Biological structure

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/4897

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De Novo Design of Light-Powered Protein Motor Domains
(2026-02-05) Demakis, Cullen William; Baker, David
A grand challenge in protein design is the design of molecular machines. These machines integrate energy inputs to undergo cycles of conformational change and perform work at the molecular scale. Such machines are critical in nature–transporting cargo, driving the formation of chemical gradients, and generating tissue-level motions. However, such machines have long been beyond the reach of protein design. Combining challenges in multi-state design with the necessity for energetic inputs and coordination between domains, complex functions are difficult to design for directly. In this work the problem is simplified–designing the motor domains that translate energetic inputs into motion. The design of de novo proteins with large, rigid-body conformational changes is described and functionalization of these protein switches as light-powered motors is detailed. This work establishes both a toolset of components for machine design and methods for generation of additional motor domains with altered characteristics, enabling protein design to approach the capabilities of natural machines and tailor de novo machines to problems not yet addressed by nature.
The role of V4 in object motion processing
(2023-04-17) Bigelow, Anthony; Pasupathy, Anitha
We rely heavily on our visual system for many functions. More specifically, the ability to process and track moving objects is of utmost importance. Much of the literature and conventional teachings in neuroscience hypothesize that the visual system has two functionally distinct pathways: a dorsal visual pathway for motion processing and a ventral visual pathway for form processing. However, this functional dichotomy is slowly eroding – specifically, many neurons along the ventral visual pathway demonstrate the ability to process the motion of objects. We focused our attention on ventral visual area V4, the most interconnected visual area of the visual system. We sought to understand how motion processing in V4 contrasts and complements motion processing in key dorsal stream areas, such as areas MT and MST. Many studies have shown the critical role that the dorsal visual stream plays in processing and perceiving motion information yet gaps remain in our knowledge of visual information. We find that neurons in V4 show selectivity for the motion of objects, rather than motion behind an aperture (i.e., V4 neurons preferentially encode the motion of stimuli with explicit boundaries translating across the visual field rather than static stimuli). These results demonstrate a novel and complementary motion processing mechanism in the ventral stream.
An ancient neurotrophin code in zebrafish; the Runx/Cbfβ complex specifies the fate switch from TrkB+ to TrkC+ nociceptors and sensory receptor expression
(2016-07-14) Gau, Philia; Dhaka, Ajay; Raible, David
The somatosensory system is a critical interface to the environment. It contains a highly diverse group of sensory neurons that are responsible for sensing touch, temperature, pain, and proprioception. The Runx transcription factors have been identified as key factors in generating some somatosensory neuron diversity. However, it is not well understood how these factors function. This work establishes the functional significance of the zebrafish somatosensory system by focusing on the nociceptive ion channel TRPV1 and explores the interaction of the Runx transcription factors with their cofactor Cbfβ during somatosensory neuron development. This work demonstrates that Cbfβ is an obligate cofactor for the Runx transcription factors and is required for somatosensory neuron specification, suggests that Runx1 and Runx3 perform the same function in fish, and uncovers an ancient neurotrophin code in zebrafish.
Structural and biochemical studies of protein poly(ADP-ribosyl)ation
(2013-04-17) Wang, Zhizhi; Xu, Wenqing
Protein poly(ADP-ribosyl)ation (PARylation) has been found to be involved in various cellular processes, such as DNA damage recognition and repair, chromatin structure dynamics, gene transcription and poly(ADP-ribose)-dependent cell death. Most recently, PARylation has been shown to control the polyubiquitination and degradation of Axin, a key regulator of the Wnt signaling pathway. RNF146, which contains a WWE domain and a RING domain, is the only proven E3 ubiquitin ligase to date that requires PARylation of the substrate for subsequent polyubiquitination. The RNF146 WWE domain has been shown to bind poly(ADP-ribose) (PAR). In this thesis, first I review the current knowledge on protein PARylation. Then I describe my doctoral research on the structural and biochemical studies on how WWE domain recognizes PAR polymer. My studies reveal the structural basis of the RNF146 WWE domain/iso-ADP-ribose (iso-ADPR, the internal unit of PAR) interaction and, for the first time, define the PAR/iso-ADPR binding as a bona fide function of the WWE domain family. This suggests that PAR may be a signal for protein ubiquitination and this signal is decoded by WWE domain-containing E3 ubiquitin ligases. In Chapter 3, I describe my structural studies on poly(ADP-ribose) glycohydrolase (PARG), which is the principal enzyme responsible for the degradation of PAR polymers in vivo. By solving the structures of the mouse PARG catalytic domain and its complex with its inhibitor ADP-HPD, I reveal how the PAR polymer as a substrate is recognized by PARG. The structures also suggest how the N terminal flexible peptide preceding the PARG catalytic domain regulates the enzymatic activity of PARG. This study helps the understanding of PARG catalytic and regulatory mechanisms as well as the rational design of PARG inhibitors.
