Aß40 and Aß42], total tau, and phosphorylated tau [p-tau181 and p-tau217]), cognition (Joggle battery, NIH Toolbox for Assessment of Neurological and Behavioral Function, and Cognitive Failures Questionnaire), stress and burden, health, and mood (depression and anxiety). Persons with dementia will complete 1 week of actigraphy at each time point. RESULTS: Recruitment procedures started in February 2022. All data are expected to be collected by 2026. Full trial results are planned to be published by 2027. Secondary analyses of baseline data will be subsequently published. CONCLUSIONS: This randomized controlled trial tests NiteCAPP CARES, a web-based CBT-I for rural caregivers. The knowledge obtained will address not only what outcomes improve but also how and why they improve and for how long, which will help us to modify NiteCAPP CARES to optimize treatment potency and support future pragmatic testing and dissemination. TRIAL REGISTRATION: ClinicalTrials.gov NCT04896775; https://clinicaltrials.gov/ct2/show/NCT04896775. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/37874.

Transdermal Delivery of High Molecular Weight Antibiotics to Deep Tissue Infections via Droplette Micromist Technology Device (DMTD).

Wound infection by multidrug-resistant (MDR) bacteria is a major disease burden. Systemic administration of broad-spectrum antibiotics colistin methanesulfonate (CMS) and vancomycin are the last lines of defense against deep wound infections by MDR bacteria. However, systemic administration of CMS and vancomycin are linked to life-threatening vital organ damage. Currently there are no effective topical application strategies to deliver these high molecular weight antibiotics across the stratum corneum. To overcome this difficulty, we tested if high molecular weight antibiotics delivered by Droplette micromist technology device (DMTD), a transdermal delivery device that generates a micromist capable of packaging large molecules, could attenuate deep skin tissue infections. Using green fluorescent protein-tagged E. coli and live tissue imaging, we show that (1) the extent of attenuation of deep-skin E. coli infection was similar when treated with topical DMTD- or systemic IP (intraperitoneal)-delivered CMS; (2) DMTD-delivered micromist did not spread the infection deeper; (3) topical DMTD delivery and IP delivery resulted in similar levels of vancomycin in the skin after a 2 h washout period; and (4) IP-delivered vancomycin was about 1000-fold higher in kidney and plasma than DMTD-delivered vancomycin indicating systemic toxicity. Thus, topical DMTD delivery of these antibiotics is a safe treatment for the difficult-to-treat deep skin tissue infections by MDR bacteria.

Deletion of Specific Conserved Motifs from the N-Terminal Domain of αB-Crystallin Results in the Activation of Chaperone Functions.

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54-61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54-61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21-28 residues from the activated αBΔ54-61 (to get αBΔ21-28, Δ54-61) on the structure-function of recombinant αBΔ21-28, Δ54-61. The αBΔ21-28, Δ54-61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21-28, ∆54-61 was found to prevent ß-amyloid (Aß1-42) fibril formation in vitro and suppressed Aß1-42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54-61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54-61 in oxidatively stressed cells. Our study shows that the residues 21-28 and 54-61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21-28 residues further potentiates the activated αBΔ54-61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21-28, Δ54-61.

Integrative proteomics and phosphoproteomics reveals phosphorylation networks involved in the maintenance and expression of embryogenic competence in sugarcane callus.

