The opportunistic human pathogen, A. baumannii, senses and responds to light using the blue light sensing A (BlsA) photoreceptor protein. BlsA is a blue-light-using flavin adenine dinucleotide (BLUF) protein that is known to regulate a wide variety of cellular functions through interactions with different binding partners. Using immunoprecipitation of tagged BlsA in A. baumannii lysates, we observed a number of proteins that interact with BlsA, including several transcription factors. In addition to a known binding partner, the iron uptake regulator Fur, we identified the biofilm response regulator BfmR as a putative BlsA-binding partner. Using microscale thermophoresis, we determined that both BfmR and Fur bind to BlsA with nanomolar binding constants. To better understand how BlsA interacts with and regulates these transcription factors, we solved the X-ray crystal structures of BlsA in both a ground (dark) state and a photoactivated light state. Comparison of the light- and dark-state structures revealed that, upon photoactivation, the two α-helices comprising the variable domain of BlsA undergo a distinct conformational change. The flavin-binding site, however, remains largely unchanged from dark to light. These structures, along with docking studies of BlsA and Fur, reveal key mechanistic details about how BlsA propagates the photoactivation signal between protein domains and on to its binding partner. Taken together, our structural and biophysical data provide important insights into how BlsA controls signal transduction in A. baumannii and provides a likely mechanism for blue-light-dependent modulation of biofilm formation and iron uptake.

Mutations in leucine‐rich repeat kinase 2 (lrrk2) are the most common genetic cause of Parkinson's disease. Difficulty in elucidating the pathogenic mechanisms resulting from disease‐associated Lrrk2 variants stems from the complexity of Lrrk2 function and activities. Lrrk2 contains multiple protein–protein interacting domains, a GTPase domain, and a kinase domain. Lrrk2 is implicated in many cellular processes including vesicular trafficking, autophagy, cytoskeleton dynamics, and Wnt signaling. Here, we generated a zebrafish lrrk2 allelic series to study the requirements for Lrrk2 during development and to dissect the importance of its various domains. The alleles are predicted to encode proteins that either lack all functional domains (lrrk2sbu304), the GTPase, and kinase domains (lrrk2sbu71) or the kinase domain (lrrk2sbu96). All three lrrk2 mutants are viable, morphologically normal, and display wild‐type‐like locomotion. Because Lrrk2 modulates Wnt signaling in some contexts, we assessed Wnt signaling in all three mutant lines. Analysis of Wnt signaling by studying the expression of target genes using whole mount RNA in situ hybridization and a transgenic Wnt reporter revealed wild‐type domains of Wnt activity in each of the mutants. However, we found that Wnt pathway activation is attenuated in lrrk2sbu304/sbu304, which lacks both scaffolding and catalytic domains, but not in the other alleles during late embryogenesis. This supports a model in which Lrrk2 scaffolding functions are key to a context‐dependent role in promoting canonical Wnt signaling.

No abstract

Despite the poor long-term survival of patients with pancreatic ductal adenocarcinoma (PDA), there are significant survival probabilities in subgroups of patients that cannot be predicted by surgical, pathological, and clinical features, including stage, surgical margins and perineural invasion. Only in the last year, more than a handful papers reported advanced genomic analyses on multiple components of tumors to dissect molecular heterogeneities that may provide a better understanding of gene expression signatures to predict survival probabilities. Although gene-mutation and expression “omic” signatures hold promise, they are difficult to translate into clinical practice. Therefore, we initiated a two phase unbiased and systematic study to first identify and validate a prognostic biomarker by screening multiple independent RNASeq databases and performing survival analyses, on a total of 493 cases. Second, we performed immunohistochemical analyses on 117 cases to validate the in silico analysis results using a pathology based scoring system. We identified in the discovery dataset that keratin 17 (K17) expression discriminated between rapid and slower progressing PDA among patients with localized disease. K17-positive cases had four times the likelihood of dying from this disease compared to stage-matched K17-negative cases (P= 0.0094; median survival 1.64 years compared to >5 years, respectively). In addition, we found that positive-K17 patients have almost twice the probability of recurrence after chemotherapy, compared to matched stage, negative-K17 counterparts. These critical observations were validated in four independent PDA patient cohorts, indicating the negative prognostic value of K17 in this devastating disease, independent of the patient’s age, sex, and tumor stage and grade. Furthermore, we found that pathology-based quantification of K17 expression by immunohistochemistry in malignant cells was associated with poor-patient outcome (P= 0.0026). Based on the recent molecular “omics” subtyping PDA identified by Bailey et al., 2016 and Moffitt et al., 2015, we found that K17 expression can be used to distinguish the “squamous” and “basal” subtypes presented in these reports, respectively. Equally important, in addition to PDA, we found that high expression of K17 is strongly associated with decreased patient survival in a number of carcinomas, including but not limited to cancers of the lung, breast, liver, stomach. Results consistently indicated that K17 expression is associated with shortened interval to death (Hazard Ratio >2). Collectively, these results suggest that activation of K17 expression across multiple primary tumors is a conserved molecular event in tumors with shared clinical behavior and not anatomic location. Based on mechanistic approaches, we know that K17 is a regulator of cell-cycle progression and directly acts to enhance pancreatic tumor growth, providing insight into the mechanisms that drive PDA tumor biology as a major cause of patient mortality. In conclusion, K17 was identified as a robust and independent clinically relevant prognostic biomarker that may be used to better stratify clinical outcome at the time of initial diagnosis. Citation Format: Luisa Escobar-Hoyos, Elizabeth A. Vanner, Shula Schechter, Lucia Roa-Pena, Jinyu Li, Jinelle Wint, Scott Powers, Ali Akalin, Jela Bandovic, Peter Bailey, David K. Chang, Richard A. Moffit, Jen Jen Yeh, Andrew Biankin, Kenneth R. Shroyer.{Authors}. Translating keratin 17 status to stratify clinically relevant pancreatic cancer heterogeneity and survival. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B76.