Notch signaling in cell-fate specification and maintenance in the developing and adult mammalian inner ear
(2009) Hartman, Byron H.
The inner ear contains the auditory and vestibular organs, which are sensory neuroepithelia composed of mechanosensory hair cells and glia-like supporting cells. The mammalian auditory sensory epithelium, the organ of Corti, is responsible for transduction of sound stimuli into electrical signals for hearing function. Hair cells are susceptible to damage from noise, toxins, and ageing. Mammals do not regenerate hair cells after damage, but nonmammalian vertebrates are capable of hair cell regeneration. Replacement of lost hair cells in lower vertebrates occurs through plasticity of supporting cells, which directly change fate into hair cells or proliferate to produce new cells through a recapitulation of developmental mechanisms. However, supporting cells in the mature mammalian cochlea do not exhibit the potential to change fate or proliferate in order to regenerate lost sensory cells. However, during embryonic development, hair cells and supporting cells develop from common precursors and the fate of individual cells is regulated through intercellular signaling and genetic mechanisms. The overall goal of this research is to further understand the mechanisms of specification and maintenance of sensory cell fates in the developing and adult mammalian inner ear. Understanding the way in which cell types are developed and maintained is a crucial step in overcoming the barriers to regeneration in the mammalian inner ear. A key regulator of development in the inner ear is the Notch signaling pathway, a highly conserved cell signaling system present in most multicellular organisms. In this dissertation, after a review of relevant literature, I will describe our investigation of Notch signaling in the developing and adult mammalian inner ear. We conducted comprehensive gene expression analyses and describe novel expression patterns of two Notch ligands, Dll3 and DNER, and a key Notch effector, Hes5. We found that the Notch pathway is highly active during cochlear development but appears to be absent from the adult cochlea. We performed several transgenic gain-of-function experiments, which show that Notch signal activation during development is capable of conferring prosensory character to regions of nonsensory epithelia. However, constitutive activation of Notch in mature cochlear supporting cells was not sufficient to confer regeneration potential.
Development of the lateral musculature in the teleost, Brachydanio rerio;: a fine structural study
(1967) Waterman, Robert Earle, 1940-
Cell adhesion molecules in human hair follicle morphogenesis
(1996) Kaplan, Elizabeth Danford
Hair follicle formation in fetal development is characterized morphologically by invagination and elongation into the dermis of an epidermal cell collective in association with a follicle-specific population of mesenchymal cells. The process is thought to involve cell proliferation and migration, mediated by molecules such as NCAM and E-cadherin, which govern intercellular adhesion, and the extracellular matrix molecules tenascin-C (TN-C) and chondroitin sulfate proteoglycan, which affect cell-substrate adhesion. In research presented here, immunohistochemical analysis was used to examine their distribution patterns, along with those of the adhesion modulating molecules ICAM-1, alpha-2 beta-1 integrin, hyaluronan, versican and perlecan, in relation to human hair follicle morphogenesis. Alternative splicing of a central domain of TN-C has differential effects on cell adhesion and is associated with increased cell proliferation, so molecular probes were developed and used with isoform-specific antibodies for molecular and biochemical analyses of TN-C expression in relation to cell proliferation. Initiation of hair follicle morphogenesis was distinguished by discrete placodes of a small TN-C isoform which lacked the alternatively spliced domain, and which were present at the dermal-epidermal interface. The alternatively spliced domain was detected later in follicle development, and was distributed in the follicle epithelium, basement membrane, and the extracellular matrix of the follicle-specific mesenchyme. Northern and Western blot analysis resolved three mRNA transcripts of 6, 7, and 8 kb in length, and three protein isoforms of approximately $\rm220\times10\sp3,\ 250\times10\sp3\ and\ 280-300\times10\sp3$ kD. These showed temporally distinct expression patterns, and initiation was marked by maximal levels of the 6 kb mRNA at the same time as TN-C transcript was detected by in situ hybridization in collections of epidermal cells. Mesenchymal cell expression was not detected until later stages of development.Invagination was characterized by the transient presence of ICAM-1 on follicle epithelial cells, and by the overlapping distribution of TN-C, versican and hyaluronan in the basement membrane and follicle-associated mesenchyme, where cells were enriched in NCAM immunostaining. TN-C expression was associated with proliferation of epithelial and mesenchymal cells at the periphery of the developing appendage, while the core and base of the developing structure formed a non-proliferative zone. The distributions of versican and hyaluronan, although more widespread than that of TN-C, overlapped with TN-C in the proliferative region, and the three molecules were diminished or absent from the mitotically inactive regions. Preliminary observations included here suggest that TN-C and versican interact in vitro and may imparting a macromolecular organization to the follicle-specific extracellular matrix in vivo.