Plant embryogenic cell culture allows mass propagation and genetic manipulation, but the mechanisms that determine the fate of these totipotent cells in somatic embryos have not yet been elucidated. Here, we performed label-free quantitative proteomics and phosphoproteomics analyses to determine signaling events related to sugarcane somatic embryo differentiation, especially those related to protein phosphorylation. Embryogenic calli were compared at multiplication (EC0, dedifferentiated cells) and after 14 days of maturation (EC14, onset of embryo differentiation). Metabolic pathway analysis showed enriched lysine degradation and starch/sucrose metabolism proteins during multiplication, whereas the differentiation of somatic embryos was found to involve the enrichment of energy metabolism, including the TCA cycle and oxidative phosphorylation. Multiplication-related phosphoproteins were associated with transcriptional regulation, including SNF1 kinase homolog 10 (KIN10), SEUSS (SEU), and LEUNIG_HOMOLOG (LUH). The regulation of multiple light harvesting complex photosystem II proteins and phytochrome interacting factor 3-LIKE 5 were predicted to promote bioenergetic metabolism and carbon fixation during the maturation stage. A motif analysis revealed 15 phosphorylation motifs. The [D-pS/T-x-D] motif was overrepresented during somatic embryo differentiation. A protein-protein network analysis predicted interactions among SNF1-related protein kinase 2 (SnRK2), abscisic acid-responsive element-binding factor 2 (ABF2), and KIN10, which indicated the role of these proteins in embryogenic competence. The predicted interactions between TOPLESS (TPL) and histone deacetylase 19 (HD19) may be involved in posttranslational protein regulation during somatic embryo differentiation. These results reveal the protein regulation dynamics of somatic embryogenesis and new players in somatic embryo differentiation, including their predicted phosphorylation motifs and phosphosites.

Label-Free Quantitative Phosphoproteomics Reveals Signaling Dynamics Involved in Embryogenic Competence Acquisition in Sugarcane.

In this study, a label-free quantitative phosphoproteomic analysis was performed to identify and quantify signaling events related to the acquisition of embryogenic competence in sugarcane. Embryogenic and nonembryogenic calli were compared at the multiplication phase, resulting in the identification of 163 phosphoproteins unique to embryogenic calli, 9 unique to nonembryogenic calli, and 51 upregulated and 40 downregulated in embryogenic calli compared to nonembryogenic calli. Data are available via ProteomeXchange with identifier PXD018054. Motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx], and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. The embryogenic-related phosphoproteins (those unique and upregulated in embryogenic calli) identified in the present study are related to abscisic acid-induced signaling and abiotic stress response; they include OSK3, ABF1, LEAs, and RD29Bs. On the other hand, the nonembryogenic-related phosphoproteins EDR1 and PP2Ac-2 are negative regulators of abscisic acid signaling, suggesting a relationship between phosphoproteins involved in the abscisic acid and stress responses in the acquisition of embryogenic competence. Moreover, embryogenic-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TOPLESS, and with RNA metabolism, including AGO1, DEAH5, SCL30, UB2C, and SR45, were identified to play potential roles in embryogenic competence. These results reveal novel phosphorylation sites for several proteins and identify potential candidate biomarkers for the acquisition of embryogenic competence in sugarcane.

Quantitative Proteomic Analysis of Chikungunya Virus-Infected Aedes aegypti Reveals Proteome Modulations Indicative of Persistent Infection.

The mosquito-borne chikungunya virus (CHIKV) poses a threat to human health in tropical countries throughout the world. The molecular interactions of CHIKV with its mosquito vector Aedes aegypti are not fully understood. Following oral acquisition of CHIKV via salinemeals, we analyzed changes in the proteome of Ae. aegypti in 12 h intervals by label-free quantification using a timsTOF Pro mass spectrometer. For each of the seven time points, between 2647 and 3167 proteins were identified among CHIKV-infected and noninfected mosquito samples, and fewer than 6% of those identified proteins were affected by the virus. Functional enrichment analysis revealed that the three pathways, Endocytosis, Oxidative phosphorylation, and Ribosome biogenesis, were enriched during CHIKV infection. On the other hand, three pathways of the cellular RNA machinery and five metabolism related pathways were significantly attenuated in the CHIKV-infected samples. Furthermore, proteins associated with cytoskeleton and vesicular transport, as well as various serine-type endopeptidases and metallo-proteinases, were modulated in the presence of CHIKV. Our study reveals biological pathways and novel proteins interacting with CHIKV in the mosquito. Overall, CHIKV infection caused minor changes to the mosquito proteome demonstrating a high level of adaption between the vector and the virus, essentially coexisting in a nonpathogenic relationship. The mass spectrometry data have been deposited to the MassIVE repository (https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=abfd14f7015243c69854731998d55df1) with the data set identifier MSV000085115.

