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STAT

An MUSC blog
Keyword: nephrology

  The Structure of the Myo1c-Neph1 Complex

 

 

 

 

 

 

 

 

 

 Summary: Researchers at the MUSC used small-angle X-ray scattering to determine the full structure of the motor protein Myo1c and it complex with Neph1, a protein crucial for kidney filtration. Their findings suggest that Myo1c uses the actin cytoskeleton as a “track” for Neph1 transport—a finding with translational relevance for glomerular diseases such as diabetic nephropathy, as movement of Neph1 to and from the surface membrane triggers the injury/recovery response.

The motor protein Myo1c binds to Neph1, a protein crucial for ensuring effective filtration by the kidney, and serves as one mode of its cellular transport, according to findings by investigators at the Medical University of South Carolina (MUSC) and their collaborators report in an article in press at Molecular and Cellular Biology.

Neph1 is essential for the maintenance of podocytes—neuron-like cells with long finger-like projections that wrap around the glomerular capillaries, serving as the final barrier between the blood and the urine. When podocyte structure fails, the kidney is no longer able to act as an effective filter, and excessive protein escapes the capillaries into the urine. The resulting proteinuria can lead to kidney failure over time. Faulty podocyte structure and filtration also characterize diabetic nephropathy and glomerular diseases (e.g., focal and segmental glomerular sclerosis), which were once considered orphan diseases but have become more prevalent in recent years as sedentary lifestyles and diets high in processed foods have become commonplace.

The MUSC investigators and their collaborators used small-angle X-ray scattering (SAXS) to determine the full structure of Myo1c and its complex with Neph1. The crystallographic structure of two of Myo1c’s three segments had previously been determined but never that of the entire protein or the complex it forms with another protein.

 “Having a 3D crystal structure is more like a snapshot of the protein in three-dimensional space but of more physiological relevance is understanding how the proteins behave in solution,” says senior author Deepak Nihalani, Ph.D., an Associate Professor in the College of Medicine, Division of Nephrology, at MUSC. “We did a solution-based structure where we could actually get the structure of the entire protein and study that structure in complex with a cargo protein (Neph1).”

Nihalani’s team sent solutions of purified Myo1c to Brookhaven National Laboratory for SAXS analysis, examined the resulting SAXS intensity profiles to determine the structure of Myo1c in solution, and used molecular modeling to fill in the gaps of the 3D crystal structure. They also showed that Neph1 binds to the C tail region of Myo1c.

These findings suggest that Myo1c uses the actin cytoskeleton as a “track” for cellular transport of its Neph1 cargo (bound to its C tail). The movement of Neph1 through cellular space is known to be linked to the injury/recovery response. When injury occurs, Neph1 and other surface proteins are dislodged into the cytoplasm and must find their way back to the surface of the cell membrane to trigger the events needed for recovery.

As a means of transport for Neph1, Myo1c likely plays an important role in the injury/recovery response as well. However, it is unclear whether Myo1c favors renal protection or injury since it is not yet known in which direction Myo1c transports Neph1.

If it carries Neph1 from the surface into the cytoplasm and perinuclear space, it could be associated with more severe glomerular disease. In that case, Myo1c could be an attractive therapeutic target, as inhibiting it would prevent these proteins from leaving the cell membrane, an event that triggers the injury response. Over time, the inflammation associated with the injury response can lead to renal damage. If instead it ferries Neph1 back to the cell membrane, it could be essential in recovery after injury.

“These are early findings, but they show that Myo1c is critical to the transport of Neph1. Understanding whether that transport contributes to protection or injury could have translational importance for the treatment of glomerular diseases,” says Nihalani.

To solve that mystery, Nihalani and his collaborators have developed a mouse model in which Myo1c is knocked out in the podocytes and are conducting experiments to better understand the effects of Myo1c knockout on the injury/recovery response. They also hope to study whether Myo1c forms complexes with other proteins key for the maintenance of healthy renal function.

Image Caption: The Structural Domain of the Myo1c-Neph1 Complex. Image courtesy of  Dr. Deepak Nihalani. Reproduced from Molecular and Cellular Biology (10.1128/MCB.00020-16), with permission of the American Society of Microbiology.

Aberrant phenotypes in zebrafish after Tuba knockdown

Image Caption: Aberrant phenotypes resulting from Tuba knockdown in zebrafish. Image courtesy of Dr. Joshua Lipschutz.