Regulation of retinal endothelial cells and pericytes by VEGF, TGF-beta1, and SPARC
(1998) Yan, Qi, 1962-
Ocular diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. In an effort to understand the components of the retinal angiogenesis and its regulation by various molecules, I developed a procedure to isolate retinal microvascular endothelial cells (EC) and pericytes from the primate and bovine species. This was important because retinal capillary EC are different from EC derived from other vascular beds. Retinal EC expressed von Willebrand factor, acetylated low-density lipoprotein, CD31, and did not contain smooth muscle a-actin. Monkey retinal EC could be maintained to passage 3, whereas bovine retinal EC could be maintained to passage 8. Subconfluent cultures of monkey retinal EC secreted extracellular matrix proteins that included fibronectin, laminin, collagen types I and IV, and SPARC. VEGF and bFGF both stimulated retinal EC replication, which was also elevated under hypoxia. The synergism of VEGF and bFGF on retinal EC proliferation and cell invasion was observed in a 3-dimensional assay but not in 2-dimensional dish culture.Unlike VEGF and bFGF, the role of transforming growth factor (TGF)-$\beta1$ in angiogenesis has been controversial. The effects of TGF-$\beta1$ on retinal microvascular cells are not fully defined. I have found that TGF-$\beta1$ inhibited the proliferation of both retinal EC and pericytes in a concentration-dependent manner. Moreover, TGF-$\beta1$ induced specifically apoptotic cell death in retinal EC but not in pericytes. Apoptosis of retinal EC mediated by TGF-$\beta1$ was associated with a decreased level of the cyclin-dependent kinase inhibitor p21$\rm\sp{waf1/cip1},$ compared with that observed in the apoptosis-resistant cells. In contrast, the translation product of the tumor suppressor gene p53 was increased in the TGF-$\beta1$-treated apoptotic cells, suggesting p53 mediated retinal EC apoptosis is independent of p21$\rm\sp{waf1/cip1}.$ Thus, I have proposed that p21$\rm\sp{waf1/cip1}$ and p53 function in distinct pathways that are protective or permissive, respectively, for the apoptotic signals mediated by TGF-$\beta1.$SPARC is expressed in a variety of tissues during embryogenesis and remodeling, and is believed to regulate vascular morphogenesis and cellular differentiation. Although usually limited in normal adult tissues, SPARC is expressed at significant levels in the adult bovine retina, whereas newborn calf retina showed a similar expression pattern with a greatly reduced level. Strong reactivity with anti-SPARC antibody was found in the soma of ganglion cells and their axons. SPARC was also present in retinal astrocytes but not in Mueller cells, capillaries of the inner retina but not large vessels. To determine the significance of SPARC expression in retina, I test two hypotheses. (1) SPARC has counteradhesive properties that can cause cell rounding, and would thus induce retinal EC apoptosis in vitro; (2) SPARC is one of the endogenous inhibitors that either maintain the quiescence of retinal EC under normal physiological conditions or inhibit retinal neovascularization. My conclusions are that SPARC or its peptides did not induce retinal EC apoptosis in vitro under the experimental conditions used; SPARC and its peptides were potent inhibitors of retinal EC replication, as they effectively inhibited EC proliferation induced by retinal extract. Future study will focus on testing the role of SPARC in retinal neovascularization through the use of wild-type vs. SPARC-null mice in an animal model of the retinopathy, and on delineating mechanisms whereby SPARC regulates retinal pericyte function.

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