Kilodalton Protein Research Articles

Activation of the NLRP3 inflammasome by human adenovirus type 7 L4 100-kilodalton protein.

Human adenovirus type 7 (HAdV-7) is a significant viral pathogen that causes respiratory infections in children. Currently, there are no specific antiviral drugs or vaccines for children targeting HAdV-7, and the mechanisms of its pathogenesis remain unclear. The NLRP3 inflammasome-driven inflammatory cascade plays a crucial role in the host's antiviral immunity. Our previous study demonstrated that HAdV-7 infection activates the NLRP3 inflammasome. Building upon this finding, our current study has identified the L4 100 kDa protein encoded by HAdV-7 as the primary viral component responsible for NLRP3 inflammasome activation. By utilizing techniques such as co-immunoprecipitation, we have confirmed that the 100 kDa protein interacts with the NLRP3 protein and facilitates the assembly of the NLRP3 inflammasome by binding specifically to the NACHT and LRR domains of NLRP3. These insights offer a deeper understanding of HAdV-7 pathogenesis and contribute to the development of novel antiviral therapies.

Phosphorylation regulates viral biomolecular condensates to promote infectious progeny production

Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral BMCs to limit viral multiplication.

Molecular-based study of scrub typhus in Kerala, South India from 2014 to 2021: a laboratory-based study.

The online version contains supplementary material available at 10.1007/s00580-023-03443-8.

Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates

Periplasmic chaperones 17-kilodalton protein (Skp) and survival factor A (SurA) are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA associations with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy-entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multifaceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.

Thymidine Kinase 2 and Mitochondrial Protein COX I in the Cerebellum of Patients with Spinocerebellar Ataxia Type 31 Caused by Penta-nucleotide Repeats (TTCCA)n

Spinocerebellar ataxia type 31 (SCA31), an autosomal-dominant neurodegenerative disorder characterized by progressive cerebellar ataxia with Purkinje cell degeneration, is caused by a heterozygous 2.5–3.8 kilobase penta-nucleotide repeat of (TTCCA)n in intron 11 of the thymidine kinase 2 (TK2) gene. TK2 is an essential mitochondrial pyrimidine-deoxyribonucleoside kinase. Bi-allelic loss-of-function mutations of TK2 lead to mitochondrial DNA depletion syndrome (MDS) in humans through severe (~ 70%) reduction of mitochondrial electron-transport-chain activity, and tk2 knockout mice show Purkinje cell degeneration and ataxia through severe mitochondrial cytochrome-c oxidase subunit I (COX I) protein reduction. To clarify whether TK2 function is altered in SCA31, we investigated TK2 and COX I expression in human postmortem SCA31 cerebellum. We confirmed that canonical TK2 mRNA is transcribed from exons far upstream of the repeat site, and demonstrated that an extended version of TK2 mRNA (“TK2-EXT”), transcribed from exons spanning the repeat site, is expressed in human cerebellum. While canonical TK2 was conserved among vertebrates, TK2-EXT was specific to primates. Reverse transcription-PCR demonstrated that both TK2 mRNAs were preserved in SCA31 cerebella compared with control cerebella. The TK2 proteins, assessed with three different antibodies including our original polyclonal antibody against TK2-EXT, were detected as ~ 26 kilodalton proteins on western blot; their levels were similar in SCA31 and control cerebella. COX I protein level was preserved in SCA31 compared to nuclear DNA-encoded protein. We conclude that the expression and function of TK2 are preserved in SCA31, suggesting a mechanism distinct from that of MDS.

P-001: Modulation of soluble B-cell maturation antigen levels in patients with relapsed and/or refractory multiple myeloma after treatment with teclistamab and talquetamab