Quantitative Proteomics Reveals Docosahexaenoic Acid-Mediated Neuroprotective Effects in Lipopolysaccharide-Stimulated Microglial Cells.

The high levels of docosahexaenoic acid (DHA) in cell membranes within the brain have led to a number of studies exploring its function. These studies have shown that DHA can reduce inflammatory responses in microglial cells. However, the method of action is poorly understood. Here, we report the effects of DHA on microglial cells stimulated with lipopolysaccharides (LPSs). Data were acquired using the parallel accumulation serial fragmentation method in a hybrid trapped ion mobility-quadrupole time-of-flight mass spectrometer. Over 2800 proteins are identified using label-free quantitative proteomics. Cells exposed to LPSs and/or DHA resulted in changes in cell morphology and expression of 49 proteins with differential abundance (greater than 1.5-fold change). The data provide details about pathways that are influenced in this system including the nuclear factor κ-light-chain-enhancer of the activated B cells (NF-κB) pathway. Western blots and enzyme-linked immunosorbent assay studies are used to help confirm the proteomic results. The MS data are available at ProteomeXchange.

Defects in long-chain 3-hydroxy acyl-CoA dehydrogenase lead to hepatocellular carcinoma: A novel etiology of hepatocellular carcinoma.

The incidence of both nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) have been increasing at an alarming rate. Little is known about NAFLD without cirrhosis as a risk for HCC. Here we report, for the first time, generation of a mouse model with a defect in long-chain 3-hydoxy acyl-CoA dehydrogenase (LCHAD). The LCHAD exon 15 deletion was embryonic lethal to the homozygous mice whereas heterozygous mice (HT) develop significant hepatic steatosis starting at young age (3 months old) and HCC at older age (>13 months old) without any evidence of fibrosis or cirrhosis. None of the wild-type (WT) mice developed steatosis and HCC (n = 39), whereas HT-LCHAD mice (n = 41) showed steatosis and ~20% (8/41) developed liver masses with histological features of HCC. Proteomic analysis of liver tissues from WT-mice and HT-mice with no signs of HCC was conducted. Proteins with significant changes in abundance were identified by mass spectrometry. Abundance of 24 proteins was significantly different (p < 0.01) between WT and HT-LCHAD mice. The proteins found to vary in abundance are associated with different cellular response processes ranging from intermediary metabolism of carbohydrate, protein and lipid to oxidative stress, signal transduction and the process of tumorigenesis. Protein expression pattern of the HT-LCHAD mouse liver indicates predisposition to HCC and suggests that impaired hepatic mitochondrial fatty acid oxidation plays an important role in the development and progression of HCC. To assess the implication of these studies in human disease, we demonstrated significant downregulation of HADHA transcripts in HCC patients.

Cell surface Thomsen-Friedenreich proteome profiling of metastatic prostate cancer cells reveals potential link with cancer stem cell-like phenotype.

