The discovery and optimization of biomolecules that reliably function in metazoan cells is imperative for both the study of basic biology and the treatment of disease. We describe the development, ...characterization, and proof-of-concept application of a platform for directed evolution of diverse biomolecules of interest (BOIs) directly in human cells. The platform relies on a custom-designed adenovirus variant lacking multiple genes, including the essential DNA polymerase and protease genes, features that allow us to evolve BOIs encoded by genes as large as 7 kb while attaining the mutation rates and enforcing the selection pressure required for successful directed evolution. High mutagenesis rates are continuously attained by trans-complementation of a newly engineered, highly error-prone form of the adenoviral polymerase. Selection pressure that couples desired BOI functions to adenoviral propagation is achieved by linking the functionality of the encoded BOI to the production of adenoviral protease activity by the human cell. The dynamic range for directed evolution can be enhanced to several orders of magnitude via application of a small-molecule adenoviral protease inhibitor to modulate selection pressure during directed evolution experiments. This platform makes it possible, in principle, to evolve any biomolecule activity that can be coupled to adenoviral protease expression or activation by simply serially passaging adenoviral populations carrying the BOI. As proof-of-concept, we use the platform to evolve, directly in the human cell environment, several transcription factor variants that maintain high levels of function while gaining resistance to a small-molecule inhibitor. We anticipate that this platform will substantially expand the repertoire of biomolecules that can be reliably and robustly engineered for both research and therapeutic applications in metazoan systems.
Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered. Here, we demonstrate that ...the presence or absence of a single cysteine in type-I collagen's C-propeptide domain is a key factor governing the ability of a given collagen polypeptide to stably homotrimerize. We also identify a critical role for Ca
in non-covalent collagen C-propeptide trimerization, thereby priming the protein for disulfide-mediated covalent immortalization. The resulting cysteine-based code for stable assembly provides a molecular model that can be used to predict, a priori, the identity of not just collagen homotrimers, but also naturally occurring 2:1 and 1:1:1 heterotrimers. Moreover, the code applies across all of the sequence-diverse fibrillar collagens. These results provide new insight into how evolution leverages disulfide networks to fine-tune protein assembly, and will inform the ongoing development of designer proteins that assemble into specific oligomeric forms.
The dorsal fin is one of the most varied swimming structures in Acanthomorpha, the spiny‐finned fishes. This fin can be present as a single contiguous structure supported by bony spines and soft ...lepidotrichia, or it may be divided into an anterior, spiny dorsal fin and a posterior, soft dorsal fin. The freshwater fish family Percidae exhibits especially great variation in dorsal fin spacing, including fishes with separated fins of varying gap length and fishes with contiguous fins. We hypothesized that fishes with separated dorsal fins, especially those with large gaps between fins, would have stiffened fin elements at the leading edge of the soft dorsal fin to resist hydrodynamic loading during locomotion. For 10 percid species, we measured the spacing between dorsal fins and calculated the second moment of area of selected spines and lepidotrichia from museum specimens. There was no significant relationship between the spacing between dorsal fins and the second moment of area of the leading edge of the soft dorsal fin.
In fishes the dorsal fin can be a single contiguous structure (red) or it may be separated (blue) into an anterior, spiny dorsal fin and a posterior, soft dorsal fin. We hypothesized that fishes with separated dorsal fins would have stiffened fin elements at the leading edge of the soft dorsal fin in order to resist hydrodynamic loading during swimming. Our micro‐CT‐data showed no significant difference in cross‐sectional shape or size between the leading edge of the soft dorsal fin in fishes with separated fins and the equivalent fin element in fishes with contiguous fins.
Background
Defective clearance of tau by neuronal autophagy has been implicated in the accumulation of toxic forms of tau in Alzheimer’s disease (AD). A novel, endogenous activator of ...chaperone‐mediated autophagy (CMA‐modulator) was identified in cell culture experiments (Cuervo laboratory, unpublished data). It is unclear what role this endogenous CMA‐modulator plays in AD. We hypothesized that loss of the CMA‐modulator in neurons could lead to decreased clearance of abnormal tau monomers, promoting the accumulation of toxic forms of tau. This study examined the proportion of neurons with CMA‐modulator in postmortem brain tissue from subjects with a range of AD neuropathologic changes as well as both amnestic and non‐amnestic AD presentation.
Method
We used multiplex immunofluorescence (CMA‐modulator, NeuN for neurons, 1F3C for acetyl‐tau, AT100 for phospho‐tau) on tissue microarrays in the inferior temporal gyrus of 17 subjects (Table 1) and for each subject quantified neurons within a gray matter area of 7.4 ‐ 7.6 mm2 using digital pathology. To analyze the change in percentage of neurons positive for CMA‐modulator across Braak stages, we fit a linear regression model.
Result
In amnestic AD, the percentage of neurons positive for CMA‐modulator declined linearly with advancing Braak stage, but this trend was not statistically significant (slope = 2.42% loss per Braak stage, p = 0.086). The predicted percentage of positive neurons was 22.8% at Braak stage 0 and 8.25% at Braak stage 6 and amnestic presentation. All non‐amnestic cases were Braak stage 6, and the proportion of neurons positive for the CMA‐modulator varied greatly from 0.2% to 77.1%. There was no significant association between the percentage of CMA‐modulator positive neurons and either age or gender. Quantification for the acetyl‐tau and phospho‐tau markers is ongoing.
Conclusion
There may be a gradual loss of the endogenous CMA‐modulator across Braak stages in amnestic AD, but this study was underpowered to draw definitive conclusions. We are increasing the sample size.
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