<h3>Background</h3> B-cell maturation antigen (BCMA, CD269), a single transmembrane protein, can be shed as a soluble 6 kilodalton protein fragment (sBCMA) upon cleavage at the transmembrane domain by γ-secretase. Given its selective expression in the B-cell lineage, BCMA is a validated target for multiple myeloma. Teclistamab and talquetamab are CD3 bispecific antibodies developed to recruit CD3+ T cells to BCMA+ or G protein-coupled receptor family C group 5 member D (GPRC5D)+ multiple myeloma (MM) cells, respectively. Levels of sBCMA after teclistamab or talquetamab treatment were evaluated in patients with relapsed and/or refractory MM (RRMM). <h3>Methods</h3> Serum samples from patients with RRMM in teclistamab and talquetamab phase 1 studies (64007957MMY1001 and 64407564MMY1001, respectively) were collected at various timepoints between baseline and cycle 4 or end of treatment and analyzed for sBCMA by an electrochemiluminescence ligand binding assay. Quantitative analyses of sBCMA data were conducted with reference to a patient's tumor burden and response, and to pharmacokinetic data. <h3>Results</h3> Teclistamab and talquetamab modulated levels of sBCMA in patients with high (≥50%) and low (<50%) frequency of tumor plasma cells (TPCs) and in high and low risk cytogenetic groups. In cycle 3, a majority of the responders showed sBCMA reductions (teclistamab: 88% [50/57]; talquetamab: 98% [49/50]) compared with baseline. In contrast, non-responders (progressive disease/ stable disease/ minimal response) showed an increase in sBCMA (teclistamab: 80% [33/41]; talquetamab: 49% [24/49]) compared with baseline. A higher magnitude of sBCMA reductions was noted in patients with deep responses. In a few patients who initially responded to teclistamab or talquetamab and then relapsed, sBCMA levels trended toward an initial reduction followed by an increase. Levels of sBCMA correlated with % bone marrow TPCs. A majority of patients with plasmacytoma (based on limited data) appeared to have high sBCMA levels, suggesting that sBCMA could be a comprehensive marker for tumor burden. Teclistamab preliminary population pharmacokinetic analysis showed that sBCMA did not appear to impact teclistamab exposure, suggesting that sBCMA does not act as a sink for teclistamab. <h3>Conclusions</h3> Changes in the levels of sBCMA upon treatment with teclistamab and talquetamab correlated with clinical activity, supporting further clinical development. The findings support BCMA as a potential surrogate marker of myeloma tumor burden and as a valuable marker for response in patients with MM.

Structural Analysis of the cl-Par-4 Tumor Suppressor as a Function of Ionic Environment

Prostate apoptosis response-4 (Par-4) is a proapoptotic tumor suppressor protein that has been linked to a large number of cancers. This 38 kilodalton (kDa) protein has been shown to be predominantly intrinsically disordered in vitro. In vivo, Par-4 is cleaved by caspase-3 at Asp-131 to generate the 25 kDa functionally active cleaved Par-4 protein (cl-Par-4) that inhibits NF-κB-mediated cell survival pathways and causes selective apoptosis in tumor cells. Here, we have employed circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) to assess the effects of various monovalent and divalent salts upon the conformation of cl-Par-4 in vitro. We have previously shown that high levels of sodium can induce the cl-Par-4 fragment to form highly compact, highly helical tetramers in vitro. Spectral characteristics suggest that most or at least much of the helical content in these tetramers are non-coiled coils. Here, we have shown that potassium produces a similar effect as was previously reported for sodium and that magnesium salts also produce a similar conformation effect, but at an approximately five times lower ionic concentration. We have also shown that anion identity has far less influence than does cation identity. The degree of helicity induced by each of these salts suggests that the “Selective for Apoptosis in Cancer cells” (SAC) domain—the region of Par-4 that is most indispensable for its apoptotic function—is likely to be helical in cl-Par-4 under the studied high salt conditions. Furthermore, we have shown that under medium-strength ionic conditions, a combination of high molecular weight aggregates and smaller particles form and that the smaller particles are also highly helical, resembling at least in secondary structure, the tetramers found at high salt.

A phase I open-label study to investigate safety and tolerability, efficacy, pharmacokinetics, pharmacodynamics, and immunogenicity of MT-5111 in patients with HER2-positive tumors.

