General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.

Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with kmax/KM = 2.1 × 103 and 3.7 × 103 M-1 s-1, respectively, and an identical pKa = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design.

V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein.

Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini-mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this study, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔGf°) of -4.4 and -6.9 kcal mol-1 for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔVf) was significantly larger for V67 (71 ± 2 mL mol-1) than for L67 (58 ± 3 mL mol-1) inteins. The measured difference in ΔVf (13 ± 3 mL mol-1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. It is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.

A Case of Metastatic Urothelial Carcinoma-Associated Acute Esophageal Necrosis.

Acute esophageal necrosis is a rare syndrome with endoscopic findings of a diffuse circumferential pattern of black mucosa. Although underlying pathogenesis is unclear, it is known to have associations with malignancy. We present a rare case of a patient with a history of metastatic urothelial carcinoma who was found to have acute esophageal necrosis.

A Case of Pancreatic Ductal Adenocarcinoma in an Elderly Adult With Heterotaxy Syndrome.

Heterotaxy is a syndrome characterized by a spectrum of anatomical anomalies in organ lateralization due to embryological errors. It frequently involves intrathoracic organs, especially the heart, leading to congenital abnormalities. Abdominal organs can also be affected, causing clinical features such as sepsis from asplenia or intestinal volvulus; however, these are less studied. Currently, there is no data on the relationship between heterotaxy and malignancy. We present an interesting case of an elderly adult admitted for a workup of newly diagnosed pancreatic ductal carcinoma, who was found to have heterotaxy of the stomach and spleen, with eventual tumor invasion of these organs. This case suggests that heterotaxy may increase the risk of gastrointestinal malignancy and result in a poorer prognosis due to the complexity of tumor resection involving additional organs.

A Case of Primary Lung Adenocarcinoma With Metastasis to Colon Harboring EGFR Exon 19 Deletion.

Lung cancer metastasizing to the colon is exceedingly rare and can present similarly to colorectal cancer. It is crucial to conduct further evaluations using immunohistochemical (IHC) stains and genomic testing to differentiate between the two and provide appropriate treatment without delay. Lung cancer generally has a poor prognosis, especially in cases with distant metastases. Although gastrointestinal (GI) metastases from lung cancer have been reported, cases of lung cancer manifesting as colon metastasis are extremely rare, with only a few instances documented.

A Case of Inadequately Treated Bacterial Pneumonia Leading to Bronchopleural Fistula.

Bronchopulmonary fistula (BPF) is an abnormal connection between the pleural space and bronchial tree, potentially leading to fatal outcomes if untreated. While BPF commonly arises following lung surgery, it can also be linked to infections. This report details the case of a 47-year-old male with recent untreated bacterial pneumonia, who developed bilateral pneumothoraces with persistent air leaks, Pseudomonas and Aspergillus empyema, culminating in a right-sided BPF necessitating video-assisted thoracic surgery (VATS) decortication. The agenda of this presentation is to enhance early recognition of BPF, which can be presented subtly, to avert severe complications.