The tumor-associated Thomsen-Friedenreich glycoantigen (TF-Ag) plays an important role in hematogenous metastasis of multiple cancers. The LTQ Orbitrap LC-MS/MS mass spectrometry analysis of cell surface TF-Ag proteome of metastatic prostate cancer cells reveals that several cell surface glycoproteins expressing this carbohydrate antigen in prostate cancer (CD44, α2 integrin, ß1 integrin, CD49f, CD133, CD59, EphA2, CD138, transferrin receptor, profilin) are either known as stem cell markers or control important cancer stem-like cell functions. This outcome points to a potential link between TF-Ag expression and prostate cancer stem-like phenotype. Indeed, selecting prostate cancer cells for TF-Ag expression resulted in the enrichment of cells with stem-like properties such as enhanced clonogenic survival and growth, prostasphere formation under non-differentiating and differentiating conditions, and elevated expression of stem cell markers such as CD44 and CD133. Further, the analysis of the recent literature demonstrates that TF-Ag is a common denominator for multiple prostate cancer stem-like cell populations identified to date and otherwise characterized by distinct molecular signatures. The current paradigm suggests that dissemination of tumor cells with stem-like properties to bone marrow that occurred before surgery and/or radiation therapy is largely responsible for disease recurrence years after radical treatment causing a massive clinical problem in prostate cancer. Thus, developing means for destroying disseminated prostate cancer stem-like cells is an important goal of modern cancer research. The results presented in this study suggest that multiple subpopulation of putative prostate cancer stem-like cells characterized by distinct molecular signatures can be attacked using a single target commonly expressed on these cells, the TF-Ag.

Controlled modification of biomolecules by ultrashort laser pulses in polar liquids.

Targeted chemical modification of peptides and proteins by laser pulses in a biologically relevant environment, i.e. aqueous solvent at room temperature, allows for accurate control of biological processes. However, the traditional laser methods of control of chemical reactions are applicable only to a small class of photosensitive biomolecules because of strong and ultrafast perturbations from biomolecule-solvent interactions. Here, we report excitation of harmonics of vibration modes of solvent molecules by femtosecond laser pulses to produce controlled chemical modifications of non-photosensitive peptides and proteins in polar liquids under room conditions. The principal modifications included lysine formylation and methionine sulfoxidation both of which occur with nearly 100% yield under atmospheric conditions. That modification occurred only if the laser irradiance exceeded certain threshold level. The threshold, type, and extent of the modifications were completely controlled by solvent composition, laser wavelength, and peak irradiance of ultrashort laser pulses. This approach is expected to assist in establishing rigorous control over a broad class of biological processes in cells and tissues at the molecular level.

Quantitative Proteomics of Zea mays Hybrids Exhibiting Different Levels of Heterosis.

Maize hybrids exhibiting heterosis (hybrid vigor) were generated from inbred parents with increasing genetic distance. B73 was used as the common female parent in crosses with N192 (low heterosis), MO17 (high-heterosis 1), and NC350 (high-heterosis 2). Total and mitochondria-enriched proteomes were analyzed from ear shoots of field-grown hybrids and their inbred parents. GeLCMS (1D SDS-PAGE fractionation, trypsin digestion, LTQ Orbitrap nano-RP-LC MS/MS) was used to analyze proteins, and spectral counting was used for quantitation. In total, 3,568 proteins were identified and quantified in hybrids including 2,489 in the mitochondria-enriched fraction and 2,162 in the total protein fraction. Sixty-one proteins were differentially abundant (p < 0.05) in one or both of the high-heterosis hybrids compared with the low-heterosis hybrid. For the total proteome, eight of these showed similar trends in abundance in both of the higher-heterosis hybrids. Nine proteins showed this heterosis-correlated pattern in the mitochondrial proteome, including a mitochondria-associated target of rapamycin (TOR) protein. Although differentially abundant proteins belong to various pathways, protein, and RNA metabolism, and stress responsive proteins were the major classes changed in response to increasing heterosis.

Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission.

Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity.

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization.

Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide-mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways.

A novel regulatory mechanism based upon a dynamic core structure for the mitochondrial pyruvate dehydrogenase complex?