TPS258 Background: Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action &amp; are capable of forcing internalization, self-routing through intracellular compartments to the cytosol &amp; inducing potent cell-kill via the enzymatic &amp; permanent inactivation of ribosomes. MT-5111 is a de-immunized ETB targeting HER2+ solid tumors. Its novel mechanism of action, via enzymatic ribosome inactivation, may not be subject to resistance mechanisms that exist for tyrosine kinase inhibitors, antibody-drug conjugates, or antibody modalities. MT-5111 binds an epitope on HER2, distinct from trastuzumab or pertuzumab, that may provide for combination potential with other HER2-targeting agents. MT-5111 is a 55 kilodalton protein &amp; may have improved tumor penetration capability. The objective of this trial will be to determine the safety, tolerability, &amp; maximum tolerated dose (MTD) of MT-5111 in patients (pts) with advanced HER2+ solid tumors. Methods: This Phase 1, first-in-human, open-label, dose escalation &amp; expansion study will evaluate MT-5111 monotherapy in pts with HER2-positive solid tumors. The primary objective is to determine the MTD; secondary objectives include pharmacokinetics, tumor response &amp; immunogenicity. Part 1 consists of MT-5111 dose escalation (0.5, 1.0, 2.0, 3.0, 4.5, 6.75, 10µg/kg/dose) based on a modified 3+3 design (n≤42 pts); Part 2 (dose expansion) will evaluate MT-5111 at the MTD in ≤98 pts. All pts will be administered MT-5111 over 30 min via IV infusion on Days 1, 8, &amp; 15 of each 21-day cycle until disease progression, unacceptable toxicity, death, withdrawal of consent, or another reason for withdrawal. Part 1 will include pts with any HER2+ solid cancers. Part 2 will enroll 3 expansion cohorts: HER2+ breast (BC), HER2+ gastric or gastroesophageal junction adenocarcinomas (collectively referred as gastroesophageal adenocarcinomas [GEA]) &amp; other HER2+ solid cancers. Immunohistochemistry (IHC) status must be 2+ or 3+, regardless of in situ hybridization (ISH) results; if no IHC is available for pts with BC or GEA, ISH criteria per the American Society of Clinical Oncology College of American Pathologists guidelines will be used. In metastatic cases, HER2 positivity must be demonstrated on metastatic lesions. Pts with HER2+ BC should have had ≥2 lines of HER2-directed therapy; pts with HER2+ GEA should have received or been intolerant to trastuzumab. Pts with evaluable disease may be included in Part 1; in Part 2, all pts must have ≥1 measurable lesion per Response Evaluation Criteria in Solid Tumors v1.1. Further details can be found on clinicaltrials.gov (NCT04029922). Enrollment, which began in September 2019, is ongoing. Clinical trial information: NCT04029922.

The Sensitivity of the Pair-Angle Distribution Function to Protein Structure

The continued development of X-ray free-electron lasers and serial crystallography techniques has opened up new experimental frontiers. Nanoscale dynamical processes such as crystal growth can now be probed at unprecedented time and spatial resolutions. Pair-angle distribution function (PADF) analysis is a correlation-based technique that has the potential to extend the limits of current serial crystallography experiments, by relaxing the requirements for crystal order, size and number density per exposure. However, unlike traditional crystallographic methods, the PADF technique does not recover the electron density directly. Instead it encodes substantial information about local three-dimensional structure in the form of three- and four-body correlations. It is not yet known how protein structure maps into the many-body PADF correlations. In this paper, we explore the relationship between the PADF and protein conformation. We calculate correlations in reciprocal and real space for model systems exhibiting increasing degrees of order and secondary structural complexity, from disordered polypeptides, single alpha helices, helix bundles and finally a folded 100 kilodalton protein. These models systems inform us about the distinctive angular correlations generated by bonding, polypeptide chains, secondary structure and tertiary structure. They further indicate the potential to use angular correlations as a sensitive measure of conformation change that is complementary to existing structural analysis techniques.

Rec A disruption unveils cross talk between DNA repair and membrane damage, efflux pump activity, biofilm formation in Mycobacterium smegmatis

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has emerged in recent decades as one of the leading causes of mortality worldwide. The burden of TB is alarmingly high, with one third affected global population as reported by WHO. Short-course treatment with an antibiotic is a powerful weapon to treat infection of susceptible MTB strain, however; MTB has developed resistance to anti-TB drugs, which is an escalating global health crisis. Thus there is urgent need to identify new drug targets. RecA is a 38 kilodalton protein required for the repair and maintenance of DNA and regulation of the SOS response. The objective of this study is to understand the effect of disruption of RecA gene (deletion mutant ΔdisA from previous study) in a surrogate model for MTB, Mycobacterium smegmatis. This study demonstrated that disruption of RecA causes enhanced susceptibility towards rifampicin and generation of ROS leading to lipid peroxidation and impaired membrane homeostasis as depicted by altered cell membrane permeability and efflux pump activity. Mass spectrometry based lipidomic analysis revealed decreased mycolic acid moieties, phosphatidylinositol mannosides (PIM), Phthiocerol dimycocerosate (DIM). Furthermore, biofilm formation was considerably reduced. Additionally, we have validated all the disrupted phenotypes by RT-PCR which showed a good correlation with the biochemical assays. Lastly, RecA mutant displayed reduced infectivity in Caenorhabditis elegans illustrating its vulnerability as antimycobacterial target. Together, present study establishes a link between DNA repair, drug efflux and biofilm formation and validates RecA as an effective drug target. Intricate studies are needed to further understand and exploit this therapeutic opportunity.