Summary: Zebrafish help investigators at the Medical University of South Carolina shed light on the mechanisms underlying cilia dysfunction in polycystic kidney disease and other ciliopathies

In an article published online ahead of print on February 19, 2015 in the Journal of Biological Chemistry (JBC), investigators at the Medical University of South Carolina (MUSC) and the Ralph H. Johnson VA Medical Center report findings from in vitro and in vivo studies that elucidate the mechanisms underlying the impaired ciliogenesis and abnormal kidney development characteristic of polycystic kidney disease (PKD). Depletion of dynamin-binding protein or Tuba, a guanine nucleotide exchange factor, disrupted renal ciliogenesis in cell culture and led to abnormal kidney morphology in a Tuba knockdown zebrafish model of PKD.

Currently, no drug has been approved by the U.S. Food and Drug Administration to treat autosomal dominant PKD, which affects a half million Americans and more than 12 million people worldwide. The disease is characterized by the development of fluid-filled cysts in both kidneys, leading to end-stage renal disease, usually around age 50 to 60. In PKD, it is speculated that dysfunctional cilia are unable to detect the presence of urine flow, triggering reactivation of developmental pathways, which lead to the uncontrolled production of cysts that eventually destroy the kidney.

Cilia, the finger-like protrusions on most epithelial cells, were not so long ago thought to be as irrelevant to cell biology as the appendix is to physiology, a vestigial remnant of a long ago evolutionary past.  Today, they are recognized as essential chemo-mechanical sensors that monitor and regulate what crosses into and out of a cell. Dysfunctional cilia are now known to be implicated in not only PKD but a wide range of diseases affecting the eyes, ears, heart, and other organs. Understanding how cilia become dysfunctional in these diseases could provide insight into how to better treat or prevent them.  

“How are cilia made? If you know that, you can figure out what goes wrong in ciliopathies, including polycystic kidney disease,” says nephrologist Joshua H. Lipschutz, M.D., the senior author on the article, who holds a dual appointment at MUSC and the Ralph H. Johnson VA Medical Center. 

Much must go right for ciliogenesis to occur. Proteins necessary for ciliogenesis are manufactured in the endoplasmic reticulum before traveling to the trans-Golgi network to be sorted into “zip-coded packages” or vesicles for transport to the cilia. Lipschutz and others previously showed that the exocyst, a protein targeting complex, plays a crucial role in receiving these “zip-coded packages” containing ciliary proteins.  The GTPase Cdc42 regulates the exocyst, which is the mailbox where these “packages” are received in the kidney.  Renal ciliogenesis occurs only when the packaged proteins are delivered to the Cdc42-activated exocyst complex. Depleting either the exocyst or Cdc42 disrupts renal ciliogenesis.

In the JBC article, Lipschutz and his MUSC coauthors go a step further—showing in cell culture and a zebrafish model that depletion of Tuba, a guanine nucleotide exchange factor required for Cdc42 activation, also disrupts renal ciliogenesis. Tuba is thought to ensure that the Cdc42/exocyst mailbox is in place at the base of the cilia and ready to receive the packaged proteins. Without Tuba, Cdc42 is not appropriately activated, and the exocyst is mislocalized, so the undelivered packages continue to pile up, perhaps playing a role in the uncontrolled production of renal cysts in PKD.

When grown in a collagen gel, Madin-Darby canine kidney (MDCK) cells form into cysts, and the orientation of proteins, called polarity, are abnormal following Tuba knockdown.  Specifically, apical proteins that would normally face the urinary space are mislocalized throughout the cell. 

In zebrafish, injection of low doses of both Tuba and Cdc42 antisense morpholinos, which had no effect when administered separately, led to severe phenotypes similar to those seen following knockdown of other ciliary proteins. This is called genetic synergy and provides further evidence that both Tuba and Cdc42 are part of the same pathway. Because knockdown of Tuba in zebrafish affects cilia in a number of organs, including the brain, a variety of aberrant phenotypes were seen in the Tuba knockdown zebrafish model.

Lipschutz, who directs the zebrafish core at MUSC along with co-author Seok-Hyung Kim, Ph.D., is well aware of the advantages of the zebrafish for research—its genome is well characterized, it can be bred rapidly and inexpensively, and its transparent body enables easy visualization of aberrations under microscopy. However, the next step in this line of research will be to study the effects of Tuba depletion in the kidneys of mice, since murine kidneys are more like human kidneys than those of zebrafish.

Once the pathways underlying impaired ciliogenesis in PKD are more fully understood, therapeutic interventions can be designed to disrupt those pathways.  As Lipschutz notes, “We do this research to help our patients. Further elucidating the pathways that underlie impaired ciliogenesis is an essential step in beginning to develop treatment options for PKD and other ciliopathies.”

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