The Arabidopsis thaliana genome includes three genes for mitochondrial dihydrolipoamide acetyltransferase, the E2-component of the mitochondrial pyruvate dehydrogenase complex (PDC). Two genes encode E2-proteins with a single lipoyl domain, while the third has a two-lipoyl domain structure. Transcripts for each E2 protein were expressed in all plant organs. Each recombinant AtmtE2 can individually form an icosahedral PDC core structure, and results from bimolecular fluorescence complementation assays are consistent with formation of hetero-core structures from all permutations of the AtmtE2 proteins. We propose a unique regulatory mechanism involving dynamic formation of hetero-core complexes that include both mono- and di-lipoyl forms of AtmtE2.

Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.

Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit-subunit interaction sites in cataract-causing mutant αA-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker-containing peptides. In this study, we cross-linked wild-type (αA-WT) and mutant (αA-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d0 and d4) cross-linker-BS²G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88-K99 in the ß5 strand of the mutant αA-G98R crystallin that is not found in wild-type αA-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly.

The αA66-80 peptide interacts with soluble α-crystallin and induces its aggregation and precipitation: a contribution to age-related cataract formation.

Formation of protein aggregates in the aging eye lens has been shown to correlate with progressive accumulation of specific low-molecular weight (LMW) peptides derived from crystallins. Prominent among the LMW fragments is αA66-80, a peptide derived from αA-crystallin and present at higher concentrations in the water-insoluble nuclear fractions of the aging lens. The αA66-80 peptide has amyloid-like properties and preferentially insolubilizes α-crystallin from soluble lens fractions. However, the specific interactions and mechanisms by which the peptide induces α-crystallin aggregation have not been delineated. To gain insight into the mechanisms of peptide-induced aggregation, we investigated the interactions of the peptide with α-crystallin by various biochemical approaches. The peptide weakens α-crystallin chaperone ability and drastically promotes α-crystallin aggregation via the formation of insoluble peptide-protein complexes through transient intermediates. 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid studies suggest that the peptide induces changes in the hydrophobicity of α-crystallin that could trigger the formation and growth of aggregates. The peptide-α-crystallin aggregates were found to be resistant to dissociation by high ionic strengths, whereas guanidinium hydrochloride and urea were effective dissociating agents. We conclude that the αA66-80 peptide forms a hydrophobically driven, stable complex with α-crystallin and reduces its solubility. Using isotope-labeled chemical cross-linking and mass spectrometry, we show that the peptide binds to multiple sites, including the chaperone site, the C-terminal extension, and subunit interaction sites in αB-crystallin, which may explain the antichaperone property of the peptide and the consequential age-related accumulation of aggregated proteins. Thus, the α-crystallin-derived peptide could play a role in the pathogenesis of cataract formation in the aging lens.

Specific changes in total and mitochondrial proteomes are associated with higher levels of heterosis in maize hybrids.

The phenomenon of hybrid vigor (heterosis) has long been harnessed by plant breeders to improve world food production. However, the changes that are essential for heterotic responses and the mechanisms responsible for heterosis remain undefined. Large increases in biomass and yield in high-heterosis hybrids suggest that alterations in bioenergetic processes may contribute to heterosis. Progeny from crosses between various inbred lines vary in the extent of vigor observed. Field-grown maize F1 hybrids that consistently exhibited either low or high heterosis across a variety of environments were examined for changes in proteins that may be correlated with increased plant vigor and yield. Unpollinated ears at the time of flowering (ear shoots) were selected for the studies because they are metabolically active, rich in mitochondria, and the sizes of the ears are diagnostic of yield heterosis. Total protein and mitochondrial proteomes were compared among low- and higher-heterosis hybrids. Two-dimensional difference gel electrophoresis was used to identify allelic and/or isoform differences linked to heterosis. Identification of differentially regulated spots by mass spectrometry revealed proteins involved in stress responses as well as primary carbon and protein metabolism. Many of these proteins were identified in multiple spots, but analysis of their abundances by label-free mass spectrometry suggested that most of the expression differences were due to isoform variation rather than overall protein amount. Thus, our proteomics studies suggest that expression of specific alleles and/or post-translational modification of specific proteins correlate with higher levels of heterosis.