Abstract P1-18-35: MT-5111, a novel HER2 targeting engineered toxin body, under clinical development to overcome mechanisms of resistance to existing HER2 targeted therapies

Abstract Engineered toxin bodies (ETBs) are differentiated, targeted therapeutics comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLTA) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forcing internalization, self-routing through intracellular compartments to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes. MT-5111 is a de-immunized ETB targeting HER2 for solid tumors. MT-5111 works through a novel mechanism of direct cell-kill, via enzymatic ribosome inactivation, and may not be subject to resistance mechanisms that exist for TKI, ADC, or antibody modalities. MT-5111 is a 55 kilodalton protein and may have improved tumor penetration features in the solid tumor settings. MT-5111 has been designed to bind a HER2 domain distinct from the trastuzumab and pertuzumab binding sites and retains binding to HER2 and cell-kill activity even in the presence of these monoclonal antibodies. As such, MT-5111 may have the potential to be combined with other HER2 targeted therapies. MT-5111 specifically binds and kills HER2 expressing cells in a manner consistent with SLTA-mediated cellular cytotoxicity. MT-5111 effectively killed eight of nine cell lines with moderate to high HER2 surface expression, five of which were breast cancer cell lines. No cytotoxicity was observed on multiple HER2-negative cell lines at MT-5111 levels &amp;gt;500 fold higher than IC50 levels in HER2-positive cells. The cytotoxic activity of MT-5111 (picomolar range) is dependent on the presence of the HER2 receptor, and efficient killing is observed prior to receptor saturation. As a large molecule protein, MT-5111 is not a substrate of drug efflux transporters such as MDR1 which has been demonstrated to be one of the primary mechanisms of resistance to the antibody drug conjugate T-DM1. Specifically, T-DM1 resistant cell lines with moderate HER2 expression (JIMT-1 breast cancer and SNU-216 gastric cancer lines) were sensitive to MT-5111 but were not effectively killed by T-DM1, highlighting the benefit of a novel MOA to treat resistant disease. Further, MT-5111 has demonstrated effective cell-killing in vitro against trastuzumab resistant cell lines (HCC1954 breast cancer). MT-5111 binds both human and cynomolgus monkey HER2 protein similarly. From repeat dose studies in non-human primates, the serum exposure of MT-5111 was used to model the pharmacokinetics of MT-5111 dosing in human. This modelling suggests that MT-5111 can be administered at doses in humans above the IC50 required for HER2-specific cellular cytotoxicity in vitro. MT-5111 was designed to achieve a short half-life to allow for efficient tumor cell targeting but minimal serum exposure time to avoid systemic effects over time. This short half-life was confirmed in primates (t1/2 of ~2 to 5 hours). A Phase 1, first in human, open-label dose escalation, and expansion study of MT-5111 (NCT04029922) in subjects with HER2-positive solid tumors whose disease has progressed after treatment with other approved therapies is open for enrollment. In conclusion, MT-5111 represents a novel HER2 targeted therapy which could provide benefit in subjects with HER2-positive cancers and potentially overcome mechanisms of tumor resistance to existing HER2 targeted therapies. Citation Format: Jack P Higgins, Asis Sarkar, Eric T Williams, Aimee Iberg, Roger Waltzman, Erin K Willert. MT-5111, a novel HER2 targeting engineered toxin body, under clinical development to overcome mechanisms of resistance to existing HER2 targeted therapies [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-35.

MT-5111: A novel HER2 targeting engineered toxin body in clinical development.

433 Background: Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered Shiga-like Toxin A subunit genetically fused to antibody-like binding domains. MT-5111 is a de-immunized ETB targeting HER2 for solid tumors. MT-5111 works through a novel mechanism of direct cell-kill, via enzymatic ribosome inactivation, and may not be subject to resistance mechanisms that exist for TKI, ADC, or antibody modalities. MT-5111 binds an epitope on HER2 distinct from trastuzumab or pertuzumab, that may provide for combination potential with other HER2 targeting agents. MT-5111 is a 55 kilodalton protein and may have improved tumor penetration capability in solid tumor settings. Methods: HER2 expression and activity of MT-5111 was assessed in vitro by flow cytometry and cell viability assays. Serum exposure and tolerability of MT-5111 was measured in non-human primate (NHP) studies. Results: MT-5111 effectively kills 8/9 cell lines (2 gastric) with moderate to high HER2 surface expression, as well as two additional gastric cell lines with lower HER2 expression. No cytotoxicity is observed on multiple HER2- cell lines. As a protein, MT-5111 is not a substrate of drug efflux transporters that limit efficacy of ADCs. MT-5111 demonstrates effective cell-killing in vitro against cell lines expressing HER2 but resistant to trastuzumab (HCC1954) or T-DM1 (JIMT-1 and gastric SNU-216), highlighting the benefit of a novel mechanism of action to treat resistant disease. MT-5111 binds human and NHP HER2 protein. Based on serum exposure of MT-5111 in NHPs used to model pharmacokinetics, planned MT-5111 dosing in humans is above the IC50 required for HER2-specific cellular cytotoxicity in vitro. MT-5111 has a short half-life that, while allowing for efficient tumor cell targeting, minimizes serum exposure to avoid systemic effects over time. Conclusions: A Phase 1, first in human, open-label dose escalation and expansion study of MT-5111 (NCT04029922) in subjects with HER2+ solid tumors whose disease has progressed after treatment with other approved therapies is open for enrollment. MT-5111 represents a novel HER2 targeted therapy for patients with HER2+ cancers with potential to overcome mechanisms of tumor resistance to existing therapies.

Structures of Unfolded Outer Membrane Proteins in Complex with Chaperones

The viability of Gram-negative bacteria is ensured by the presence of outer membrane proteins (OMPs) acting as channels, porins, lipases and receptors. They are considered as interesting drug targets and thus obtaining a molecular perspective about various stages of their biogenesis is of great interest. However, the biogenesis of OMPs is biologically puzzling because it is not driven by ATP. We lack a detailed understanding of the various steps involved in this process. Unlike other proteins, OMPs adopt an active conformation only on reaching the outer membrane and thus have to be transferred from the cytoplasm through the periplasm in a folding competent state. Chaperones like Seventeen kilodalton protein (Skp) and Survival Factor A (SurA) protect these nascent hydrophobic proteins against aggregation. Yet, it remains elusive which conformations and interaction dynamics occur in the chaperone and unstructured OMP interplay. Using single molecule Förster Resonance Energy Transfer (smFRET), we investigate the structure of unfolded OMPs in chaperone-bound and unbound states. We find that both OMPs adopt a rather compact conformation in a low denaturant buffer. Remarkably, when bound with periplasmic chaperones, we observe that this compact state is dissolved by the chaperones differently for both OMPs. The small 8 beta-stranded OmpX shows heterogeneous conformations with sub-millisecond dynamics, while the 12 beta-stranded Outer membrane phospholipase A (OmpLA) appears to be pulled and pushed upon by the chaperones in expanded conformations. In both the cases, the chaperone-OMP interactions were remarkably long-lasting for hours, even below the dissociation constant. Our findings hint towards mechanisms by which OMPs are arranged in a folding competent state before reaching the outer membrane and shed light on the elusive mechanism of chaperone-OMP interaction in periplasm.

Nomenclature of factor XI and the contact system

Nomenclature of factor XI and the contact system

Abstract 1768: Biochemical and structural analysis of the Neurofibromin (NF1) protein and a potential role for protein destabilization in Rasopathy diseases

Abstract Neurofibromin is the 320 kilodalton protein product of the NF1 gene which is mutated in the Rasopathy disease Neurofibromatosis Type I. Defects in NF1 lead to abrogation of the GTPase activating activity of the protein, leading to aberrant signaling through the RAS-MAPK pathway. Very little is known about the function of the majority of the NF1 protein—to date only the GAP domain and a region containing a Sec-PH motif have detailed structural information. To better understand the role of this large protein, we have carried out a series of biochemical and biophysical studies which clearly show that full length NF1 protein exists primarily as a dimer. Data from in vivo experiments confirmed that this dimerization also occurs in cells, and presumably has biological implications. A model was generated from negative-stain EM data which suggests that NF1 monomers form both intermolecular contacts to form dimers and intramolecular contacts which stabilize the individual monomers. Deletion analysis permitted us to map these two interacting domains to different parts of the NF1 protein, and crosslinking studies coupled with mass spectrometry helped to identify regions of possible interaction. In addition, introduction of point mutations observed in patients with NF diseases caused a dramatic decrease in the stability of proteins both in vitro and in vivo, potentially arguing for a rationale for loss of NF1 activity by these single mutations Citation Format: Mukul Sherekar, Sae-Won Han, Simon Messing, Matthew Drew, William Gillette, Claire Lorenzo, Frank McCormick, Dominic Esposito. Biochemical and structural analysis of the Neurofibromin (NF1) protein and a potential role for protein destabilization in Rasopathy diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1768.

Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication.

Here, we show that the cellular DNA replication protein and ATR substrate SMARCAL1 is recruited to viral replication centers early during adenovirus infection and is then targeted in an E1B-55K/E4orf6- and cullin RING ligase-dependent manner for proteasomal degradation. In this regard, we have determined that SMARCAL1 is phosphorylated at S123, S129, and S173 early during infection in an ATR- and CDK-dependent manner, and that pharmacological inhibition of ATR and CDK activities attenuates SMARCAL1 degradation. SMARCAL1 recruitment to viral replication centers was shown to be largely dependent upon SMARCAL1 association with the RPA complex, while Ad-induced SMARCAL1 phosphorylation also contributed to SMARCAL1 recruitment to viral replication centers, albeit to a limited extent. SMARCAL1 was found associated with E1B-55K in adenovirus E1-transformed cells. Consistent with its ability to target SMARCAL1, we determined that E1B-55K modulates cellular DNA replication. As such, E1B-55K expression initially enhances cellular DNA replication fork speed but ultimately leads to increased replication fork stalling and the attenuation of cellular DNA replication. Therefore, we propose that adenovirus targets SMARCAL1 for degradation during infection to inhibit cellular DNA replication and promote viral replication.IMPORTANCE Viruses have evolved to inhibit cellular DNA damage response pathways that possess antiviral activities and utilize DNA damage response pathways that possess proviral activities. Adenovirus has evolved, primarily, to inhibit DNA damage response pathways by engaging with the ubiquitin-proteasome system and promoting the degradation of key cellular proteins. Adenovirus differentially regulates ATR DNA damage response signaling pathways during infection. The cellular adenovirus E1B-55K binding protein E1B-AP5 participates in ATR signaling pathways activated during infection, while adenovirus 12 E4orf6 negates Chk1 activation by promoting the proteasome-dependent degradation of the ATR activator TOPBP1. The studies detailed here indicate that adenovirus utilizes ATR kinase and CDKs during infection to promote the degradation of SMARCAL1 to attenuate normal cellular DNA replication. These studies further our understanding of the relationship between adenovirus and DNA damage and cell cycle signaling pathways during infection and establish new roles for E1B-55K in the modulation of cellular DNA replication.

Free and Chaperone Bound Unfolded States of Outer Membrane Proteins

Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with chaperones. In the case of bacterial outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because they remain unfolded for extended periods of time during their biogenesis and rely on interactions with periplasmic chaperones to prevent aggregation and support correct folding. Here, we study the unfolded-state properties of outer-membrane proteins. Using a combination of ensemble and single-molecule spectroscopy techniques including single-molecule FRET, we find that under strongly denaturing conditions and in the absence of chaperones, OMPs adopt conformationally heterogeneous unfolded states that lack the fast chain reconfiguration motions expected for an unstructured, fully unfolded chain. Interestingly, when complexed with periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA), we observe for the protein OmpLA adopts an expanded, yet squished conformational ensemble, in which the overall broad distribution of unfolded states is preserved. Differently sized OMPs like OmpX (8 β-strands) and OmpF (16 β-strands) allow a broader perspective on chaperone enabled conformations of OMPs. These findings indicate that periplasmic chaperones Skp and SurA prestructure OMPs for their next binding partner (e.g., the β-barrel assembly machinery (BAM)) to facilitate insertion and proper folding into the outer membrane.

Immunization With Skp Delivered on Outer Membrane Vesicles Protects Mice Against Enterotoxigenic Escherichia coli Challenge.

Outer membrane vesicles (OMVs) are promising vaccine components because they combine antigen and adjuvant in a single formulation. Detoxified Salmonella enterica strains that express penta-acylated lipid A retain OMV immunogenicity but with reduced reactogenicity. We have previously shown that a recombinant form of the enterotoxigenic Escherichia coli (ETEC) 17 kilodalton protein (Skp) protects mice in a pulmonary challenge model, when fused to the glutathione-S-transferase (GST) epitope and combined with cholera toxin. Here we compared directly the efficacy of expressing Skp in detoxified Salmonella OMVs to GST-Skp for their ability to protect mice against ETEC challenge. We observed that the display of Skp on OMVs, in the absence of exogenous adjuvant, protects the mice as well as the recombinant GST-Skp with adjuvant, showing that we can achieve protection when antigen and adjuvant are administered as a single formulation. Collectively, these data demonstrate the utility of using OMVs for the expression and display of antigens for use in vaccine development and validate previously published work demonstrating that immunization with Skp is efficacious in protecting mice against ETEC challenge.

The Long and Short of Synaptonemal Complex Assembly: Investigating the genesis and functional relevance of a smaller Zip1 isoform

The proper segregation of homologous chromosomes (homologs) during meiosis is a paramount task for living cells. Homologs successfully disjoin from one another on the meiosis I spindle so long as they have undergone prior alignment and have formed at least one crossover attachment. The Zip1 protein promotes an intimate alignment between homologs during budding yeast meiosis through its role as a building block of the synaptonemal complex (SC), a proteinaceous structure that assembles along the full length of aligned partner chromosomes. Independent of its role in building SC, Zip1 also plays a critical role in promoting interhomolog crossover recombination, although the molecular underpinnings of this activity are unclear. Through a structure‐function study to identify residues that are essential for Zip1's crossover recombination or SC assembly functions, we identified two zip1 mutants that produce an unexpectedly smaller form of Zip1. In meiotic cells that express either a zip1 allele in which six contiguous residues are changed to alanine, or an allele in which only two of the aforementioned six residues are changed to alanine, a large fraction of Zip1 migrates on a protein gel as if it has lost ten kilodaltons of its wild‐type molecular weight. The change in apparent molecular weight of Zip1 observed in our mutants raises the unanticipated possibility that most Zip1 proteins within the wild‐type meiotic cell are constitutively modified, perhaps by a ten kilodalton protein, and that this post‐translational modification is dependent on the residues that are altered in our two zip1 mutant alleles. Here we describe the results of two experiments designed to ask whether Zip1 in wild‐type meiotic nuclei is constitutively modified by the SUMO protein. First, we will examine the apparent size of Zip1 protein when two SUMO ligase proteins, Siz1 and Siz2, are simultaneously removed. Second, we will determine whether Zip1's molecular weight appears increased in a strain that expresses a larger molecular weight version of SUMO, 3xHA‐SUMO. Finally, we will examine SC assembly and crossover recombination in the two zip1 mutant strains that produce the putative unmodified form of Zip1, in order to determine a function for this potential post‐translational modification.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Clinical Relevance of Cystatin C as a Renal Marker in Dogs with Chronic Mitral Valve Insufficiency

Cystatin C is a low molecular weight 13 kilodalton protein. It is known to be a more sensitive marker of glomerular filtration rate than creatinine in humans. The purpose of the present study was to demonstrate the changes of renal markers including cystatin C according to the severity of chronic mitral valve insufficiency (CMVI) and to investigate the clinical relevance of cystatin C as an early renal marker in dogs with CMVI. A retrospective study was performed to assess renal function according to International Small Animal Cardiac Health Council (ISACHC) system classification of heart failure in dogs with CMVI. Thirty seven dogs were divided into a group 1 (healthy dogs ; n = 10), a group 2 (ISACHC I ; n = 10) and a group 3 (ISACHC II-III ; n = 17). In all dogs, serum concentrations of bun (sUr), creatinine (sCr) and cystatin C (sCys-C) were measured with an automated analyzer. In dogs with CMVI, sCys-C concentrations were significantly correlated with sCr concentrations and were independent of age, BW, SBP, and sex. Renal dysfunction tended to occur more frequently as the severity of CMVI increases. In dogs with mild CMVI, only sCys-C concentrations were statistically higher than in healthy dogs. This study demonstrates the clinical relevance of sCys-C. sCys-C may be a valuable renal marker for early diagnosis of renal dysfunction in dogs with CMVI.